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Sample records for preadipocyte cell line

  1. Cell line models of differentiation: preadipocytes and adipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The intense study of adipocyte biology spurred by interest in regulating body composition and metabolism has given rise to a number of in vitro cell models. These in vitro models have been invaluable in determining the mechanisms involved in adipocyte differentiation. In addition in vitro cell sys...

  2. Hydroxytyrosol Inhibits Cannabinoid CB1 Receptor Gene Expression in 3T3-L1 Preadipocyte Cell Line.

    PubMed

    Tutino, Valeria; Orlando, Antonella; Russo, Francesco; Notarnicola, Maria

    2016-02-01

    The 3T3-L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3-L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR-γ receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity-associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3-L1 pre-adipocytes the effects of n-3-PUFA, α-Linolenic acid (OM-3), n-6-PUFA, Linoleic acid (OM-6), and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis-related genes PPAR-γ, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3-L1 cell differentiation by down-regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti-adipogenic effects, whereas OM-3 and OM-6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM-3 and OM-6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription. PMID:26189725

  3. Soluble HMGB1 Is a Novel Adipokine Stimulating IL-6 Secretion through RAGE Receptor in SW872 Preadipocyte Cell Line: Contribution to Chronic Inflammation in Fat Tissue

    PubMed Central

    Nativel, Brice; Gunasekaran, Manoj Kumar; Andries, Jessica; Stanislas, Giovédie; Planesse, Cynthia; Da Silva, Christine Robert; Césari, Maya; Iwema, Thomas; Gasque, Philippe; Viranaicken, Wildriss

    2013-01-01

    Low-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto- and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes. PMID:24073286

  4. Characterization of ScAP-23, a new cell line from murine subcutaneous adipose tissue, identifies genes for the molecular definition of preadipocytes.

    PubMed

    Kim, Ji Young; Wu, Yu; Smas, Cynthia M

    2007-10-22

    The 3T3-L1 model of in vitro adipogenesis has provided key insights into the molecular nature of this process. However, given that 3T3-L1 are of an embryonic origin, it is not clear to what extent they represent adipogenesis as it occurs in white adipose tissue (WAT). With the goal of better defining preadipocytes and adipogenesis in WAT, we have generated a new cell culture model from adipocyte precursors present in C57BL/6 mouse subcutaneous WAT. ScAP-23 preadipocytes show fibroblastic morphology, and on treatment with dexamethasone, 3-methylisobutylxanthine, insulin, and indomethacin, convert to nearly 100% adipocyte morphology. ScAP-23 adipocytes contain abundant lipid droplets and express transcripts for PPAR gamma, C/EBP family, and SREBP-1c transcription factors, SCD1, aFABP, ATGL, GLUT4, FAS, LDL, and GPDH, and are insulin responsive. Differential screening of 1,176 genes using nylon DNA arrays identified 10 transcripts enriched in ScAP-23 adipocytes vs. preadipocytes and 26 transcripts enriched in ScAP-23 preadipocytes vs. adipocytes. Semiquantitative or real-time PCR analyses identified a common cohort of 14 transcripts markedly downregulated in both ScAP-23 and 3T3-L1 adipogenesis. These included catenin-beta1, chemokine ligand-2, serine or cysteine peptidase inhibitor f1, aurora kinase B, thrombospondin2, and solute carrier-7a5. Five of these transcripts (Ccl2, Serpinf1, Aurkb, Thbs2, and Slc7a5) demonstrated at least a twofold increase in WAT from obese (ob/ob) mice compared with that of wild-type mice. This suggests that comparative gene expression studies of ScAP-23 and 3T3-L1 adipogenesis may be particularly fruitful in identifying preadipocyte-expressed genes that play a role in adipose tissue physiology and/or pathophysiology. PMID:17609412

  5. Targeting exosomes from preadipocytes inhibits preadipocyte to cancer stem cell signaling in early-stage breast cancer

    PubMed Central

    Gernapudi, Ramkishore; Yao, Yuan; Zhang, Yongshu; Wolfson, Benjamin; Roy, Sanchita; Duru, Nadire; Eades, Gabriel; Yang, Peixin

    2015-01-01

    The tumor microenvironment plays a critical role in regulating breast tumor progression. Signaling between preadipocytes and breast cancer cells has been found to promote breast tumor formation and metastasis. Exosomes secreted from preadipocytes are important components of the cancer stem cell niche. Mouse preadipocytes (3T3L1) are treated with the natural antitumor compound shikonin (SK) and exosomes derived from mouse preadipocytes are co-cultured with MCF10DCIS cells. We examine how preadipocyte-derived exosomes can regulate early-stage breast cancer via regulating stem cell renewal, cell migration, and tumor formation. We identify a critical miR-140/SOX2/SOX9 axis that regulates differentiation, stemness, and migration in the tumor microenvironment. Next, we find that the natural antitumor compound SK can inhibit preadipocyte signaling inhibiting nearby ductal carcinoma in situ (DCIS) cells. Through co-culture experiments, we find that SK-treated preadipocytes secrete exosomes with high levels of miR-140, which can impact nearby DCIS cells through targeting SOX9 signaling. Finally, we find that preadipocyte-derived exosomes promote tumorigenesis in vivo, providing strong support for the importance of exosomal signaling in the tumor microenvironment. Our data also show that targeting the tumor microenvironment may assist in blocking tumor progression. PMID:25783182

  6. Targeting exosomes from preadipocytes inhibits preadipocyte to cancer stem cell signaling in early-stage breast cancer.

    PubMed

    Gernapudi, Ramkishore; Yao, Yuan; Zhang, Yongshu; Wolfson, Benjamin; Roy, Sanchita; Duru, Nadire; Eades, Gabriel; Yang, Peixin; Zhou, Qun

    2015-04-01

    The tumor microenvironment plays a critical role in regulating breast tumor progression. Signaling between preadipocytes and breast cancer cells has been found to promote breast tumor formation and metastasis. Exosomes secreted from preadipocytes are important components of the cancer stem cell niche. Mouse preadipocytes (3T3L1) are treated with the natural antitumor compound shikonin (SK) and exosomes derived from mouse preadipocytes are co-cultured with MCF10DCIS cells. We examine how preadipocyte-derived exosomes can regulate early-stage breast cancer via regulating stem cell renewal, cell migration, and tumor formation. We identify a critical miR-140/SOX2/SOX9 axis that regulates differentiation, stemness, and migration in the tumor microenvironment. Next, we find that the natural antitumor compound SK can inhibit preadipocyte signaling inhibiting nearby ductal carcinoma in situ (DCIS) cells. Through co-culture experiments, we find that SK-treated preadipocytes secrete exosomes with high levels of miR-140, which can impact nearby DCIS cells through targeting SOX9 signaling. Finally, we find that preadipocyte-derived exosomes promote tumorigenesis in vivo, providing strong support for the importance of exosomal signaling in the tumor microenvironment. Our data also show that targeting the tumor microenvironment may assist in blocking tumor progression. PMID:25783182

  7. 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.

  8. Expression Profiling of Preadipocyte MicroRNAs by Deep Sequencing on Chicken Lines Divergently Selected for Abdominal Fatness

    PubMed Central

    Wang, Weishi; Du, Zhi-Qiang; Cheng, Bohan; Wang, Yuxiang; Yao, Jing; Li, Yumao; Cao, Zhiping; Luan, Peng; Wang, Ning; Li, Hui

    2015-01-01

    Through posttranscriptional gene regulation, microRNA (miRNA) is linked to a wide variety of biological processes, including adipogenesis and lipid metabolism. Although miRNAs in mammalian adipogenesis have been worked on extensively, their study in chicken adipogenesis is still very limited. To find miRNAs potentially important for chicken preadipocyte development, we compared the preadipocyte miRNA expression profiles in two broiler lines divergently selected for abdominal fat content, by sequencing two small RNA libraries constructed for primary preadipocytes isolated from abdominal adipose tissues. After bioinformatics analyses, from chicken miRNAs deposited in miRBase 20.0, we identified 225 miRNAs to be expressed in preadipocytes, 185 in the lean line and 200 in the fat line (derived from 208 and 203 miRNA precursors, respectively), which corresponds to 114 miRNA families. The let-7 family miRNAs were the most abundant. Furthermore, we validated the sequencing results of 15 known miRNAs by qRT-PCR, and confirmed that the expression levels of most miRNAs correlated well with those of Solexa sequencing. A total of 33 miRNAs was significantly differentially expressed between the two chicken lines (P<0.05). Gene ontology analysis revealed that they could target genes enriched in the regulation of gene transcription and chromatin function, response to insulin stimulation, and IGF-1 signaling pathways, which could have important roles in preadipocyte development. Therefore, a valuable information and resource of miRNAs on chicken adipogenesis were provided in this study. Future functional investigations on these miRNAs could help explore related genes and molecular networks fundamental to preadipocyte development. PMID:25675096

  9. Adipose tissue stem cells meet preadipocyte commitment: going back to the future[S

    PubMed Central

    Cawthorn, William P.; Scheller, Erica L.; MacDougald, Ormond A.

    2012-01-01

    White adipose tissue (WAT) is perhaps the most plastic organ in the body, capable of regeneration following surgical removal and massive expansion or contraction in response to altered energy balance. Research conducted for over 70 years has investigated adipose tissue plasticity on a cellular level, spurred on by the increasing burden that obesity and associated diseases are placing on public health globally. This work has identified committed preadipocytes in the stromal vascular fraction of adipose tissue and led to our current understanding that adipogenesis is important not only for WAT expansion, but also for maintenance of adipocyte numbers under normal metabolic states. At the turn of the millenium, studies investigating preadipocyte differentiation collided with developments in stem cell research, leading to the discovery of multipotent stem cells within WAT. Such adipose tissue-derived stem cells (ASCs) are capable of differentiating into numerous cell types of both mesodermal and nonmesodermal origin, leading to their extensive investigation from a therapeutic and tissue engineering perspective. However, the insights gained through studying ASCs have also contributed to more-recent progress in attempts to better characterize committed preadipocytes in adipose tissue. Thus, ASC research has gone back to its roots, thereby expanding our knowledge of preadipocyte commitment and adipose tissue biology. PMID:22140268

  10. Ramie Leaf Extracts Suppresses Adipogenic Differentiation in 3T3-L1 Cells and Pig Preadipocytes

    PubMed Central

    Lee, Joomin; Kim, Ah-Ra; Lee, Jae-Joon

    2016-01-01

    The present study was carried out to evaluate the anti-obesity effect of different concentrations of extracts of hot air-dried ramie leaf (HR) and freeze-dried ramie leaf (FR) in 3T3-L1 cells and pig preadipocytes. To analyze the effect on cell proliferation, cells were treated with 25 μg/mL or 100 μg/mL HR or FR extract for 2 days. Cell differentiation was evaluated by measuring glycerol-3-phosphate dehydrogenase and lipoprotein lipase (LPL) activities and intracellular triglyceride content. Treatment with either HR or FR extracts inhibited the proliferation of 3T3-L1 cells and pig preadipocytes in a dose-dependent manner. HR extract treatment inhibited the differentiation of both cell types more effectively than FR treatment. The extent of triglyceride accumulation decreased significantly in both cells following either HR or FR treatment. Furthermore, LPL activity significantly decreased after treatment with HR or FR extract. These results indicated that HR and FR extracts may inhibit proliferation and differentiation of 3T3-L1 cells and pig preadipocytes. Further studies are needed to explore the anti-obesity effect of HR and FR extracts. PMID:26954122

  11. Ramie Leaf Extracts Suppresses Adipogenic Differentiation in 3T3-L1 Cells and Pig Preadipocytes.

    PubMed

    Lee, Joomin; Kim, Ah-Ra; Lee, Jae-Joon

    2016-09-01

    The present study was carried out to evaluate the anti-obesity effect of different concentrations of extracts of hot air-dried ramie leaf (HR) and freeze-dried ramie leaf (FR) in 3T3-L1 cells and pig preadipocytes. To analyze the effect on cell proliferation, cells were treated with 25 μg/mL or 100 μg/mL HR or FR extract for 2 days. Cell differentiation was evaluated by measuring glycerol-3-phosphate dehydrogenase and lipoprotein lipase (LPL) activities and intracellular triglyceride content. Treatment with either HR or FR extracts inhibited the proliferation of 3T3-L1 cells and pig preadipocytes in a dose-dependent manner. HR extract treatment inhibited the differentiation of both cell types more effectively than FR treatment. The extent of triglyceride accumulation decreased significantly in both cells following either HR or FR treatment. Furthermore, LPL activity significantly decreased after treatment with HR or FR extract. These results indicated that HR and FR extracts may inhibit proliferation and differentiation of 3T3-L1 cells and pig preadipocytes. Further studies are needed to explore the anti-obesity effect of HR and FR extracts. PMID:26954122

  12. 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

  13. 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.

  14. Cell-Autonomous Brown-Like Adipogenesis of Preadipocytes From Retinoblastoma Haploinsufficient Mice.

    PubMed

    Petrov, Petar D; Palou, Andreu; Bonet, M Luisa; Ribot, Joan

    2016-09-01

    Mechanisms behind the emergence of brown adipocyte-like (brite or beige) adipocytes within white adipose tissue (WAT) are of interest. Retinoblastoma protein gene (Rb) haploinsufficiency associates in mice with improved metabolic regulation linked to a greater capacity for fatty acid oxidation and thermogenesis in WAT. We aimed to explain a feasible mechanism of WAT-to-BAT remodeling in this model. Differentiated primary adipocytes and Sca1-positive preadipocytes derived from adipose depots of Rb(+/-) mice and wild-type siblings were compared. Primary white Rb(+/-) adipocytes displayed under basal conditions increased glucose uptake and an enhanced expression of brown adipocyte-related genes (Pparg, Ppargc1a, Ppargc1b, Prdm16, Cpt1b) but not of purported beige/brite transcriptional markers (Cd137, Tmem26, Tbx1, Slc27a1, Hoxc9, Shox2). Lack of induction of beige markers phenocopied results in WAT of adult Rb(+/-) mice. Flow cytometry analysis evidenced an increased number of preadipocytes in WAT depots of Rb(+/-) mice. Sca1(+) preadipocytes from WAT of Rb(+/-) mice displayed increased gene expression of several transcription factors common to the brown and beige adipogenic programs (Prdm16, Pparg, Ppargc1a) and of receptors of bone morphogenetic proteins (BMPs); however, among the recently proposed beige markers, only Tbx1 was upregulated. Adult Rb(+/-) mice had increased circulating levels of BMP7. These results indicate that preadipose cells resident in WAT depots of Rb(+/-) mice retain an increased capacity for brown-like adipogenesis that appears to be different from beige adipogenesis, and suggest that the contribution of these precursors to the Rb(+/-) adipose phenotype is driven, at least in part, by interaction with BMP7 pathways. J. Cell. Physiol. 231: 1941-1952, 2016. © 2016 Wiley Periodicals, Inc. PMID:26727985

  15. Preadipocyte factor 1 induces pancreatic ductal cell differentiation into insulin-producing cells

    PubMed Central

    Rhee, Marie; Lee, Seung-Hwan; Kim, Ji-Won; Ham, Dong-Sik; Park, Heon-Seok; Yang, Hae Kyung; Shin, Ju-Young; Cho, Jae-Hyoung; Kim, Young-Bum; Youn, Byung-Soo; Sul, Hei Sook; Yoon, Kun-Ho

    2016-01-01

    The preadipocyte factor 1 (Pref-1) is involved in the proliferation and differentiation of various precursor cells. However, the intracellular signaling pathways that control these processes and the role of Pref-1 in the pancreas remain poorly understood. Here, we showed that Pref-1 induces insulin synthesis and secretion via two independent pathways. The overexpression of Pref-1 activated MAPK signaling, which induced nucleocytoplasmic translocation of FOXO1 and PDX1 and led to the differentiation of human pancreatic ductal cells into β-like cells and an increase in insulin synthesis. Concurrently, Pref-1 activated Akt signaling and facilitated insulin secretion. A proteomics analysis identified the Rab43 GTPase-activating protein as a downstream target of Akt. A serial activation of both proteins induced various granular protein syntheses which led to enhanced glucose-stimulated insulin secretion. In a pancreatectomised diabetic animal model, exogenous Pref-1 improved glucose homeostasis by accelerating pancreatic ductal and β-cell regeneration after injury. These data establish a novel role for Pref-1, opening the possibility of applying this molecule to the treatment of diabetes. PMID:27044861

  16. Preadipocyte factor 1 induces pancreatic ductal cell differentiation into insulin-producing cells.

    PubMed

    Rhee, Marie; Lee, Seung-Hwan; Kim, Ji-Won; Ham, Dong-Sik; Park, Heon-Seok; Yang, Hae Kyung; Shin, Ju-Young; Cho, Jae-Hyoung; Kim, Young-Bum; Youn, Byung-Soo; Sul, Hei Sook; Yoon, Kun-Ho

    2016-01-01

    The preadipocyte factor 1 (Pref-1) is involved in the proliferation and differentiation of various precursor cells. However, the intracellular signaling pathways that control these processes and the role of Pref-1 in the pancreas remain poorly understood. Here, we showed that Pref-1 induces insulin synthesis and secretion via two independent pathways. The overexpression of Pref-1 activated MAPK signaling, which induced nucleocytoplasmic translocation of FOXO1 and PDX1 and led to the differentiation of human pancreatic ductal cells into β-like cells and an increase in insulin synthesis. Concurrently, Pref-1 activated Akt signaling and facilitated insulin secretion. A proteomics analysis identified the Rab43 GTPase-activating protein as a downstream target of Akt. A serial activation of both proteins induced various granular protein syntheses which led to enhanced glucose-stimulated insulin secretion. In a pancreatectomised diabetic animal model, exogenous Pref-1 improved glucose homeostasis by accelerating pancreatic ductal and β-cell regeneration after injury. These data establish a novel role for Pref-1, opening the possibility of applying this molecule to the treatment of diabetes. PMID:27044861

  17. Cell Lines

    PubMed Central

    Cherbas, Lucy; Gong, Lei

    2014-01-01

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

  18. 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.

  19. Shp2 suppresses the adipogenic differentiation of preadipocyte 3T3-L1 cells at an early stage

    PubMed Central

    Tao, J; Zheng, L; Meng, M; Li, Y; Lu, Z

    2016-01-01

    Tyrosine phosphatase protein Shp2 is a potential therapeutic target for obesity. However, the mechanism of Shp2 during adipogenesis is not fully understood. The present study investigated the role of Shp2 in the terminal differentiation of preadipocytes. The results showed that Shp2 suppressed adipocyte differentiation in 3T3-L1 cells; overexpression of Shp2 reduced lipid droplet production in 3T3-L1 cells, whereas Shp2 knockdown increased lipid droplet production in 3T3-L1 cells. Furthermore, inhibition of Shp2 activity also enhanced adipocyte differentiation. Interestingly, Shp2 expression was specifically decreased early during differentiation in response to stimulation with the dexamethasone–methylisobutylxanthine–insulin (DMI) hormone cocktail. During the first 2 days of differentiation, Shp2 overexpression impaired the DMI-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) in 3T3-L1 cells and blocked the peak expression of CCAAT/enhancer-binding proteins β and δ during preadipocyte differentiation. In conclusion, Shp2 downregulated the early stages of hormone-induced differentiation of 3T3-L1 cells and inhibited the expression of the first wave of transcription factors by suppressing the DMI-induced STAT3 signaling pathway. These discoveries point to a novel role of Shp2 during adipogenesis and support the hypothesis that Shp2 could be a therapeutic target for the control of obesity. PMID:27551539

  20. PAZ6 Cells Constitute a Representative Model for Human Brown Pre-Adipocytes

    PubMed Central

    Kazantzis, Melissa; Takahashi, Virginia; Hinkle, Jessica; Kota, Smitha; Zilberfarb, Vladimir; Issad, Tarik; Abdelkarim, Mouaadh; Chouchane, Lotfi

    2012-01-01

    The role of brown adipose tissue (BAT) in human metabolism and its potential as an anti-obesity target organ have recently received much renewed attention. Following radiological detection of substantial amounts of BAT in adults by several independent research groups, an increasing number of studies are now dedicated to uncover BAT’s genetic, developmental, and environmental determinants. In contrast to murine BAT, human BAT is not present as a single major fat depot in a well-defined location. The distribution of BAT in several areas in the body significantly limits its availability to research. A human brown adipocyte cell line is therefore critical in broadening the options available to researchers in the field. The human BAT-cell line PAZ6 was created to address such a need and has been well characterized by several research groups around the world. In the present review, we discuss their findings and propose potential applications of the PAZ6 cells in addressing the relevant questions in the BAT field, namely for future use in therapeutic applications. PMID:22649407

  1. 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.

  2. Search for the preadipocyte progenitor proceeds to a new dimension

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increases in adipocyte number represent a major contribution to the expansion in adipose tissue mass characteristic of obesity induced by either diet, genetics or hormones in man and animals. The identity and regulation of the adipocyte precursor cell, or preadipocyte, and the preadipocyte precursor...

  3. Real-time monitoring of 3T3-L1 preadipocyte differentiation using a commercially available electric cell-substrate impedance sensor system.

    PubMed

    Kramer, Adam H; Joos-Vandewalle, Julia; Edkins, Adrienne L; Frost, Carminita L; Prinsloo, Earl

    2014-01-24

    Real-time analysis offers multiple benefits over traditional end point assays. Here, we present a method of monitoring the optimisation of the growth and differentiation of murine 3T3-L1 preadipocytes to adipocytes using the commercially available ACEA xCELLigence Real-Time Cell Analyser Single Plate (RTCA SP) system. Our findings indicate that the ACEA xCELLigence RTCA SP can reproducibly monitor the primary morphological changes in pre- and post-confluent 3T3-L1 fibroblasts induced to differentiate using insulin, dexamethasone, 3-isobutyl-1-methylxanthine and rosiglitazone; and may be a viable primary method of screening compounds for adipogenic factors. PMID:24388983

  4. 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.

  5. Effects of rosiglitazone on proliferation and differentiation of duck preadipocytes.

    PubMed

    Ding, Fang; Qiu, Jiamin; Li, Qingqing; Hu, Jiwei; Song, Chenling; Han, Chunchun; He, Hua; Wang, Jiwen

    2016-02-01

    Rosiglitazone (RSG), one member of the thiazolidinediones (TZDs), is a type of anti-diabetic drug in diabetic humans and animal models, whose function remains unknown in waterfowl. In this study, effects of RSG on duck preadipocyte differentiation were investigated. We detected cell viability using CCK method and measured the mRNA expression of key genes and protein contents involved in preadipocyte differentiation via qRT-PCR and ELISA kits, respectively. Lipid accumulation was determined via Oil Red O staining extraction, and lipolysis was measured by free fatty acid release in the culture medium. Results showed that high concentrations of RSG (50, 100 μM) significantly decreased cell viability. RSG (0-10 μM) enhanced preadipocyte differentiation in a dose-dependent manner and thus promoted lipid accumulation. With increasing RSG concentrations, cellular lipid content gradually decreased and preadipocyte differentiation was suppressed. mRNA expression of key genes involved in preadipocyte differentiation including FAS, ACC, SCD1, LPL, PLIN, SREBP1c, and ATGL were significantly upregulated by RSG, and the protein content of FAS, ACC, and ATGL were also increased in response to RSG. Meanwhile, RSG exposure increased free fatty acid release in the culture medium. Similar results were obtained in response to RSG plus oleate that was used to induce cell differentiation. These findings suggest that RSG does not promote duck preadipocyte viability, but it does induce duck preadipocyte differentiation, which might influence both lipogenesis and lipolysis pathways. PMID:26487429

  6. 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

  7. Differentiation of preadipocytes and mature adipocytes requires PSMB8.

    PubMed

    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

  8. Platycodon grandiflorum A. De Candolle Ethanolic Extract Inhibits Adipogenic Regulators in 3T3-L1 Cells and Induces Mitochondrial Biogenesis in Primary Brown Preadipocytes.

    PubMed

    Kim, Hye-Lin; Park, Jinbong; Park, Hyewon; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; Jeong, Mi-Young; Um, Jae-Young

    2015-09-01

    This study was designed to evaluate the effects of Platycodon grandiflorum A. DC. ethanolic extract (PG) on obesity in brown/white preadipocytes. The effect of PG on the differentiation and mitochondrial biogenesis of brown adipocytes is still not examined. An in vivo study showed that PG induced weight loss in mice with high-fat-diet-induced obesity. PG successfully suppressed the differentiation of 3T3-L1 cells by down-regulating cellular induction of the peroxisome proliferators activated receptor γ (PPARγ), CCAAT enhancer binding protein α (C/EBPα), lipin-1, and adiponectin but increasing expression of silent mating type information regulation 2 homologue 1 (SIRT1) and the phosphorylation of AMP-activated protein kinase α (AMPKα). The effect of PG on the adipogenic factors was compared with that of its bioactive compound platycodin D. In addition, PG increased expressions of mitochondria-related genes, including uncoupling protein 1 (UCP1), peroxisome proliferator activated receptor-coactivator 1 α (PGC1α), PR domain containing 16 (PRDM16), SIRT3, nuclear respiratory factor (NRF), and cytochrome C (CytC) in primary brown adipocytes. These results indicate that PG stimulates the differentiation of brown adipocytes through modulation of mitochondria-related genes and could offer clinical benefits as a supplement to treat obesity. PMID:26244589

  9. Thyroid Hormone Receptor Sumoylation Is Required for Preadipocyte Differentiation and Proliferation*

    PubMed Central

    Liu, Yan-Yun; Ayers, Stephen; Milanesi, Anna; Teng, Xiaochun; Rabi, Sina; Akiba, Ysutada; Brent, Gregory A.

    2015-01-01

    Thyroid hormone and thyroid hormone receptor (TR) play an essential role in metabolic regulation. However, the role of TR in adipogenesis has not been established. We reported previously that TR sumoylation is essential for TR-mediated gene regulation and that mutation of either of the two sites in TRα or any of the three sites in TRβ reduces TR sumoylation. Here, we transfected TR sumoylation site mutants into human primary preadiocytes and the mouse 3T3L1 preadipocyte cell line to determine the role of TR sumoylation in adipogenesis. Reduced sumoylation of TRα or TRβ resulted in fewer and smaller lipid droplets and reduced proliferation of preadipocytes. TR sumoylation mutations, compared with wild-type TR, results in reduced C/EBP expression and reduced PPARγ2 mRNA and protein levels. TR sumoylation mutants recruited NCoR and disrupted PPARγ-mediated perilipin1 (Plin1) gene expression, associated with impaired lipid droplet formation. Expression of NCoRΔID, a mutant NCoR lacking the TR interaction domain, partially “rescued” the delayed adipogenesis and restored Plin1 gene expression and adipogenesis. TR sumoylation site mutants impaired Wnt/β-catenin signaling pathways and the proliferation of primary human preadipocytes. Expression of the TRβ K146Q sumoylation site mutant down-regulated the essential genes required for canonical Wnt signal-mediated proliferation, including Wnt ligands, Fzds, β-catenin, LEF1, and CCND1. Additionally, the TRβ K146Q mutant enhanced the canonical Wnt signaling inhibitor Dickkopf-related protein 1 (DKK1). Our data demonstrate that TR sumoylation is required for activation of the Wnt canonical signaling pathway during preadipocyte proliferation and enhances the PPARγ signaling that promotes differentiation. PMID:25572392

  10. Thyroid hormone receptor sumoylation is required for preadipocyte differentiation and proliferation.

    PubMed

    Liu, Yan-Yun; Ayers, Stephen; Milanesi, Anna; Teng, Xiaochun; Rabi, Sina; Akiba, Ysutada; Brent, Gregory A

    2015-03-20

    Thyroid hormone and thyroid hormone receptor (TR) play an essential role in metabolic regulation. However, the role of TR in adipogenesis has not been established. We reported previously that TR sumoylation is essential for TR-mediated gene regulation and that mutation of either of the two sites in TRα or any of the three sites in TRβ reduces TR sumoylation. Here, we transfected TR sumoylation site mutants into human primary preadiocytes and the mouse 3T3L1 preadipocyte cell line to determine the role of TR sumoylation in adipogenesis. Reduced sumoylation of TRα or TRβ resulted in fewer and smaller lipid droplets and reduced proliferation of preadipocytes. TR sumoylation mutations, compared with wild-type TR, results in reduced C/EBP expression and reduced PPARγ2 mRNA and protein levels. TR sumoylation mutants recruited NCoR and disrupted PPARγ-mediated perilipin1 (Plin1) gene expression, associated with impaired lipid droplet formation. Expression of NCoRΔID, a mutant NCoR lacking the TR interaction domain, partially "rescued" the delayed adipogenesis and restored Plin1 gene expression and adipogenesis. TR sumoylation site mutants impaired Wnt/β-catenin signaling pathways and the proliferation of primary human preadipocytes. Expression of the TRβ K146Q sumoylation site mutant down-regulated the essential genes required for canonical Wnt signal-mediated proliferation, including Wnt ligands, Fzds, β-catenin, LEF1, and CCND1. Additionally, the TRβ K146Q mutant enhanced the canonical Wnt signaling inhibitor Dickkopf-related protein 1 (DKK1). Our data demonstrate that TR sumoylation is required for activation of the Wnt canonical signaling pathway during preadipocyte proliferation and enhances the PPARγ signaling that promotes differentiation. PMID:25572392

  11. Blasted Cell Line Names

    PubMed Central

    Wang, Jing; Byers, Lauren A.; Yordy, John S.; Liu, Wenbin; Shen, Li; Baggerly, Keith A.; Giri, Uma; Myers, Jeffrey N.; Ang, K. Kian; Story, Michael D.; Girard, Luc; Minna, John D.; Heymach, John V.; Coombes, Kevin R.

    2010-01-01

    Background: While trying to integrate multiple data sets collected by different researchers, we noticed that the sample names were frequently entered inconsistently. Most of the variations appeared to involve punctuation, white space, or their absence, at the juncture between alphabetic and numeric portions of the cell line name. Results: Reasoning that the variant names could be described in terms of mutations or deletions of character strings, we implemented a simple version of the Needleman-Wunsch global sequence alignment algorithm and applied it to the cell line names. All correct matches were found by this procedure. Incorrect matches only occured when a cell line was present in one data set but not in the other. The raw match scores tended to be substantially worse for the incorrect matches. Conclusions: A simple application of the Needleman-Wunsch global sequence alignment algorithm provides a useful first pass at matching sample names from different data sets. PMID:21082038

  12. 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

  13. 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}.

  14. 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

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

    PubMed Central

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

    2016-01-01

    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

  16. 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. PMID:24534601

  17. Osteodifferentiation of human preadipocytes induced by strontium released from hydrogels.

    PubMed

    Nardone, Valeria; Fabbri, Sergio; Marini, Francesca; Zonefrati, Roberto; Galli, Gianna; Carossino, Annamaria; Tanini, Annalisa; Brandi, Maria Luisa

    2012-01-01

    In recent years, there has been an increasing interest in interactive application principles of biology and engineering for the development of valid biological systems for tissue regeneration, such as for the treatment of bone fractures or skeletal defects. The application of stem cells together with biomaterials releasing bioactive factors promotes the formation of bone tissue by inducing proliferation and/or cell differentiation. In this study, we used a clonal cell line from human adipose tissue-derived mesenchymal stem cells (hADSCs or preadipocytes), named PA2-E12, to evaluate the effects of strontium (Sr(2+)) released in the culture medium from an amidated carboxymethylcellulose (CMCA) hydrogel enriched with different Sr(2+) concentrations on osteodifferentiation. The osteoinductive effect was evaluated through both the expression of alkaline phophatase (ALP) activity and the hydroxyapatite (HA) production during 42 days of induction. Present data have shown that Sr(2+) released from CMCA promotes the osteodifferentiation induced by an osteogenic medium as shown by the increase of ALP activity at 7 and 14 days and of HA production at 14 days. In conclusion, the use of biomaterials able to release in situ osteoinductive agents, like Sr(2+), could represent a new strategy for future applications in bone tissue engineering. PMID:22927856

  18. 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

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

    PubMed

    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

  20. Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells.

    PubMed

    Post, S; Abdallah, B M; Bentzon, J F; Kassem, M

    2008-07-01

    Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for

  1. Soy pinitol acts partly as an insulin sensitizer or insulin mediator in 3T3-L1 preadipocytes

    PubMed Central

    Do, Gyeong-Min; Choi, Myung-Sook; Kim, Hye-Jin; Woo, Myung-Nam; Lee, Mi-Kyung

    2007-01-01

    The blood glucose-lowering property of pinitol is mediated via the insulin signaling pathway. This study was carried out to evaluate the effects of soy pinitol on adipogenesis in a 3T3-L1 cell line; 3T3-L1 preadipocytes were treated with pinitol (0–1 mM) together with insulin for 9 days. The regulation of lipid metabolism was assessed by oil-red-O staining of intracellular lipids and real-time PCR of adipogenesis-related factors. The inhibition of cell proliferation was estimated by MTT assay. Pinitol treatment did not inhibit lipid accumulation, nor did it affect expression of adipogenesis-related factors, including ADD1, aP2 and FAS, in a dose-dependent manner. Expression of adiponectin, GLUT4, IRS, C/EBPα and PPARγ mRNAs, however, increased in cells treated with 0.5 mM and/or 1 mM pinitol. Pinitol treatment did not affect the inhibition of cell growth and proliferation in a dose-dependent manner. Accordingly, we suggest that pinitol is nontoxic to this cell line, and that it enhances adipogenesis by acting as an insulin sensitizer or insulin mediator via the upregulation of adiponectin, GLUT4, IRS, C/EBPα and PPARγ in 3T3-L1 preadipocytes. PMID:18850231

  2. 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

  3. Macrophage-secreted factors impair human adipogenesis: involvement of proinflammatory state in preadipocytes.

    PubMed

    Lacasa, Danièle; Taleb, Soraya; Keophiphath, Mayoura; Miranville, Alexandra; Clement, Karine

    2007-02-01

    Obesity is considered a chronic low-grade inflammatory state. The white adipose tissue produces a variety of inflammation-related proteins whose expression is increased in obese subjects. The nonadipose cell fraction, which includes infiltrated macrophages, is a determinant source of inflammation-related molecules within the adipose tissue. Our working hypothesis is that macrophage infiltration affects fat expansion through a paracrine action on adipocyte differentiation. Human primary preadipocytes were then differentiated in the presence of conditioned media obtained from macrophages differentiated from blood monocytes. Preadipocytes treated by macrophage-conditioned medium displayed marked reduction of adipogenesis as assessed by decreased cellular lipid accumulation and reduced gene expression of adipogenic and lipogenic markers. In addition to this effect, the activation of macrophages by lipopolysaccharides stimulated nuclear factor kappaB signaling, increased gene expression and release of proinflammatory cytokines and chemokines, and induced preadipocyte proliferation. This phenomenon was associated with increased cyclin D1 gene expression and maintenance of the fibronectin-rich matrix. Anti-TNFalpha neutralizing antibody inhibits the inflammatory state of preadipocytes positioning TNFalpha as an important mediator of inflammation in preadipocytes. Strikingly, conditioned media produced by macrophages isolated from human adipose tissue exerted comparable effects with activated macrophages, i.e. decreased adipogenesis and increased inflammatory state in the preadipocytes. These data show that macrophage-secreted factors inhibit the formation of mature adipocytes, suggesting possible role in limiting adipose tissue expansion in humans. PMID:17082259

  4. Asymmetry in traction forces produced by migrating preadipocytes is bounded to 33.

    PubMed

    Abuhattum, Shada; Weihs, Daphne

    2016-09-01

    Wound healing by gap closure is accomplished by the migratory cells within tissues. In fat tissue, the preadipocytes, stem cells committed to the adipose (fat) lineage, typically migrate into the wound area, and then differentiate into mature adipocytes to facilitate tissue repair and regeneration. While cell migration has previously been characterized, typically for fibroblasts, little is known about the dynamic, mechanical interactions of migrating cells with their microenvironment; cells crawl on a two-dimensional (2D) substrate by attaching and applying forces that allow them to extend leading edges and retract their rear. Moreover, preadipocytes, the highly migratory precursors of fat cells, have not been studied from this aspect. Here, we have evaluated the migration of preadipocytes, through their speed and directionality as well as the magnitude of the lateral forces applied during their migration on a 2D gel with Young's modulus of 2.44 kPa. We have found that the preadipocytes migrate non-directionally in the absence of chemoattractant, at an average rate of 0.27 µm/min, similar to fibroblasts. The preadipocytes exhibited a wide range of total traction forces (100-800 nN), and migrated along the long axis of their elongated morphology. Interestingly, we have observed an asymmetry in the location of force application between the lead and rear of the cells that was bounded in magnitude, where cells applied only up to 33% more force on either side; cell sides were defined relative to the minor axis of a bounding ellipse. These quantitative mechanobiological aspects of natural preadipocyte migration may shed light on the wound healing processes occurring in adipose tissue. PMID:27340101

  5. 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.

  6. Thyroid cell lines in research on goitrogenesis.

    PubMed

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

    1991-12-01

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

  7. Clonal analyses and gene profiling identify genetic biomarkers of human brown and white preadipocyte thermogenic potential

    PubMed Central

    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

    2015-01-01

    Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. Both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here, we demonstrated the generated clones of brown and white preadipocytes from human neck fat of four individuals and characterized their adipogenic differentiation and thermogenic function. Combining an uncoupling protein 1(UCP1) reporter system and expression profiling, we defined novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated in culture. Knocking out the positive UCP1 regulators identified by this approach, 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 cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer the identification of possible biomarkers of thermogenically competent preadipocytes. PMID:26076036

  8. 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

  9. Zeranol stimulates proliferation and aromatase activation in human breast preadipocytes.

    PubMed

    Zhong, Saiyi; Liu, Shouchun; Chen, Suhua; Lin, Huajuan; Wang, Weimin; Qin, Xiaoming

    2016-07-01

    Aromatase is a crucial enzyme for the biosynthesis of estrogens and is involved in the process of breast carcinogenesis. Concerns have been raised regarding the effects of environmental estrogens as potential regulators of aromatase expression in human breast cells. Zeranol is a non‑steroidal agent with potent estrogenic activity, which is widely used as a growth promoter for cattle in certain countries. The present study hypothesized that aromatase expression and activity may be elevated by low dose zeranol exposure, providing a source of estrogens that may stimulate cell proliferation. In the present study, primary cultured human breast preadipocytes were used as an in vitro model. The effects of zeranol on cell proliferation were measured using the MTS assay, aromatase expression levels were determined by immunocytochemical staining and reverse transcription‑polymerase chain reaction, and aromatase enzyme activity and estrogen production were analyzed using corresponding assay kits. The results demonstrated that low dose zeranol (2‑50 nM) was able to significantly promote cell proliferation, aromatase mRNA expression, aromatase activity and estrogen production in primary cultured human breast preadipocytes, thus suggesting that zeranol may act as an aromatase activator. The findings of the present study suggest that zeranol promotes breast cancer cell growth by stimulating aromatase activation and increasing estrogen biosynthesis in adipose tissue. PMID:27220457

  10. Substance P promotes expansion of human mesenteric preadipocytes through proliferative and antiapoptotic pathways

    PubMed Central

    Gross, Kara; Karagiannides, Iordanes; Thomou, Thomas; Koon, Hon Wai; Bowe, Collin; Kim, Ho; Giorgadze, Nino; Tchkonia, Tamara; Pirtskhalava, Tamara; Kirkland, James L.; Pothoulakis, Charalabos

    2009-01-01

    White adipose tissue is intimately involved in the regulation of immunity and inflammation. We reported that human mesenteric preadipocytes express the substance P (SP)-mediated neurokinin-1 receptor (NK-1R), which signals proinflammatory responses. Here we tested the hypothesis that SP promotes proliferation and survival of human mesenteric preadipocytes and investigated responsible mechanism(s). Preadipocytes were isolated from mesenteric fat biopsies during gastric bypass surgery. Proliferative and antiapoptotic responses were delineated in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), bromodeoxyuridine (BrdU), caspase-3, and TUNEL assays, as well as Western immunoanalysis. SP (10−7 M) increased MTS and proliferation (BrdU) and time dependently (15–30 min) induced Akt, EGF receptor, IGF receptor, integrin αVβ3, phosphatidylinositol 3-kinase, and PKC-θ phosphorylation. Furthermore, pharmacological antagonism of Akt and PKC-θ activation significantly attenuated SP-induced preadipocyte proliferation. Exposure of preadipocytes to the proapoptotic Fas ligand (FasL, 100 μM) resulted in nuclear DNA fragmentation (TUNEL assay), as well as increased cleaved poly (ADP-ribose) polymerase, cleaved caspase-7, and caspase-3 expression. Cotreatment with SP almost completely abolished these responses in a NK-1R-dependent fashion. SP (10−7 M) also time dependently stimulated expression 4E binding protein 1 and phosphorylation of p70 S6 kinase, which increased protein translation efficiency. SP increases preadipocyte viability, reduces apoptosis, and stimulates proliferation, possibly via cell cycle upregulation and increased protein translation efficiency. SP-induced proliferative and antiapoptotic pathways in fat depots may contribute to development of the creeping fat and inflammation characteristic of Crohn's disease. PMID:19282377

  11. PPARy and GLUT-4 expression as developmental regulators/markers for preadipocyte differentiation into an adipocyte

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this document, we have integrated knowledge about two major cellular markers found in cells of the adipocyte lineage. The first factor is PPARy, which has been identified as an important adipogenic regulator. PPARy plays an important role in converting adipofibroblasts, fibroblasts or preadipocyt...

  12. 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.

  13. Human Adrenocortical Carcinoma Cell Lines

    PubMed Central

    Wang, Tao; Rainey, William E.

    2011-01-01

    Summary The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide an important tool to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein we discuss the human adrenocortical cell lines and their use as model systems for adrenal studies. PMID:21924324

  14. In Vitro Effects of Strontium on Proliferation and Osteoinduction of Human Preadipocytes

    PubMed Central

    Nardone, V.; Zonefrati, R.; Mavilia, C.; Romagnoli, C.; Ciuffi, S.; Fabbri, S.; Palmini, G.; Galli, G.; Tanini, A.; Brandi, M. L.

    2015-01-01

    Development of tools to be used for in vivo bone tissue regeneration focuses on cellular models and differentiation processes. In searching for all the optimal sources, adipose tissue-derived mesenchymal stem cells (hADSCs or preadipocytes) are able to differentiate into osteoblasts with analogous characteristics to bone marrow mesenchymal stem cells, producing alkaline phosphatase (ALP), collagen, osteocalcin, and calcified nodules, mainly composed of hydroxyapatite (HA). The possibility to influence bone differentiation of stem cells encompasses local and systemic methods, including the use of drugs administered systemically. Among the latter, strontium ranelate (SR) represents an interesting compound, acting as an uncoupling factor that stimulates bone formation and inhibits bone resorption. The aim of our study was to evaluate the in vitro effects of a wide range of strontium (Sr2+) concentrations on proliferation, ALP activity, and mineralization of a novel finite clonal hADSCs cell line, named PA20-h5. Sr2+ promoted PA20-h5 cell proliferation while inducing the increase of ALP activity and gene expression as well as HA production during in vitro osteoinduction. These findings indicate a role for Sr2+ in supporting bone regeneration during the process of skeletal repair in general, and, more specifically, when cell therapies are applied. PMID:26240575

  15. 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

  16. Oleate promotes differentiation of chicken primary preadipocytes in vitro

    PubMed Central

    Shang, Zhouchun; Guo, Lin; Wang, Ning; Shi, Hui; Wang, Yuxiang; Li, Hui

    2014-01-01

    In addition to providing energy and constituting cell membrane, fatty acids also play an important role in adipocyte differentiation and lipid metabolism. As an important member of monounsaturated fatty acids, oleate, together with other components, is widely used to induce chicken preadipocyte differentiation. However, it is not clear whether oleate alone can induce chicken preadipocyte differentiation. In the present study, four different treatments were designed to test this question: basal medium, IDX [insulin, dexamethasone and IBMX (isobutylmethylxanthine)], oleate and IDX plus oleate. Cytoplasmic lipid droplet accumulation and mRNA expression for adipogenesis-related genes were monitored. After treatment of oleate on chicken preadipocytes, apparent lipid droplet formation and lipid accumulation were observed, accompanied by increasing expression of PPARγ (peroxisome proliferator-activated receptor-γ) and AFABP (adipocyte fatty acid-binding protein), but decreasing level of GATA2 (GATA-binding protein 2). In contrast, for cells cultured in the basal medium with or without IDX supplementation, lipid droplet barely occurred. These results suggest that exogenous oleate alone can act as an inducer of preadipocyte differentiation into adipocytes. PMID:24428793

  17. Mechanism of the antiadipogenic-antiobesity effects of a rice hull smoke extract in 3T3-L1 preadipocyte cells and in mice on a high-fat diet.

    PubMed

    Kim, Sung Phil; Nam, Seok Hyun; Friedman, Mendel

    2015-09-01

    The present study investigated the inhibitory effects of a rice hull smoke extract (RHSE) against adipogenesis in 3T3-L1 preadipocyte cells and in mice fed high fat (HFD) and normal (ND) diets. At concentrations of 0.1% and 0.5%, RHSE was shown to reduce the cellular lipid content in MDI-induced 3T3-L1 cells by about 72% and 88%, respectively, compared to that in control cells without RHSE, indicating a strong antiadipogenic effect. This result was supported by the finding that the expression of the adipocyte differentiation marker adiponectin was suppressed. MTT and lactate dehydrogenase (LDH) release assays showed that RHSE doses of up to 0.5% (v/v) were not cytotoxic to the 3T3-L1 cells. RHSE activates AMP-activated protein kinase (AMPK) through raising the phosphorylated ratio during the early phase of cell differentiation, and western blot analysis showed that it dose-dependently inhibited the expression of the peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT-enhancer binding protein (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c) at the late stage of differentiation. The antiadipogenic properties of RHSE were confirmed in vivo using experimental obese mice on a high-fat diet. Dietary supplementations of 0.5% and 1% RHSE resulted in a reduction at the end of the 7-week feeding study of body weight gain of 66.9% and 72.5%, respectively, a reduction of epididymal white adipose tissue weight by up to 87.9%, restoration of elevated total cholesterol and triglyceride levels in plasma and liver to those observed in the ND-fed mice, normalization of distorted serum leptin and adiponectin levels, and restoration of liver weight and glutamate oxaloacetate transaminase/glutamate pyruvate transaminase (GOT/GPT) enzymes, blood urea, and serum creatinine to the normal control levels observed in the ND-fed mice. As was found in the 3T3-L1 cells, RHSE up-regulated AMPK phosphorylation and down-regulated PPARγ, C/EBPα and SREBP-1c protein

  18. Tissue Distribution of Porcine FTO and Its Effect on Porcine Intramuscular Preadipocytes Proliferation and Differentiation

    PubMed Central

    Chen, Xiaoling; Zhou, Bo; Luo, Yanliu; Huang, Zhiqing; Jia, Gang; Liu, Guangmang; Zhao, Hua

    2016-01-01

    The fat mass and obesity associated (FTO) gene plays an important role in adipogenesis. However, its function during porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, we prepared the antiserum against porcine FTO (pFTO), which was used to determine its subcellular localization and tissue distribution. Our data indicated that pFTO was localized predominantly in the nucleus. Real-time quantitative PCR and western blot analysis showed that pFTO was highly expressed in the lung and subcutaneous adipose tissue. Overexpression of pFTO in porcine intramuscular preadipocytes significantly promoted cell proliferation and lipid deposition. Furthermore, overexpression of pFTO in differentiating porcine intramuscular preadipocytes also significantly increased the mRNA levels of adipocyte differentiation transcription factors peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), lipoprotein lipase (LPL) and fatty acid synthase (FAS). Our findings provide the first functional evidence to reveal a role of pFTO in porcine intramuscular preadipocyte proliferation and differentiation. PMID:26964098

  19. Epac, not PKA catalytic subunit, is required for 3T3-L1 preadipocyte differentiation.

    PubMed

    Ji, Zhenyu; Mei, Fang C; Cheng, Xiaodong

    2010-01-01

    Cyclic AMP plays a critical role in adipocyte differentiation and maturation. However, it is not clear which of the two intracellular cAMP receptors, exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor or protein kinase A/cAMP-dependent protein kinase, is essential for cAMP-mediated adipocyte differentiation. In this study, we utilized a well-defined adipose differentiation model system, the murine preadipocyte line 3T3-L1, to address this issue. We showed that knocking down Epac expression in 3T3-L1 cells using lentiviral based small hairpin RNAs down-regulated peroxisome proliferator-activated receptor gamma expression and dramatically inhibited adipogenic conversion of 3T3-L1 cells while inhibiting PKA catalytic subunit activity by two mechanistically distinct inhibitors, heat stable protein kinase inhibitor and H89, had no effect on 3T3-L1 adipocyte differentiation. Moreover, cAMP analog selectively activating Epac was not able to stimulate adipogenic conversion. Our study demonstrated that while PKA catalytic activity is dispensable, activation of Epac is necessary but not sufficient for adipogenic conversion of 3T3-L1 cells. PMID:20036887

  20. Glucose- and Triglyceride-lowering Dietary Penta-O-galloyl-α-D-Glucose Reduces Expression of PPARγ and C/EBPα, Induces p21-Mediated G1 Phase Cell Cycle Arrest, and Inhibits Adipogenesis in 3T3-L1 Preadipocytes.

    PubMed

    Liu, X; Malki, A; Cao, Y; Li, Y; Qian, Y; Wang, X; Chen, X

    2015-05-01

    Plant polyphenols, such as hydrolysable tannins, are present in the human diet and known to exhibit anti-diabetic and anti-obesity activity. We previously reported that the representative hydrolysable tannin compound α-penta-galloyl-glucose (α-PGG) is a small molecule insulin mimetic that, like insulin, binds to insulin receptor (IR) and activates the IR-Akt-GLUT4 signaling pathway to trigger glucose transport and reduce blood glucose levels in db/db and ob/ob diabetic mice. However, its effects on adipogenesis and lipid metabolism were not known. In this study, high fat diet (HFD)-induced diabetic and obese mice were treated with α-PGG to determine its effects on blood glucose and triglycerides. 3T3-L1 preadipocytes were used as a cell model for identifying the anti-adipogenic activity of α-PGG at molecular and cellular levels as a first step in elucidating the mechanism of action of the compound. In vivo, oral administration of α-PGG significantly reduced levels of blood glucose, triglyceride, and insulin in HFD-induced diabetic/obese mice (P<0.05). In vitro, α-PGG inhibited the differentiation of 3T3-L1 preadipocytes into mature adipocytes. α-PGG suppressed the expression of positive adipogenic factors PPARγ C/EBPα and mTOR and augmented the negative adipogenic factor Pref-1. Furthermore, α-PGG induced upregulation of p21 and G1 phase cell cycle arrest. In contrast, adipogenic signaling pathways mediated by insulin, the cAMP response element binding protein (CREB) and glucocorticoid receptor (GR), were not inhibited. RNAi knockdown of p21 led to a 4-fold increase in triglyceride level in 3T3-L1 preadipocytes treated with MDI and α-PGG compared to regular preadipocytes. These results indicate, for the first time, that α-PGG is blood triglyceride- and glucose-lowering in HFD-induced obese and diabetic mice. It selectively inhibited some but not all major adipogenic pathways as well as the mTOR-p21-mediated cell cycle regulatory pathway. It is very

  1. IL-1β (interleukin-1β) stimulates the production and release of multiple cytokines and chemokines by human preadipocytes.

    PubMed

    Alomar, Suliman Y; Gentili, Alessandra; Zaibi, Mohamed S; Kępczyńska, Malgorzata A; Trayhurn, Paul

    2016-07-01

    The effect of IL-1β on cytokine and chemokine production by human preadipocytes has been examined. Preadipocytes were incubated with IL-1β, and cytokine and chemokine release was measured at 24 h by protein arrays, while the expression of cytokine/chemokine genes was assessed by qPCR at 4 and 24 h. IL-1β stimulated the secretion of multiple cytokines/chemokines, including IL-6, IL-8, IL-10, IL-13, MCP-4, TNFα and IP-10. IL-10 was not released by un-stimulated preadipocytes, while IL-6 exhibited the greatest response to IL-1β (453-fold increase). IL-16 and IL-12p40 did not respond to IL-1β. qPCR demonstrated that IL-1β markedly stimulated CCL3, CSF3 and CXCL10 expression at 4 h (>900-fold mRNA increase). A time-course indicated that while CCL13 (encoding MCP-4) exhibited minimal basal expression in preadipocytes, expression increased progressively following differentiation. Human preadipocytes are highly sensitive to IL-1β, the cytokine stimulating a major inflammatory response in these cells similar to that in mature adipocytes. PMID:26890442

  2. 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.

  3. Downregulated expression of the secreted glycoprotein follistatin-like 1 (Fstl1) is a robust hallmark of preadipocyte to adipocyte conversion.

    PubMed

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

    2010-04-01

    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 TNFalpha-mediated de-differentiation 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 approximately 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

  4. 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

  5. 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. PMID:25710930

  6. Aortic preadipocyte differentiation into adipocytes induced by rosiglitazone in an in vitro model.

    PubMed

    Reyes, Miguel R; Lazalde, Brissia

    2007-01-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is a key transcription factor for adipocyte differentiation. Preadipocyte differentiation into adipocytes from precursors in blood vessels is an important issue related to atherosclerotic cardiovascular disease; however, it has been poorly studied because of lack of experimental models. Our aim was to evaluate the potential of primary outgrowths derived from rat aortic rings as a model for studying the preadipocyte differentiation from aortic precursors induced by thiazolidinediones, which are exogenous ligands for PPARgamma. Cell outgrowths derived from rat aortic rings were cultured and incubated with rosiglitazone at 1-1,000 nM; presence of lipid droplets was evaluated by oil red O staining. Rosiglitazone at 100 nM exerted a clear adipogenic effect inferred from the cells filled with fine and medium size lipidic droplets; this effect was extreme at 1,000 nM with cells showing lipidic macrodroplets. These results showed that cultures derived from aortic rings are a useful model for studying arterial preadipocyte differentiation. PMID:17564754

  7. Metabolic role of dipeptidyl peptidase 4 (DPP4) in primary human (pre)adipocytes

    PubMed Central

    Zilleßen, Pia; Celner, Jennifer; Kretschmann, Anita; Pfeifer, Alexander; Racké, Kurt; Mayer, Peter

    2016-01-01

    Dipeptidyl peptidase 4 (DPP4) is the target of the gliptins, a recent class of oral antidiabetics. DPP4 (also called CD26) was previously characterized in immune cells but also has important metabolic functions which are not yet fully understood. Thus, we investigated the function of DPP4 in human white preadipocytes and adipocytes. We found that both cell types express DPP4 in high amounts; DPP4 release markedly increased during differentiation. In preadipocytes, lentiviral DPP4 knockdown caused significant changes in gene expression as determined by whole-genome DNA-array analysis. Metabolic genes were increased, e.g. PDK4 18-fold and PPARγC1α (=PGC1α) 6-fold, and proliferation-related genes were decreased (e.g. FGF7 5-fold). These effects, contributing to differentiation, were not inhibited by the PPARγ antagonist T0070907. Vice versa, the PPARγ agonist pioglitazone induced a different set of genes (mainly FABP4). DPP4 knockdown also affected growth factor signaling and, accordingly, retarded preadipocyte proliferation. In particular, basal and insulin-induced ERK activation (but not Akt activation) was markedly diminished (by around 60%). This indicates that DPP4 knockdown contributes to adipocyte maturation by mimicking growth factor withdrawal, an early step in fat cell differentiation. In mature adipocytes, DPP4 becomes liberated so that adipose tissue may constitute a relevant source of circulating DPP4. PMID:26983599

  8. 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

  9. 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.

  10. Bisphenol S Induces Adipogenesis in Primary Human Preadipocytes From Female Donors.

    PubMed

    Boucher, Jonathan G; Ahmed, Shaimaa; Atlas, Ella

    2016-04-01

    Human exposure to bisphenol A has been associated with negative health outcomes in humans and its use is now regulated in a number of countries. Bisphenol S (BPS) is increasingly used as a replacement for bisphenol A; however, its effects on cellular metabolism and potential role as an endocrine disruptor have not been fully characterized. In the current study, we evaluated the effect of BPS on adipogenesis in primary human preadipocytes. The effect of BPS on the differentiation of human preadipocytes was determined after treatment with BPS at concentrations ranging from 0.1 nM to 25 μM by quantifying lipid accumulation and mRNA and protein levels of key adipogenic markers. Treatment of preadipocytes with 25 μM BPS induced lipid accumulation and increased the mRNA and protein levels of several adipogenic markers including lipoprotein lipase and adipocyte protein 2 (aP2). Cotreatment of cells with the estrogen receptor antagonist ICI-182,780 significantly inhibited BPS-induced lipid accumulation and affected aP2 but not lipoprotein lipase protein levels. Cotreatment of cells with the glucocorticoid receptor antagonist RU486 had no effect on BPS-induced lipid accumulation or protein levels. Furthermore, reporter gene assays using a synthetic promoter containing peroxisome proliferator-activated receptor-γ (PPARG)-response elements and a PPARG-responsive human aP2 promoter region showed that BPS was able to activate PPARG. To our knowledge, this study is the first to show that BPS induces lipid accumulation and differentiation of primary human preadipocytes, and this effect may be mediated through a PPARG pathway. PMID:27003841

  11. White Tea extract induces lipolytic activity and inhibits adipogenesis in human subcutaneous (pre)-adipocytes

    PubMed Central

    Söhle, Jörn; Knott, Anja; Holtzmann, Ursula; Siegner, Ralf; Grönniger, Elke; Schepky, Andreas; Gallinat, Stefan; Wenck, Horst; Stäb, Franz; Winnefeld, Marc

    2009-01-01

    Background The dramatic increase in obesity-related diseases emphasizes the need to elucidate the cellular and molecular mechanisms underlying fat metabolism. To investigate how natural substances influence lipolysis and adipogenesis, we determined the effects of White Tea extract on cultured human subcutaneous preadipocytes and adipocytes. Methods For our in vitro studies we used a White Tea extract solution that contained polyphenols and methylxanthines. Utilizing cultured human preadipocytes we investigated White Tea extract solution-induced inhibition of triglyceride incorporation during adipogenesis and possible effects on cell viability. In vitro studies on human adipocytes were performed aiming to elucidate the efficacy of White Tea extract solution to stimulate lipolytic activity. To characterize White Tea extract solution-mediated effects on a molecular level, we analyzed gene expression of essential adipogenesis-related transcription factors by qRT-PCR and determined the expression of the transcription factor ADD1/SREBP-1c on the protein level utilizing immunofluorescence analysis. Results Our data show that incubation of preadipocytes with White Tea extract solution significantly decreased triglyceride incorporation during adipogenesis in a dose-dependent manner (n = 10) without affecting cell viability (n = 10). These effects were, at least in part, mediated by EGCG (n = 10, 50 μM). In addition, White Tea extract solution also stimulated lipolytic activity in adipocytes (n = 7). Differentiating preadipocytes cultivated in the presence of 0.5% White Tea extract solution showed a decrease in PPARγ, ADD1/SREBP-1c, C/EBPα and C/EBPδ mRNA levels. Moreover, the expression of the transcription factor ADD1/SREBP-1c was not only decreased on the mRNA but also on the protein level. Conclusion White Tea extract is a natural source that effectively inhibits adipogenesis and stimulates lipolysis-activity. Therefore, it can be utilized to modulate different

  12. Cell-host, LINE and environment

    PubMed Central

    Del Re, Brunella; Giorgi, Gianfranco

    2013-01-01

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

  13. 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

  14. Weight Loss Improves the Adipogenic Capacity of Human Preadipocytes and Modulates Their Secretory Profile

    PubMed Central

    Rossmeislová, Lenka; Mališová, Lucia; Kračmerová, Jana; Tencerová, Michaela; Kováčová, Zuzana; Koc, Michal; Šiklová-Vítková, Michaela; Viquerie, Nathalie; Langin, Dominique; Štich, Vladimír

    2013-01-01

    Calorie restriction–induced weight loss is accompanied by profound changes in adipose tissue characteristics. To determine the effect of weight loss on differentiation of preadipocytes and secretory capacity of in vitro differentiated adipocytes, we established cultures of these cells from paired subcutaneous adipose tissue biopsies obtained before and at the end of weight-reducing dietary intervention (DI) in 23 obese women. Based on lipid accumulation and the expression of differentiation markers, in vitro adipogenesis increased after weight loss and it was accompanied by enhanced expression of genes involved in de novo lipogenesis. This effect of weight loss was not driven by changes of peroxisome proliferator–activated receptor γ sensitivity to rosiglitazone. Weight loss also enhanced the expression of adiponectin and leptin while reducing that of monocyte chemoattractant protein 1 and interleukin-8 by cultured adipocytes. Thus, the weight-reducing (DI) increased adipogenic capacity of preadipocytes and shifted their secretion toward lower inflammatory profile. Reprogramming of preadipocytes could represent an adaptation to weight loss leading to partial restoration of preobese adipose tissue traits and thus contribute to the improvement of metabolic status. However, enhanced adipogenesis could also contribute to the unwanted weight regain after initial weight loss. PMID:23378611

  15. 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. PMID:24140569

  16. Cholesteryl ester transfer protein gene expression during differentiation of human preadipocytes to adipocytes in primary culture.

    PubMed

    Gauthier, B; Robb, M; McPherson, R

    1999-02-01

    The expression pattern of the CETP gene in relationship to that of LPL, adipsin, PPARgamma, C/EBPalpha, ADD1/SREBPI and actin was examined by RT-PCR during differentiation of human fibroblastic preadipocytes to adipocytes in primary culture. Preadipocytes were isolated from subcutaneous fat obtained from healthy female subjects undergoing mammary reduction procedures, and induced to differentiate in culture. Morphologically, adipogenesis was confirmed by the accumulation of lipid droplets in cells. We show that the gene encoding CETP is expressed in preadipocytes and is present throughout differentiation as compared to LPL and adipsin which were detected in the majority of samples by day 2 or 3 of adipogenesis. The transcription factors, PPARgamma, ADD1/SREBP1 and C/EBPalpha were expressed by day 2, concomitant with the appearance of LPL and adipsin but subsequent to the appearance of CETP. CETP mRNA was not detectable in human skin fibroblasts. These studies demonstrate that CETP. expression is induced at an early stage of commitment to the adipocyte lineage and may be activated by transcription factor(s), which are not members of the PPAR, ADD1/SREBP1 or C/EBP families. PMID:10030381

  17. 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

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

    PubMed

    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

  19. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line.

    PubMed

    Frandsen, Stine Krog; McNeil, Anna K; Novak, Ivana; McNeil, Paul L; Gehl, Julie

    2016-08-01

    Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment. PMID:27312328

  20. Anti-obesity effect of Blumea balsamifera extract in 3T3-L1 preadipocytes and adipocytes.

    PubMed

    Kubota, Hiroaki; Kojima-Yuasa, Akiko; Morii, Risako; Huang, Xuedan; Norikura, Toshio; Rho, Sook-Nyung; Matsui-Yuasa, Isao

    2009-01-01

    Obesity, the leading metabolic disease in the world, is a serious health problem in industrialized countries. We investigated the anti-obesity effect of Blumea balsamifera extract on adipocyte differentiation of 3T3-L1 preadipocytes and anti-obesity effect of 3T3-L1 adipocytes. We found that treatment with an extract of Blumea balsamifera suppressed lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity without affecting cell viability in 3T3-L1 preadipocytes and adipocytes. Furthermore, Blumea balsamifera extract brought significant attenuation of expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor (PPAR)gamma, CCAAT element binding protein (C/EBPs) and leptin, however, induced up-regulation of adiponectin at the protein level in 3T3-L1 preadipocytes and adipocytes. These results suggest that Blumea balsamifera extract may block adipogenesis, at least in part, by decreasing key adipogenic transcription factors in 3T3-L1 preadipocytes and may have antiatherogenic, anti-inflammatory, and antidiabetic effects through up-regulation of adiponectin in 3T3-L1 adipocytes. PMID:19885945

  1. 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.

  2. 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. PMID:26896766

  3. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

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

    2012-01-01

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

  4. 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.

  5. Glucocorticoid-induced androgen inactivation by aldo-keto reductase 1C2 promotes adipogenesis in human preadipocytes.

    PubMed

    Veilleux, Alain; Côté, Julie-Anne; Blouin, Karine; Nadeau, Mélanie; Pelletier, Mélissa; Marceau, Picard; Laberge, Philippe Y; Luu-The, Van; Tchernof, André

    2012-04-15

    Adipogenesis and lipid storage in human adipose tissue are inhibited by androgens such as DHT. Inactivation of DHT to 3α-diol is stimulated by glucocorticoids in human preadipocytes. We sought to characterize glucocorticoid-induced androgen inactivation in human preadipocytes and to establish its role in the antiadipogenic action of DHT. Subcutaneous and omental primary preadipocyte cultures were established from fat samples obtained in subjects undergoing abdominal surgeries. Inactivation of DHT to 3α/β-diol for 24 h was measured in dexamethasone- or vehicle-treated cells. Specific downregulation of aldo-keto reductase 1C (AKR1C) enzymes in human preadipocytes was achieved using RNA interference. In whole adipose tissue sample, cortisol production was positively correlated with androgen inactivation in both subcutaneous and omental adipose tissue (P < 0.05). Maximal dexamethasone (1 μM) stimulation of DHT inactivation was higher in omental compared with subcutaneous fat from men as well as subcutaneous and omental fat from women (P < 0.05). A significant positive correlation was observed between BMI and maximal dexamethasone-induced DHT inactivation rates in subcutaneous and omental adipose tissue of men and women (r = 0.24, n = 26, P < 0.01). siRNA-induced downregulation of AKR1C2, but not AKR1C1 or AKR1C3, significantly reduced basal and glucocorticoid-induced androgen inactivation rates (P < 0.05). The inhibitory action of DHT on preadipocyte differentiation was potentiated following AKR1C2 but not AKR1C1 or AKR1C3 downregulation. Specifically, lipid accumulation, G3PDH activity, and FABP4 mRNA expression in differentiated preadipocytes exposed to DHT were reduced further upon AKR1C2 siRNA transfection. We conclude that glucocorticoid-induced androgen inactivation is mediated by AKR1C2 and is particularly effective in omental preadipocytes of obese men. The interplay between glucocorticoids and AKR1C2-dependent androgen inactivation may locally modulate

  6. Breast cancer cell lines: friend or foe?

    PubMed Central

    Burdall, Sarah E; Hanby, Andrew M; Lansdown, Mark RJ; Speirs, Valerie

    2003-01-01

    The majority of breast cancer research is conducted using established breast cancer cell lines as in vitro models. An alternative is to use cultures established from primary breast tumours. Here, we discuss the pros and cons of using both of these models in translational breast cancer research. PMID:12631387

  7. TRANSFECTION OF INSECT CELL LINES USING POLYETHYLENIMINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    SciTech Connect

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

    2011-04-01

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

  9. A human gallbladder adenocarcinoma cell line.

    PubMed

    Morgan, R T; Woods, L K; Moore, G E; McGavran, L; Quinn, L A; Semple, T U

    1981-06-01

    A continuous cell line, COLO 346, was established from a liver metastasis in a patient with adenocarcinoma of the gallbladder. COLO 346 grew as an adherent monolayer of pleomorphic epithelioid cells. COLO 346 cells produced esterone, but no estradiol, progesterone, or cortisol. No adrenocorticotropic hormones, beta-subunit of human chorionic gonadotropin, carcinoembryonic antigen, or alpha-fetoprotein production by the cells was detected. Cell doubling time was 36 h. Seven allelic isozymes were assayed. COLO 346 had a chromosome mode of 74 at 21 months postestablishment with 6 marker chromosomes present in 100% of the cells analyzed. COLO 346 has been in continuous culture for over 2 yr and is available to other investigators for their studies. PMID:7262900

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

    PubMed Central

    Kumar, Arvind; Selvakumar, S.

    2015-01-01

    Background: Laryngeal cancer may also be called cancer of the larynx or laryngeal carcinoma. Conventional plants are a precious source of novel anticancer agents and are still in performance better role in health concern. The study was intended to estimation of the anticancer activity of the chloroformic extract of Tabernaemontana divaricata on the human epidermoid larynx carcinoma cell line (Hep 2). Materials and Method: The aerial parts (leaves, stem, and flowers) of T. divaricata were tested for its inhibitory effect in 96 microplate formats against Hep 2 cell line. The anticancer activity of samples on Hep 2 and Vero was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and various enzymatic parameters like catalase, reduced glutathione (GSH), GSH peroxidase, and superoxide anion scavenging activity. Viable cells were determined by the absorbance at 540 nm. Measurements were performed, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of Hep 2 and Vero cells was expressed as the % cell viability. Results: The extract on Hep 2 cell line up to 7.8 μg/ml and that IC50 value on Hep 2 cell line was 112 μg whereas 94 μg for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line. Conclusion: Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line). PMID:26109773

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

    PubMed

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

    2016-04-01

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

  12. (+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling, down-regulation of PPARγ and induction of iNOS.

    PubMed

    Freise, Christian; Trowitzsch-Kienast, Wolfram; Erben, Ulrike; Seehofer, Daniel; Kim, Ki Young; Zeitz, Martin; Ruehl, Martin; Somasundaram, Rajan

    2013-03-01

    Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC50 of 10μM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and β-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity. PMID:22818712

  13. Manganese superoxide dismutase knock-down in 3T3-L1 preadipocytes impairs subsequent adipogenesis.

    PubMed

    Krautbauer, Sabrina; Eisinger, Kristina; Hader, Yvonne; Neumeier, Markus; Buechler, Christa

    2014-08-01

    Adipogenesis is associated with the upregulation of the antioxidative enzyme manganese superoxide dismutase (MnSOD) suggesting a vital function of this enzyme in adipocyte maturation. In the current work, MnSOD was knocked-down with small-interference RNA in preadipocytes to study its role in adipocyte differentiation. In mature adipocytes differentiated from these cells, proteins characteristic for mature adipocytes, which are strongly induced in late adipogenesis like adiponectin and fatty acid-binding protein 4, are markedly reduced. Triglycerides begin to accumulate after about 6 days of the induction of adipogenesis, and are strongly diminished in cells with low MnSOD. Proteins upregulated early during differentiation, like fatty acid synthase and cytochrome C oxidase-4, are not altered. Cell viability, insulin-mediated phosphorylation of Akt, antioxidative capacity (AOC), superoxide levels, and heme oxygenase 1 with the latter being induced upon oxidative stress are not affected. L-Buthionine-(S,R)-sulfoximine (BSO) depletes glutathione and modestly lowers AOC of mature adipocytes. Addition of BSO to 3T3-L1 cells 3 days after the initiation of differentiation impairs triglyceride accumulation and expression of proteins induced in late adipogenesis. Of note, proteins that increased early during adipogenesis are also diminished, suggesting that BSO causes de-differentiation of these cells. Preadipocyte proliferation is not considerably affected by low MnSOD and BSO. These data suggest that glutathione and MnSOD are essential for adipogenesis. PMID:24740755

  14. A human gallbladder adenocarcinoma cell line.

    PubMed

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

    1989-12-01

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

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

    PubMed Central

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

    2016-01-01

    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. PMID:26907332

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

    PubMed

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

    2016-02-01

    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. PMID:26907332

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  19. 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.

  20. Detection algorithm for the validation of human cell lines.

    PubMed

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

    2012-09-15

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

  1. Characterization of a novel proinflammatory effect mediated by BK and the kinin B₂ receptor in human preadipocytes.

    PubMed

    Catalioto, Rose-Marie; Valenti, Claudio; Liverani, Luca; Giuliani, Sandro; Maggi, Carlo Alberto

    2013-08-15

    Obesity and adipose tissue contribute to local and systemic inflammation. However the role of the inflammatory mediator bradykinin (BK) in this context is not known. We therefore evaluated the effect of BK on adipokines secretion in human preadipocytes during the course of differentiation and characterized the receptors involved. Results obtained from antibody array and ELISA experiments showed that several adipokines are released by human preadipocytes under basal conditions while BK specifically stimulated the production of interleukin(IL)-6 and IL-8. The effect of BK diminished with the progression of differentiation, being almost inactive on adipocytes. In preadipocytes, BK also induced a rapid and transient [Ca²⁺](i) mobilization, a rapid and sustained increase in ERK1/2 activation and enhanced forskolin-stimulated cAMP accumulation. BK was without effect on cell proliferation and viability as assessed by bromodeoxyuridine incorporation, WST-1 conversion, or lactate dehydrogenase leakage and was without effect on adipogenesis as measured by triglyceride accumulation, GPDH activity and leptin release. The B₁ receptor agonist, Lys-[des-Arg⁹]-BK, displayed poor activity or was without effect while overall BK effects were prevented by the selective B₂ receptor antagonist, fasitibant chloride, but not by the B₁ selective antagonist, Lys-[Leu⁸][des-Arg⁹]-BK. Immunoblot analysis and immunofluorescence studies showed that the kinin B₂ receptor was essentially expressed at the beginning of the differentiation program. In conclusion, human preadipocytes expressed kinin B₂ receptors linked to multiple signaling pathways, IL-6 and IL-8 production, and BK proinflammatory response in adipose tissue could be prevented by fasitibant chloride. PMID:23796753

  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. PMID:27044015

  3. 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. PMID:26218273

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

    PubMed Central

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

    2013-01-01

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

  5. Gemcitabine induces cell senescence in human pancreatic cancer cell lines.

    PubMed

    Song, Yao; Baba, Tomohisa; Mukaida, Naofumi

    2016-08-26

    Patients with pancreatic ductal adenocarcinoma (PDAC) commonly require chemotherapy because they frequently develop metastatic disease or locally advanced tumors. Gemcitabine, an analogue of cytosine arabinoside, is commonly used for PDAC treatment. We observed that gemcitabine induced senescence phenotypes characterized by enhanced senescence-associated β-galactosidase (SA β-Gal) staining and increased expression of senescence-associated molecules in two human pancreatic cancer cell lines, Miapaca-2 and Panc-1, which exhibit resistance to gemcitabine but not L3.pl cells with a high sensitivity to gemcitabine. Gemcitabine-induced cell senescence can be inhibited by reactive oxygen species inhibitor, N-acetyl cysteine. Although gemcitabine also enhanced CXCL8 expression, anti-CXCL8 antibody failed to reduce gemcitabine-induced increases in SA β-Gal-positive cell numbers. These observations would indicate that cell senescence can proceed independently of CXCL8 expression, a characteristic feature of senescence-associated secretion phenotype. PMID:27311854

  6. Differential Chemokine Signature between Human Preadipocytes and Adipocytes

    PubMed Central

    Ignacio, Rosa Mistica C.; Gibbs, Carla R.; Lee, Eun-Sook

    2016-01-01

    Obesity is characterized as an accumulation of adipose tissue mass represented by chronic, low-grade inflammation. Obesity-derived inflammation involves chemokines as important regulators contributing to the pathophysiology of obesity-related diseases such as cardiovascular disease, diabetes and some cancers. The obesity-driven chemokine network is poorly understood. Here, we identified the profiles of chemokine signature between human preadipocytes and adipocytes, using PCR arrays and qRT-PCR. Both preadipocytes and adipocytes showed absent or low levels in chemokine receptors in spite of some changes. On the other hand, the chemokine levels of CCL2, CCL7-8, CCL11, CXCL1-3, CXCL6 and CXCL10-11 were dominantly expressed in preadipocytes compared to adipocytes. Interestingly, CXCL14 was the most dominant chemokine expressed in adipocytes compared to preadipocytes. Moreover, there is significantly higher protein level of CXCL14 in conditioned media from adipocytes. In addition, we analyzed the data of the chemokine signatures in adipocytes obtained from healthy lean and obese postmenopausal women based on Gene Expression Omnibus (GEO) dataset. Adipocytes from obese individuals had significantly higher levels in chemokine signature as follows: CCL2, CCL13, CCL18-19, CCL23, CCL26, CXCL1, CXCL3 and CXCL14, as compared to those from lean ones. Also, among the chemokine networks, CXCL14 appeared to be the highest levels in adipocytes from both lean and obese women. Taken together, these results identify CXCL14 as an important chemokine induced during adipogenesis, requiring further research elucidating its potential therapeutic benefits in obesity. PMID:27340388

  7. Differential Chemokine Signature between Human Preadipocytes and Adipocytes.

    PubMed

    Ignacio, Rosa Mistica C; Gibbs, Carla R; Lee, Eun-Sook; Son, Deok-Soo

    2016-06-01

    Obesity is characterized as an accumulation of adipose tissue mass represented by chronic, low-grade inflammation. Obesity-derived inflammation involves chemokines as important regulators contributing to the pathophysiology of obesity-related diseases such as cardiovascular disease, diabetes and some cancers. The obesity-driven chemokine network is poorly understood. Here, we identified the profiles of chemokine signature between human preadipocytes and adipocytes, using PCR arrays and qRT-PCR. Both preadipocytes and adipocytes showed absent or low levels in chemokine receptors in spite of some changes. On the other hand, the chemokine levels of CCL2, CCL7-8, CCL11, CXCL1-3, CXCL6 and CXCL10-11 were dominantly expressed in preadipocytes compared to adipocytes. Interestingly, CXCL14 was the most dominant chemokine expressed in adipocytes compared to preadipocytes. Moreover, there is significantly higher protein level of CXCL14 in conditioned media from adipocytes. In addition, we analyzed the data of the chemokine signatures in adipocytes obtained from healthy lean and obese postmenopausal women based on Gene Expression Omnibus (GEO) dataset. Adipocytes from obese individuals had significantly higher levels in chemokine signature as follows: CCL2, CCL13, CCL18-19, CCL23, CCL26, CXCL1, CXCL3 and CXCL14, as compared to those from lean ones. Also, among the chemokine networks, CXCL14 appeared to be the highest levels in adipocytes from both lean and obese women. Taken together, these results identify CXCL14 as an important chemokine induced during adipogenesis, requiring further research elucidating its potential therapeutic benefits in obesity. PMID:27340388

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

    PubMed

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

    2010-04-01

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

  9. 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. PMID:24297409

  10. 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

  11. Cell line banks and their role in cancer research.

    PubMed

    Hay, R J; Reid, Y A; McClintock, P R; Chen, T R; Macy, M L

    1996-01-01

    The utility of centralized cell banks in providing reference cultures for cancer research is reviewed. Procedures applied at The American Type Culture Collection in development, maintenance and expansion of such a resource are discussed for example, with emphasis on human tumor cell lines. The various categories of cell-line holdings are explained, and status with regard both to the numbers of lines available and distribution experienced are documented. The locations of other national cell repositories plus contact data are provided. PMID:8806094

  12. 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.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

    The GnRH receptor (GnRHR) mediates the pituitary functions of GnRH, as well as its anti-proliferative effects in sex hormone-dependent cancer cells. Here we compare the signaling of GnRHR in pituitary gonadotrope cell lines vs. prostate cancer cell lines. We first noticed that the expression level of PKCα, PKCβII and PKCε is much higher in αT3-1 and LβT2 gonadotrope cell lines vs. LNCaP and DU-145 cell lines, while the opposite is seen for PKCδ. Activation of PKCα, PKCβII and PKCε by GnRH is relatively transient in αT3-1 and LβT2 gonadotrope cell lines and more prolonged in LNCaP and DU-145 cell lines. On the otherhand, the activation and re-distribution of the above PKCs by PMA was similar for both gonadotrope cell lines and prostate cancer cell lines. Activation of ERK1/2 by GnRH and PMA was robust in the gonadotrope cell lines, with a smaller effect observed in the prostate cancer cell lines. The Ca2+ ionophore A23187 stimulated ERK1/2 in gonadotrope cell lines but not in prostate cancer cell lines. GnRH, PMA and A23187 stimulated JNK activity in gonadotrope cell lines, with a more sustained effect in prostate cancer cell lines. Sustained activation of p38 was observed for PMA and A23187 in Du-145 cells, while p38 activation by GnRH, PMA and A23187 in LβT2 cells was transient. Thus, differential expression and re-distribution of PKCs by GnRH and the transient vs. the more sustained nature of the activation of the PKC-MAPK cascade by GnRH in gonadotrope cell lines vs. prostate cancer cell lines respectively, may provide the mechanistic basis for the cell context-dependent differential biological responses observed in GnRH interaction with pituitary gonadotropes vs. prostate cancer cells. PMID:23380421

  16. Analysis of three marine fish cell lines by rapd assay.

    PubMed

    Guo, H R; Zhang, S C; Tong, S L; Xiang, J H

    2001-01-01

    We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines. PMID:11573817

  17. Establishment and characterization of unique human gallbladder cancer cell lines.

    PubMed

    Ghosh, Mila; Koike, Naoto; Yanagimoto, Go; Tsunoda, Shin-Ichi; Kaul, Sunil; Hirano, Takashi; Emura, Fabian; Kashiwagi, Hironobu; Kawamoto, Toru; Ohkohchi, Nobuhiro; Saijo, Kaoru; Ohno, Tadao; Miwa, Masanao; Todoroki, Takeshi

    2004-05-01

    Gallbladder cancer has a dismal prognosis. Understanding the disease at the biological, genetic, molecular, cellular, and clinical level is essential for effective diagnostics and therapeutics. However, the currently established gallbladder cell lines are insufficient for better understanding and further research. The aim of our present study was to establish and characterize human gallbladder cancer cell lines. We established 5 cell lines from resected specimens of gallbladder cancers. These cell lines revealed typical tumor histopathological characteristics. We examined growth characteristics and the colony-forming ability of established cell lines in terms of their cell cycle parameters, expression of tumor markers (carcinoembryonic antigen; CEA, carbohydrated antigen 19-9; CA19-9, MUC-1 and c-kit) and the oncogene c-erbB2 by flow cytometer. Comparative genomic hybridization (CGH) analysis with specific gene probes was performed to detect changes in the gene copy numbers. Human origin of cell lines was confirmed by chromosomal analysis. Cells maintained differentiation characteristics of the original tumors. The doubling time of different cell lines varied from 30 to 96 h. All 5 cell lines formed colonies in the colony forming assays and expressed CEA, CA19-9, MUC-1 and the oncogene c-erbB2 and showed chromosomal aneuploidy. CGH analysis demonstrated gain of chromosomal region bearing SRC, RAB1, and PAP in all cell lines and hTERT in 4 cell lines. These newly established cell lines might serve as a useful model for studying the molecular pathogenesis of gallbladder cancer. Furthermore, they may serve as a model for testing new therapeutics against gallbladder cancer. These chromosomal aberrations and imbalances provide a starting point for molecular analyses of genomic regions and genes in gallbladder carcinogenesis. PMID:15067341

  18. 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.

  19. 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.

  20. The pursuit of ES cell lines of domesticated ungulates

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features.

    PubMed

    Dangles-Marie, Virginie; Pocard, Marc; Richon, Sophie; Weiswald, Louis-Bastien; Assayag, Franck; Saulnier, Patrick; Judde, Jean-Gabriel; Janneau, Jean-Louis; Auger, Nathalie; Validire, Pierre; Dutrillaux, Bernard; Praz, Françoise; Bellet, Dominique; Poupon, Marie-France

    2007-01-01

    Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease. PMID:17210723

  2. Palmitate Antagonizes Wnt/Beta-catenin Signaling in 3T3-L1 Pre-adipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Long chain saturated free fatty acids such as palmitate (PA) produce insulin resistance, endoplasmic reticulum stress, and apoptosis in mature adipocytes and pre-adipocytes. In pre-adipocytes, saturated free fatty acids also promote adipogenic induction in the presence of adipogenic hormones. Wnt/be...

  3. High incidence of TERT mutation in brain tumor cell lines.

    PubMed

    Johanns, Tanner M; Fu, Yujie; Kobayashi, Dale K; Mei, Yu; Dunn, Ian F; Mao, Diane D; Kim, Albert H; Dunn, Gavin P

    2016-07-01

    TERT promoter gene mutations are highly recurrent in malignant glioma. However, little information exists regarding their presence in experimental brain tumor models. To better characterize systems in which TERT mutation studies could be appropriately modeled experimentally, the TERT promoter was examined by conventional sequencing in primary brain tumor initiating cells (BTIC), two matched recurrent BTIC lines, a panel of established malignant glioma cell lines, and two meningioma cell lines. Telomerase gene expression was examined by quantitative PCR. We found that all glioblastoma BTIC lines harbored a TERT mutation, which was retained in two patient-matched recurrent BTIC. The TERT C228T or C250T mutation was found in 33/35 (94 %) of established malignant glioma cell lines and both meningioma cell lines examined. Brain tumor cell lines expressed variably high telomerase levels. Thus, a high percentage of glioma cell lines, as well as two meningioma cell lines, harbors TERT mutations. These data characterize tractable, accessible models with which to further explore telomerase biology in these tumor types. PMID:26960334

  4. Authentication of the R06E Fruit Bat Cell Line

    PubMed Central

    Jordan, Ingo; Munster, Vincent J.; Sandig, Volker

    2012-01-01

    Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery. PMID:22754654

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

    PubMed Central

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

    2013-01-01

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

  6. miR-375 negatively regulates porcine preadipocyte differentiation by targeting BMPR2.

    PubMed

    Liu, Siyuan; Sun, Guangjie; Yuan, Bao; Zhang, Lianjiang; Gao, Yan; Jiang, Hao; Dai, Lisheng; Zhang, Jiabao

    2016-05-01

    The differentiation of preadipocytes into adipose tissues is tightly regulated by various factors including miRNAs and cytokines. In this study, taking advantage of isolated porcine primary preadipocytes, we showed that ectopic expression of miR-375 could change preadipocyte differentiation. In addition, bone morphogenetic protein receptor 2 (BMPR2) was identified as a direct target of miR-375. Silencing BMPR2 had the same inhibition effects as overexpressing miR-375 on the preadipocyte differentiation. Together, we demonstrated that miR-375 is a negative regulator of adipogenic differentiation using porcine primary preadipocytes. These results clarified the role of miR-375 in ex vivo adipogenic differentiation. PMID:27059117

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

    PubMed Central

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

    2016-01-01

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

  8. Permissiveness of human hepatoma cell lines for HCV infection

    PubMed Central

    2012-01-01

    Background Although primary and established human hepatoma cell lines have been evaluated for hepatitis C virus (HCV) infection in vitro, thus far only Huh7 cells have been found to be highly permissive for infectious HCV. Since our understanding of the HCV lifecycle would benefit from the identification of additional permissive cell lines, we assembled a panel of hepatic and non-hepatic cell lines and assessed their ability to support HCV infection. Here we show infection of the human hepatoma cell lines PLC/PRF/5 and Hep3B with cell culture-derived HCV (HCVcc), albeit to lower levels than that achieved in Huh7 cells. To better understand the reduced permissiveness of PLC and Hep3B cells for HCVcc infection, we performed studies to evaluate the ability of each cell line to support specific steps of the viral lifecycle (i.e. entry, replication, egress and spread). Results We found that while the early events in HCV infection (i.e. entry plus replication initiation) are cumulatively equivalent or only marginally reduced in PLC and Hep3B cells, later steps of the viral life cycle such as steady-state replication, de novo virus production and/or spread are impaired to different degrees in PLC and Hep3B cultures compared to Huh7 cell cultures. Interestingly, we also observed that interferon stimulated gene (i.e. ISG56) expression was significantly and differentially up-regulated in PLC and Hep3B cells following viral infection. Conclusions We conclude that the restrictions observed later during HCV infection in these cell lines could in part be attributed to HCV-induced innate signaling. Nevertheless, the identification of two new cell lines capable of supporting authentic HCVcc infection, even at reduced levels, expands the current repertoire of cell lines amendable for the study of HCV in vitro and should aid in further elucidating HCV biology and the cellular determinants that modulate HCV infection. PMID:22273112

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

    SciTech Connect

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

    1987-11-01

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

  10. Derivation of the human embryonic stem cell line RCM1.

    PubMed

    De Sousa, P A; Tye, B J; Sneddon, S; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Gardner, J; Downie, J M; Courtney, A; Brison, D R

    2016-03-01

    The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346018

  11. 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.

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

    SciTech Connect

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

    2012-01-15

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

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

    PubMed

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

    2016-08-01

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

  14. CACO-2 CELL LINES IN DRUG DISCOVERY- AN UPDATED PERSPECTIVE

    PubMed Central

    Kumar, Kalyan K.V; Karnati, Swathi; Reddy, Mamatha B; Chandramouli, R

    2010-01-01

    Cell lines are the invitro models used for the drug permeability studies in the preclinical and clinical phases of the drug discovery. Cell line models are simple and quick to use and avoids the usage of animal models for pharmacological and toxicological studies and hence cost effective, produce reliable and reproducible results for understanding and evaluating the permeability characteristics of the potential lead drug candidates. Different cell line models used in the drug permeability studies, their characteristics has been summarized emphasizing on CACO-2. By virtue of its merits, CACO-2 cell line development, transport experiments, automated assays, optimization of experimental conditions and mechanistic uses of CACO-2 cell lines dealt comprehensively in the following context. PMID:24825967

  15. Derivation of Genea057 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea057 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea057 was demonstrated with 97% of cells expressing Nanog, 81% Oct4, 75% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.59 and Novelty score of 1.32. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345782

  16. Derivation of Genea042 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345994

  17. Derivation of Genea002 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea002 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype by CGH and male Allele pattern through STR analysis. Pluripotency of Genea002 was demonstrated with 75% of cells expressing Nanog, 93% Oct4, 83% Tra1-60 and 98% SSEA4, a Pluritest pluripotency score of 24.55, Novelty score of 1.39, teratomas with tissues from all embryonic germ layers and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345802

  18. Derivation of Genea052 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345996

  19. Derivation of human embryonic stem cell line Genea023.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346015

  20. Derivation of Genea015 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346028

  1. Derivation of human embryonic stem cell line Genea022.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346017

  2. Derivation of Genea047 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345995

  3. Derivation of Genea043 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea043 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea043 was demonstrated with 92% of cells expressing Nanog, 95% Oct4, 61% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 31.74, Novelty score of 1.2 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345801

  4. Derivation of Genea016 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea016 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea016 was demonstrated with 77% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 28.4, Novelty score of 1.37 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345780

  5. Regulatory networks define phenotypic classes of human stem cell lines

    PubMed Central

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

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine1, 2 have highlighted the need for a general, reproducible method for classification of these cells3. We report here the creation and analysis of a database of global gene expression profiles (“Stem Cell Matrix”) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent, and differentiated cell types. Using an unsupervised clustering method4, 5 to categorize a collection of ~150 cell samples, we discovered that pluripotent stem cell lines group together, while other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis6 we uncovered a protein-protein network (“PluriNet”) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas, and induced pluripotent cells). Analysis of published data showed that the PluriNet appears to be a common characteristic of pluripotent cells, including mouse ES and iPS cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotence and self-renewal are under tight control by specific molecular networks. PMID:18724358

  6. 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. PMID:27322762

  7. Functional calcium imaging in zebrafish lateral-line hair cells.

    PubMed

    Zhang, Q X; He, X J; Wong, H C; Kindt, K S

    2016-01-01

    Sensory hair-cell development, function, and regeneration are fundamental processes that are challenging to study in mammalian systems. Zebrafish are an excellent alternative model to study hair cells because they have an external auxiliary organ called the lateral line. The hair cells of the lateral line are easily accessible, which makes them suitable for live, function-based fluorescence imaging. In this chapter, we describe methods to perform functional calcium imaging in zebrafish lateral-line hair cells. We compare genetically encoded calcium indicators that have been used previously to measure calcium in lateral-line hair cells. We also outline equipment required for calcium imaging and compare different imaging systems. Lastly, we discuss how to set up optimal imaging parameters and how to process and visualize calcium signals. Overall, using these methods, in vivo calcium imaging is a powerful tool to examine sensory hair-cell function in an intact organism. PMID:27263415

  8. Novel human bronchial epithelial cell lines for cystic fibrosis research

    PubMed Central

    Fulcher, M. L.; Gabriel, S. E.; Olsen, J. C.; Tatreau, J. R.; Gentzsch, M.; Livanos, E.; Saavedra, M. T.; Salmon, P.; Randell, S. H.

    2009-01-01

    Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three ΔF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Ω·cm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1β, TNF-α, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development. PMID:18978040

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

    PubMed Central

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

    2009-01-01

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

  10. Effect of ritonavir and atazanavir on human subcutaneous preadipocyte proliferation and differentiation

    PubMed Central

    Caso, Giuseppe; Mileva, Izolda; Mcnurlan, Margaret A; Mynarcik, Dennis C; Darras, Frank; Gelato, Marie C

    2010-01-01

    Protease inhibitors (PIs) have been implicated in the development of HIV-associated lipodystrophy through a reduction in the differentiation of preadipocytes. While atazanavir (ATV) is associated with fewer clinical metabolic abnormalities in the short-term, the effects of long-term exposure are not known. ATV effects on preadipocyte replication or differentiation would indicate the potential for long-term problems. This study compared ritonavir (RTV) and ATV effects on preadipocyte replication and differentiation in human primary cultures. Preadipocytes from subcutaneous fat were studied in the presence of therapeutic concentrations of RTV and ATV for replication, differentiation, and adipokine secretion. The effects of the drugs on the expression of PPARγ and related genes during differentiation were also assessed by real time quantitative PCR. RTV induced a significant inhibition of preadipocyte proliferation, differentiation and adiponectin secretion. ATV at concentrations within the range of therapeutic levels did not affect differentiation or adiponectin secretion, but did have inhibitory effects on preadipocyte proliferation. Inhibition of differentiation by PIs was associated with a decrease expression of PPARγ, C/EBPα, and aP2 genes. In summary, although ATV at therapeutic levels has a smaller impact on adipogenesis, alterations in preadipocyte proliferation suggest the potential for adverse effects with long-term use. PMID:20153378

  11. Effect of ritonavir and atazanavir on human subcutaneous preadipocyte proliferation and differentiation.

    PubMed

    Caso, Giuseppe; Mileva, Izolda; McNurlan, Margaret A; Mynarcik, Dennis C; Darras, Frank; Gelato, Marie C

    2010-05-01

    Protease inhibitors (PIs) have been implicated in the development of HIV-associated lipodystrophy through a reduction in the differentiation of preadipocytes. While atazanavir (ATV) is associated with fewer clinical metabolic abnormalities in the short-term, the effects of long-term exposure are not known. ATV effects on preadipocyte replication or differentiation would indicate the potential for long-term problems. This study compared ritonavir (RTV) and ATV effects on preadipocyte replication and differentiation in human primary cultures. Preadipocytes from subcutaneous fat were studied in the presence of therapeutic concentrations of RTV and ATV for replication, differentiation, and adipokine secretion. The effects of the drugs on the expression of PPARgamma and related genes during differentiation were also assessed by real-time quantitative PCR. RTV induced a significant inhibition of preadipocyte proliferation, differentiation and adiponectin secretion. ATV at concentrations within the range of therapeutic levels did not affect differentiation or adiponectin secretion, but did have inhibitory effects on preadipocyte proliferation. Inhibition of differentiation by PIs was associated with decreased expression of PPARgamma, C/EBPalpha, and aP2 genes. In summary, although ATV at therapeutic levels has a smaller impact on adipogenesis, alterations in preadipocyte proliferation suggest the potential for adverse effects with long-term use. PMID:20153378

  12. Investigation of Radiosensitivity Gene Signatures in Cancer Cell Lines

    PubMed Central

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

    2014-01-01

    Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins. PMID:24466029

  13. 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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-09-01

    Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014. PMID:25229492

  16. Antiproliferative effect of isopentenylated coumarins on several cancer cell lines.

    PubMed

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y; Ito, C; Furukawa, H

    2001-01-01

    33 coumarins, mainly the simple isopentenylated coumarins and derived pyrano- and furanocoumarins, were examined for their antiproliferative activity towards several cancer and normal human cell lines. The pyrano- and furanocoumarins showed strong activity against the cancer cell lines, whereas they had weak antiproliferative activity against the normal human cell lines. The decreasing rank order of potency was osthenone (10), clausarin (25), clausenidin (26), dentatin (24), nordentatin (23), imperatorin (29), seselin (27), xanthyletin (21), suberosin (17), phebalosin (8) and osthol (12). The structure-activity relationship established from the results revealed that the 1,1-dimethylallyl and isopentenyl groups have an important role for antiproliferative activity. PMID:11497276

  17. Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    PubMed Central

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

    2015-01-01

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

  18. Human papillomavirus in vulvar and vaginal carcinoma cell lines.

    PubMed Central

    Hietanen, S.; Grénman, S.; Syrjänen, K.; Lappalainen, K.; Kauppinen, J.; Carey, T.; Syrjänen, S.

    1995-01-01

    A number of reports associate human papillomavirus (HPV) with cervical cancer and cancer cell lines derived from this tumour type. Considerably fewer reports have focused on the role of HPV in carcinomas from other sites of female anogenital squamous epithelia. In this study we have tested for the presence of HPV in eight low-passage vulvar carcinoma cell lines and one extensively passaged cell line, A431. One cell line from a primary vaginal carcinoma was included. The presence of the HPV was evaluated by the polymerase chain reaction (PCR), by Southern blot analysis and by two-dimensional gel electrophoresis. General primer-mediated PCR was applied by using primers from the L1 region, E1 region and HPV 16 E7 region. Southern blot hybridisation was performed under low-stringency conditions (Tm = -35 degrees C) using a whole genomic HPV 6/16/18 probe mixture and under high stringency conditions (Tm = -18 degrees C) with the whole genomic probes of HPV 16 and 33. HPV 16 E6-E7 mRNA was assessed by ribonuclease protection assay (RPA). HPV was found in only one vulvar carcinoma cell line, UM-SCV-6. The identified type, HPV 16, was integrated in the cell genome and could be amplified with all primers used. Also E6-E7 transcripts were found in these cells. Five original tumour biopsies were available from the HPV-negative cell lines for in situ hybridisation. All these were HPV negative with both the HPV 6/16/18 screening probe mixture under low stringency and the HPV 16 probe under high stringency. The results indicate that vulvar carcinoma cell lines contain HPV less frequently than cervical carcinoma cell lines and suggest that a significant proportion of vulvar carcinomas may evolve by an HPV-independent mechanism. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7599042

  19. CHARACTERIZATION OF A SPONTANEOUSLY TRANSFORMED CHICKEN MONONUCLEAR CELL LINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Gulevskiĭ, A K; Trifonova, A V; Lavrik, A A

    2008-01-01

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

  1. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland. PMID:12418925

  2. 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

  3. Transcriptional control of insulin-sensitive glucose carrier Glut4 expression in adipose tissue cells.

    PubMed

    Penkov, D N; Akopyan, Zh A; Kochegura, T N; Egorov, A D

    2016-03-01

    In search for new targets for obesity treatment, we have studied the effect of several transcription factors on the conversion of murine preadipocytes from the 3T3-L1 cell line into adipocytes. We have found that knockdown of Prep1 gene expression affects adipogenic differentiation and results in significant increase in the insulin-sensitive glucose carrier Glut4 gene expression. PMID:27193720

  4. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    PubMed

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  5. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease

    PubMed Central

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-01-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the ‘gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  6. 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

  7. A transient increase in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization and ATP depletion during prolonged exposure to fatty acids.

    PubMed

    Rogers, Carlyle; Davis, Barbara; Neufer, P Darrell; Murphy, Michael P; Anderson, Ethan J; Robidoux, Jacques

    2014-02-01

    Preadipocytes are periodically subjected to fatty acid (FA) concentrations that are potentially cytotoxic. We tested the hypothesis that prolonged exposure of preadipocytes of human origin to a physiologically relevant mix of FAs leads to mitochondrial inner membrane (MIM) permeabilization and ultimately to mitochondrial crisis. We found that exposure of preadipocytes to FAs led to progressive cyclosporin A-sensitive MIM permeabilization, which in turn caused a reduction in MIM potential, oxygen consumption, and ATP synthetic capacity and, ultimately, death. Additionally, we showed that FAs induce a transient increase in intramitochondrial reactive oxygen species (ROS) and lipid peroxide production, lasting roughly 30 and 120min for the ROS and lipid peroxides, respectively. MIM permeabilization and its deleterious consequences including mitochondrial crisis and cell death were prevented by treating the cells with the mitochondrial FA uptake inhibitor etomoxir, the mitochondrion-selective superoxide and lipid peroxide antioxidants MitoTempo and MitoQ, or the lipid peroxide and reactive carbonyl scavenger l-carnosine. FAs also promoted a delayed oxidative stress phase. However, the beneficial effects of etomoxir, MitoTempo, and l-carnosine were lost by delaying the treatment by 2h, suggesting that the initial phase was sufficient to prime the cells for the delayed MIM permeabilization and mitochondrial crisis. It also suggested that the second ROS production phase is a consequence of this loss in mitochondrial health. Altogether, our data suggest that approaches designed to diminish intramitochondrial ROS or lipid peroxide accumulation, as well as MIM permeabilization, are valid mechanism-based therapeutic avenues to prevent the loss in preadipocyte metabolic fitness associated with prolonged exposure to elevated FA levels. PMID:24269897

  8. Global Conservation of Protein Status between Cell Lines and Xenografts.

    PubMed

    Biau, Julian; Chautard, Emmanuel; Court, Frank; Pereira, Bruno; Verrelle, Pierre; Devun, Flavien; De Koning, Leanne; Dutreix, Marie

    2016-08-01

    Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery. PMID:27567954

  9. Development of cystic fibrosis and noncystic fibrosis airway cell lines.

    PubMed

    Zabner, Joseph; Karp, Phil; Seiler, Michael; Phillips, Stacia L; Mitchell, Calista J; Saavedra, Mimi; Welsh, Michael; Klingelhutz, Aloysius J

    2003-05-01

    In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-kappaB promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-kappaB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity. PMID:12676769

  10. 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. PMID:20623584

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

    PubMed

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

    1979-02-01

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

  12. 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.

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

    SciTech Connect

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

    2009-05-08

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

  14. DNA Fingerprinting of the NCI-60 Cell Line Panel

    PubMed Central

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

    2009-01-01

    The National Cancer Institute’s NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Since many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also demonstrate that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes. PMID:19372543

  15. DNA fingerprinting of the NCI-60 cell line panel.

    PubMed

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

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes. PMID:19372543

  16. Cold storage and cryopreservation of tick cell lines

    PubMed Central

    2010-01-01

    Background Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6°C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results Cold storage at 6°C for up to 30 days was successful in preserving R. (B.) microplus, R. (B.) decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B.) decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B.) microplus cells resumed growth during the observation period. Conclusions This constitutes the first report on successful short-term refrigeration of cells derived from R. (B.) decoloratus, R. (B.) microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance. PMID:20388200

  17. 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.

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

    PubMed

    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

  19. 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

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

    PubMed

    2015-12-01

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

  1. Measles virus persistence in an immortalized murine macrophage cell line.

    PubMed

    Goldman, M B; Buckthal, D J; Picciotto, S; O'Bryan, T A; Goldman, J N

    1995-02-20

    Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse. PMID:7871720

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

    PubMed

    Jacob, Francis; Nixdorf, Sheri; Hacker, Neville F; Heinzelmann-Schwarz, Viola A

    2014-01-01

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

  3. 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

  4. The effect of myostatin on proliferation and lipid accumulation in 3T3-L1 preadipocytes.

    PubMed

    Zhu, Hui Juan; Pan, Hui; Zhang, Xu Zhe; Li, Nai Shi; Wang, Lin Jie; Yang, Hong Bo; Gong, Feng Ying

    2015-06-01

    Myostatin is a critical negative regulator of skeletal muscle development, and has been reported to be involved in the progression of obesity and diabetes. In the present study, we explored the effects of myostatin on the proliferation and differentiation of 3T3-L1 preadipocytes by using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide spectrophotometry, intracellular triglyceride (TG) assays, and real-time quantitative RT-PCR methods. The results indicated that recombinant myostatin significantly promoted the proliferation of 3T3-L1 preadipocytes and the expression of proliferation-related genes, including Cyclin B2, Cyclin D1, Cyclin E1, Pcna, and c-Myc, and IGF1 levels in the medium of 3T3-L1 were notably upregulated by 35.2, 30.5, 20.5, 33.4, 51.2, and 179% respectively (all P<0.01) in myostatin-treated 3T3-L1 cells. Meanwhile, the intracellular lipid content of myostatin-treated cells was notably reduced as compared with the non-treated cells. Additionally, the mRNA levels of Pparγ, Cebpα, Gpdh, Dgat, Acs1, Atgl, and Hsl were significantly downregulated by 22-76% in fully differentiated myostatin-treated adipocytes. Finally, myostatin regulated the mRNA levels and secretion of adipokines, including Adiponectin, Resistin, Visfatin, and plasminogen activator inhibitor-1 (PAI-1) in 3T3-L1 adipocytes (all P<0.001). Above all, myostatin promoted 3T3-L1 proliferation by increasing the expression of cell-proliferation-related genes and by stimulating IGF1 secretion. Myostatin inhibited 3T3-L1 adipocyte differentiation by suppressing Pparγ and Cebpα expression, which consequently deceased lipid accumulation in 3T3-L1 cells by inhibiting the expression of critical lipogenic enzymes and by promoting the expression of lipolytic enzymes. Finally, myostatin modulated the expression and secretion of adipokines in fully differentiated 3T3-L1 adipocytes. PMID:25878062

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

    PubMed Central

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

    2013-01-01

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

  6. Genotypes and immunophenotypes of Hodgkin's disease-derived cell lines.

    PubMed

    Drexler, H G; Leber, B F; Norton, J; Yaxley, J; Tatsumi, E; Hoffbrand, A V; Minowada, J

    1988-06-01

    This report describes the geno- and immunophenotypic analysis of the Hodgkin's disease-derived cell lines HDLM-2, KM-H2, and L-428. The lines were all positive for the antigens CD15 (Leu-M1), CD30 (Ki-1), Hefi-1 (antigen detected by a monoclonal antibody produced against L-428), HLA class I and II, and activation/proliferation markers. The cells from all 3 cell lines lacked almost all cell lineage-associated/specific markers: HDLM-2 was only CD2+, KM-H2 was only CD9+ and CD21+, and L-428 was negative for all the specific markers tested. Genomic analysis of HDLM-2 cells revealed monoclonal rearrangements of T cell receptor beta and gamma loci and germ line configuration of immunoglobulin genes. Immunoglobulin heavy chain genes were rearranged in KM-H2 and L-428. These data suggest a possible lymphoid origin for HDLM-2, KM-H2, and L-428. Although the data presented do not provide formal proof of a lymphoid nature of Hodgkin and Reed-Sternberg cells and do not unequivocally exclude a derivation from other hematopoietic cells, extrapolation of the results from the in vitro cultures to the in vivo situation suggests a lymphoid (T or B cell) origin of these cells. PMID:3131596

  7. 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

  8. Alterations in insulin binding accompanying differentiation of 3T3-L1 preadipocytes.

    PubMed Central

    Reed, B C; Kaufmann, S H; Mackall, J C; Student, A K; Lane, M D

    1977-01-01

    Expression of the adipocyte phenotype by differentiating 3T3-L1 preadipocytes occurs upon exposure of the cells to insulin. Differentiation-linked changes in 125I-labeled insulin binding to 3T3-L1 cells were monitored and compared with those in nondifferentiating 3T3-C2 controls treated similarly. Without chronic insulin treatment, 3T3-L1 cells failed to express the adipocyte phenotype but maintained a level of 25,000-35,000 insulin-binding sites per cell. Treatment of 3T3-L1 cells with insulin resulted in an initial suppression of insulin binding followed by a 12-fold increase that paralleled the appearance of differentiated cells. A maximum of 170,000 insulin-binding sites per cell was attained for a population in which greater than 75% of the cells had differentiated. The increase of insulin receptor level appears to be differentiation-dependent and is not a general response of cells to the culture conditions. 3T3-C2 cells maintained in the presence of insulin for 30 days exhibited the undifferentiated phenotype and suppressed levels of insulin binding (35,000 sites per cell). The binding capacity of 3T3-L1 cells for epidermal growth factor remained unchanged between 25,000 and 40;000 sites per cell and was independent of the state of differentiation. Thus, induction by insulin in receptor-specific changes. Insulin receptors increase in number but epidermal growth factor receptors remain constant. PMID:303773

  9. Xenotropic retrovirus Bxv1 in human pancreatic β cell lines

    PubMed Central

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

    2016-01-01

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

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. Effect of dehydrodidemnin B on human colon carcinoma cell lines.

    PubMed

    Lobo, C; García-Pozo, S G; Núñez de Castro, I; Alonso, F J

    1997-01-01

    Didemnins are cytotoxic agents belonging to a depsipeptide family isolated from marine tunicates. In the present study, a new member, dehydrodidemnin B (DDB), isolated from the mediterranean tunicate Aplidium albicans, was used. The effect of the drug on human colon cultured cell lines was tested using multiple approaches: proliferation studies, long term survival after three hours of exposure to DDB by means of a clonogenic assay and the decrease of the protooncogen, ornithine decarboxylase, activity. A dehydrodidemnin B concentration of 10(-8) M completely inhibited cell growth. The IC50 obtained using the MTT proliferation test, indicated that the most proliferative cell line (CT-2) was the most sensitive to the drug. Using a clonogenic assay a clear dose-response was obtained for the three cell lines used; HT-29 cell line showed the minimum survival after 3 hours of dehydrodidemnin B treatment. A dose-dependent decrease in ornithine decarboxylase activity was also observed in three cell lines assayed. The data presented indicate that the dehydrodidemnin B is a potent cytotoxic agent on rapidly dividing human colon cancer cells. PMID:9066673

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

    PubMed Central

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

    2006-01-01

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

  14. Characterisation of thyroid medullary carcinoma TT cell line.

    PubMed

    Zabel, M; Grzeszkowiak, J

    1997-01-01

    TT cell line is the best known stabilized cell line derived from the human medullary thyroid carcinoma. The ultrastructural characteristics of these cells include well developed rough endoplasmic reticulum, a prominent Golgi apparatus and a considerable number of secretory granules. Numerous hormones were immunocytochemically demonstrated in TT cells of which calcitonin and calcitonin gene-related peptide (CGRP) are the products of the same gene but an alternative RNA processing. TT cells were found to produce some other hormones as well, namely ACTH, neurotensin, enkephalin, PTHrP, gastrin-releasing peptide (GRP), serotonin but also functional proteins of the chromogranin group, synaptophysin, NSE, calbindin and tyrosine hydroxylase. Some marker proteins have been detected in the cytosol (CEA) and in the cytoskeleton (alpha-tubulin, cytokeratin). The influence of numerous factors on the secretory activity of these cells has been demonstrated so far, including effects of 1,25-dihydroxycholecalciferol, glucocorticoids, sex steroids, cAMP, gastrin-releasing peptide, sodium butyrate, phorbol esters, ionomycin and forskolin. The investigators performed on the TT cell line demonstrate that this is the most reliable model system for the human parafollicular cells developed so far, in comparison to other cell lines derived from the medullary carcinoma of the thyroid. PMID:9046062

  15. Characterization of a human ovarian teratocarcinoma-derived cell line.

    PubMed

    Zeuthen, J; Nørgaard, J O; Avner, P; Fellous, M; Wartiovaara, J; Vaheri, A; Rosén, A; Giovanella, B C

    1980-01-15

    A cell line (PA I), derived from human ovarian teratocarcinoma cells, was obtained by culturing ascitic fluid cells from a patient with recurrence of malignant ovarian teratoma. During early passages the cultured cells showed a variable morphology, a long doubling time, and a low plating efficiency (2%). After about 50 passages in vitro, a cell population which was more homogeneous and resembled embryonal carcinoma cells were obtained. These cells had a shorter doubling time (26 h), and increased plating efficiency (77%). The early-passage cells were aneuploid (P 24) whereas the late-passage cells had a normal diploid karyotype with one balanced translocation between chromosomes No. 15 and No. 20 (P 224). Details of the karyotype suggest that the cells are heterozygous, i.e. derived from a stage before the first meiotic division. One of the two X chromosomes were inactive, and the cells expressed HLA antigens (A28 and B12), and beta 2-microglobulin. Expression of F9 antigen, characteristic of two-cell and later preimplantation embryos, was absent, while expression of PCC4 antigen, expressed also by blastocysts, was present. This finding suggests that the line might express some embryonic characteristics. The PA I cell line maintained in monolayer cultures showed several characteristics of malignant cells. The proportion of malignant cells increased with successive passages in vitro. The late-passage cells represented a fairly homogenous population of malignant cells similar to embryonal carcinoma cells. Late-passage PA I cells, when seeded under conditions that prevented attachment of cells to the substratum, formed embryoid bodies consisting of an inner core of cells similar to embryonal carcinoma cells, surrounded by a rind of endoderm-like cells. These two cell layers were separated by a basement membrane-like structure containing fibronectin. The core embryonal carcinoma cells expressed high alkaline phosphatase activity whereas the endoderm-like cells had low

  16. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

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

  17. Cell Line Modeling to Study Biomarker Panel in Prostate Cancer

    PubMed Central

    NickKholgh, Bita; Fang, Xiaolan; Winters, Shira M.; Raina, Anvi; Pandya, Komal S.; Gyabaah, Kenneth; Fino, Nora; Balaji, K.C.

    2016-01-01

    BACKGROUND African–American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. METHODS We assembled a PCa cell line model that included currently available African–American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. RESULTS The dysregulation of the multiplex biomarker panel in primary African–American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African–American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African–Americans and Caucasians as a prelude to future translational studies. CONCLUSION We have characterized a novel in

  18. Measurement of Acetylcholine from Cell Lines

    PubMed Central

    Lau, Jamie K.; Brown, Kathleen C.; Dasgupta, Piyali

    2016-01-01

    Cigarette smoking is the leading risk factor for the development of lung cancer. It is estimated that smoking is associated with 80–90% of lung cancer cases throughout the world (see References 1 and 2). The addictive component of cigarette smoke is nicotine. Our published data shows that nicotine promotes the production of acetylcholine (ACh) in human bronchioalveolar carcinoma cells (BACs) (Lau et al., 2013). ACh functions as a growth factor in human BACs. The following protocol is based on a published protocol by (Song et al., 2003), with some modifications (Lau et al., 2013; Song et al., 2008; Song et al., 2003; Sekhon et al., 2003). An important point to remember is that fetal bovine serum (FBS) contains a high amount of acetylcholine (ACh). Therefore, cells must be cultured in serum-free medium to measure ACh in the culture supernatant. Two aliquots of the culture supernatant are used for analysis. This protocol measures the total choline in the cell supernatent under two conditions: 1) After treatment with acetylcholinesterase (AChE), which converts the ACh to choline (also called the total choline sample) and 2) after measuring the amount of free choline in the sample. The concentration of ACh in the sample calculated by subtracting the free choline from the total choline.

  19. Role of Phosphotyrosine Interaction Domain Containing 1 in Porcine Intramuscular Preadipocyte Proliferation and Differentiation.

    PubMed

    Chen, Xiaoling; Luo, Yanliu; Huang, Zhiqing; Jia, Gang; Liu, Guangmang; Zhao, Hua

    2016-10-01

    Phosphotyrosine interaction domain containing 1 (PID1), a recently identified gene involved in obesity-associated insulin resistance, plays an important role in fat deposition. However, its effect on porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, the plasmid pcDNA3.1(+)-pPID1 was transfected into porcine intramuscular preadipocytes with Lipofectamine 3000 reagent to over-express porcine PID1 (pPID1). Over-expression of pPID1 significantly promoted porcine intramuscular preadipocyte proliferation. Expression of pPID1 mRNA was significantly increased upon porcine intramuscular preadipocyte differentiation. Indirect fluorescent immunocytochemistry demonstrated that pPID1 protein was localized predominantly in the nucleus of porcine intramuscular preadipocyte. The mRNA levels of peroxisome proliferators-activated receptor γ, CCAAT/enhancer binding protein α and lipoprotein lipase were significantly increased by pPID1 over-expression. Over-expression of pPID1 also led to an increase in lipid accumulation which was detected by Oil Red O staining, and significantly increased the intramuscular triacylglycerol content. These results indicate that pPID1 may play a role in enhancing porcine intramuscular preadipocyte proliferation and differentiation. PMID:27565873

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    1994-01-01

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

  2. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    EPA Science Inventory

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

  3. 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

  4. Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

    PubMed Central

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M. Cecilia T.; Wolf, Don; Mitalipov, Shoukhrat

    2009-01-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients. PMID:18192229

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

    PubMed

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

    2014-10-01

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

  6. Derivation of human embryonic stem cell line Genea019.

    PubMed

    Dumevska, Biljana; Peura, Teija; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346002

  7. 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

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

    PubMed

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

    2014-09-01

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

  9. Female Sex Bias in Human Embryonic Stem Cell Lines

    PubMed Central

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

    2012-01-01

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

  10. Female sex bias in human embryonic stem cell lines.

    PubMed

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

    2012-02-10

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

  11. Generation of Mouse iNKT Cell Lines

    PubMed Central

    Li, Xiangming; Tsuji, Moriya; Schneck, Jonathan; Webb, Tonya J.

    2016-01-01

    Natural killer T (NKT) cells bridge the innate and adaptive arms of the immune system, and manipulating their effector functions can have therapeutic significances in the treatment of autoimmunity, transplant biology, infectious disease and cancer. This important lymphocyte subset regulates the immune system through their potent cytokine production following the recognition of lipid antigen present in the context of the MHC class I-like CD1d molecule, in addition their ability to directly mediate cytotoxicity. Here, we describe a method of expanding mouse invariant NKT (iNKT) cell lines from mononuclear cells isolated from the thymus, spleen, or liver using bone marrow derived dendritic cells. These iNKT cell lines can be used study their co-signaling requirements, cytokine profiles and cytotoxic functions which will greatly enhance our knowledge of iNKT cell biology.

  12. JNK Inhibition Inhibits Lateral Line Neuromast Hair Cell Development

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  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. Morphology and growth of murine cell lines on model biomaterials.

    PubMed

    Godek, Marisha L; Duchsherer, Nichole L; McElwee, Quinn; Grainger, David W

    2004-01-01

    All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues. PMID:15133927

  17. Artificial islets from hybrid spheroids of three pancreatic cell lines.

    PubMed

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

    2014-05-01

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

  18. 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.

  19. Comparative proteomic profiling of Hodgkin lymphoma cell lines.

    PubMed

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

    2016-01-01

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

  20. Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Wilson, James M.; Birinyi, Louis K.; Salomon, Robert N.; Libby, Peter; Callow, Allan D.; Mulligan, Richard C.

    1989-06-01

    The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

  1. Skin Biopsy and Patient-Specific Stem Cell Lines

    PubMed Central

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

    2016-01-01

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

  2. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    SciTech Connect

    Medina, D.; Oborn, C.J. ); Li, M.L.; Bissell, M.J. )

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  3. Characterization of a Novel Radiation-Induced Sarcoma Cell Line

    PubMed Central

    Lang, J.E.; Zhu, W.; Nokes, B.T.; Sheth, G.R.; Novak, P.; Fuchs, L.; Watts, G.S.; Futscher, B.W.; Mineyev, N.; Ring, A.; LeBeau, L.; Nagle, R.; Cranmer, L.D.

    2014-01-01

    Background Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS. Methods We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS. Results Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models. Conclusion Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS. Synopsis We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a radiation-induced sarcoma (RIS). Our novel RIS cell line constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. PMID:25644184

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

    PubMed

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

    2016-05-01

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

  5. 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.

  6. 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).

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

    PubMed Central

    2010-01-01

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

  8. Simultaneous measurement of natural killer cell cytotoxicity against each of three different target cell lines.

    PubMed

    Blomberg, K

    1994-02-10

    A time-resolved fluorometric assay for the simultaneous measurement of natural killer cell activity against three different lanthanide diethylenetriaminopentaacetate (LaDTPA) labelled target cell lines is described. The target cell line K-562 was labelled with SmDTPA, the cell line Molt with TbDTPA and the cell line Raji with EuDTPA. After co-incubation of the three target cell lines with effector cells the fluorescence of the lanthanides released from the lysed target cells was measured in an enhancer solution in which they formed highly fluorescent complexes. It was possible to differentiate the specific release from the three target cell lines because the emission lines of the europium, samarium and terbium complexes formed in the enhancer solution are well separated from each other. The autofluorescence from culture media supplemented with serum was avoided by the use of time-resolved fluorometry. The results show that applying fluorometry based on the combination of spectral and temporal resolution to natural killer cell assays, makes possible the simultaneous determination of lysis in up to three target cell lines in complex culture medium. PMID:8308301

  9. Modeling adenovirus latency in human lymphocyte cell lines.

    PubMed

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

    2010-09-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection. PMID:20573817

  10. Registration of Human Embryonic Stem Cell Lines: Korea, 2010

    PubMed Central

    Lee, Ji-Yoon; Lee, Dae-Yeon; Choi, Young-Sil; Lee, Kyoung-Jae; Kim, Yong-Ou

    2011-01-01

    In an effort to increase the credibility of human embryonic stem cell (hESC) lines established in Korea, obligatory registration was introduced by the Bioethics and Safety Act 2008, effective as of January 1, 2010. The DNA fingerprint, chromosome stability, expression of pluripotency markers, and contamination of mycoplasma of the submitted lines were analyzed by Korea Centers for Disease Control and Prevention (KCDC). The characterization data and ethical aspects, such as informed consent for donation of surplus embryos, were reviewed by a 10-member advisory review committee for stem cell registry. A total of 55 domestic hESC lines were submitted for registration in 2010; among them 51 were registered. Among these submitted lines, 26 were additionally characterized by KCDC, while 25 lines previously characterized by the Ministry of Education, Science and Technology were not additionally analyzed by KCDC. Registration completed an oversight system for embryo research by registering the products of licensed embryo research projects, making embryo research more transparent in Korea. Information about hESC lines is available at the website of the Korea Stem Cell Registry (kscr.nih.go.kr). PMID:24159464

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

    PubMed Central

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

    1996-01-01

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

  12. Molecular cytogenetic analysis of breast cancer cell lines

    PubMed Central

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

    2000-01-01

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

  13. 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

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

    PubMed Central

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

    2012-01-01

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

  15. 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. PMID:27103160

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

    PubMed Central

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

    1993-01-01

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

  17. 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.

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

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; Department of Ophthalmology, Chiayi Christian Hospital, Chiayi 600, Taiwan, ROC ; 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.

  19. Generation of BAC reporter cell lines for drug discovery.

    PubMed

    Kao, Betty R; McColl, Bradley; Vadolas, Jim

    2015-01-01

    Bacterial artificial chromosome (BAC) reporter cell lines are generated through stable transfection of a BAC reporter construct wherein the gene of interest is tagged with a reporter gene such as eGFP. The large capacity of BACs (up to 350 kb of genomic sequence) enables the inclusion of all regulatory elements that ensure appropriate regulation of the gene of interest. Furthermore, the reporter gene allows the expression of the gene of interest to be readily detected by flow cytometry. Cell lines can also be easily cultured for extended periods with minimal cost. These features of BAC reporter cell lines make them highly amenable for use in high-throughput screening of large drug libraries for compounds that induce the expression of the gene of interest. This chapter describes a method for generation of BAC reporter cell lines that are suitable as cellular assay systems in high-throughput screening. Briefly, this method involves (A) generation of cell clones stably transfected with a BAC reporter construct, (B) selection of "candidate" cell clones based on the responsiveness to known inducers, (C) confirmation of the integrity of the BAC reporter construct integrated within the candidate clones, and (D) assessment of the developmental regulation of the BAC reporter construct. As an example, we describe the generation of a BAC reporter cell line containing the human β-globin locus modified to express γ-globin as eGFP for use as a cellular reporter assay for screening of drugs that can reactivate expression of developmentally silenced γ-globin for the treatment of β-hemoglobin disorders. PMID:25239756

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

    PubMed

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

    2016-01-01

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

  1. Cytolytic activity against tumor cells by macrophage cell lines and augmentation by macrophage stimulants.

    PubMed

    Taniyama, T; Holden, H T

    1980-07-15

    Previous studies have shown that macrophage cell lines retained the ability to phagocytize, to secrete lysosomal enzymes, and to function as effector cells in antibody-dependent cellular cytoxicity. In this paper, the cytolytic activity of murine macrophage cell lines against tumor target cells was assessed using an 18-h 51Cr release assay. Of the macrophage cell lines tested, RAW 264, PU5-1.8 and IC-21 had intermediate to high levels of spontaneous cytolytic activity, P388D, and J774 had low to intermediate levels, while /WEHI-3 showed little or no cytolytic activity against RBL-5, MBL-2 and TU-5 target cells. Tumor-cell killing by macrophage cell lines could be augmented by the addition of macrophage stimulants, such as bacterial lipopolysaccharide and poly I:C, indicating that the activation of macrophages by these stimulants does not require the participation of other cell types. Treatment with interferon also augmented the tumor-cell killing by macrophage cell lines. Although the mechanism by which these cell lines exert their spontaneous or boosted cytotoxic activity is not clear, it does not appear to be due to depletion of nutrients since cell lines with high metabolic and proliferative activities, such as WEHI-3 and RBL-5, showed little or no cytotoxicity and supernatants from the macrophage cell lines did not exert any cytotoxic effects in their essay. Thus, it appears that the different macrophage cell lines represent different levels of activation and/or differentiation and may be useful for studying the development of these processes as well as providing a useful tool for analyzing the mechanisms of macrophage-mediated cytolysis. PMID:6165690

  2. Genetic instability in human ovarian cancer cell lines.

    PubMed Central

    Orth, K; Hung, J; Gazdar, A; Bowcock, A; Mathis, J M; Sambrook, J

    1994-01-01

    We have analyzed the stability of microsatellites in cell lines derived from human ovarian cancers and found that 5 out of 10 of the ovarian tumor cell lines are genetically unstable at the majority of the loci analyzed. In clones and subclones derived serially from one of these cell lines (2774; serous cystadenocarcinoma), a very high proportion of microsatellites distributed in many different regions of the genome change their size in a mercurial fashion. We conclude that genomic instability in ovarian tumors is a dynamic and ongoing process whose high frequency may have been previously underestimated by PCR-based allelotyping of bulk tumor tissue. We have identified the source of the genetic instability in one ovarian tumor as a point mutation (R524P) in the human mismatch-repair gene MSH2 (Salmonella MutS homologue), which has recently been shown to be involved in hereditary nonpolyposis colorectal cancer. Patient 2774 was a 38-year-old heterozygote, and her normal tissue carried both mutant and wild-type alleles of the human MSH2 gene. However the wild-type allele was lost at some point early during tumorigenesis so that DNA isolated either from the patient's ovarian tumor or from the 2774 cell line carries only the mutant allele of the human MSH2 gene. The genetic instability observed in the tumor and cell line DNA, together with the germ-line mutation in a mismatch-repair gene, suggest that the MSH2 gene is involved in the onset and/or progression in a subset of ovarian cancer. Images PMID:7937795

  3. Hypoxic cell turnover in different solid tumor lines

    SciTech Connect

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

    2005-07-15

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

  4. 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.

  5. Cell membrane fatty acid composition differs between normal and malignant cell lines.

    PubMed

    Meng, Xialong; Riordan, Neil H; Riordan, Hugh D; Mikirova, Nina; Jackson, James; González, Michael J; Miranda-Massari, Jorge R; Mora, Edna; Trinidad Castillo, Waleska

    2004-06-01

    Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids. PMID:15377057

  6. Establishing an immortalized human osteoprecursor cell line: OPC1.

    PubMed

    Winn, S R; Randolph, G; Uludag, H; Wong, S C; Hair, G A; Hollinger, J O

    1999-10-01

    The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P

  7. Derivation of Ethnically Diverse Human Induced Pluripotent Stem Cell Lines.

    PubMed

    Chang, Eun Ah; Tomov, Martin L; Suhr, Steven T; Luo, Jiesi; Olmsted, Zachary T; Paluh, Janet L; Cibelli, Jose

    2015-01-01

    The human genome with all its ethnic variations contributes to differences in human development, aging, disease, repair, and response to medical treatments and is an exciting area of research and clinical study. The availability of well-characterized ethnically diverse stem cell lines is limited and has not kept pace with other advances in stem cell research. Here we derived xenofree ethnically diverse-human induced pluripotent stem cell (ED-iPSC) lines from fibroblasts obtained from individuals of African American, Hispanic-Latino, Asian, and Caucasian ethnic origin and have characterized the lines under a uniform platform for comparative analysis. Derived ED-iPSC lines are low passage number and evaluated in vivo by teratoma formation and in vitro by high throughput microarray analysis of EB formation and early differentiation for tri-lineage commitment to endoderm, ectoderm and mesoderm. These new xenofree ED-iPSC lines represent a well-characterized valuable resource with potential for use in future research in drug discovery or clinical investigations. PMID:26482195

  8. Establishment and applications of male germ cell and Sertoli cell lines.

    PubMed

    Wang, Hong; Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; He, Zuping

    2016-08-01

    Within the seminiferous tubules there are two major cell types, namely male germ cells and Sertoli cells. Recent studies have demonstrated that male germ cells and Sertoli cells can have significant applications in treating male infertility and other diseases. However, primary male germ cells are hard to proliferate in vitro and the number of spermatogonial stem cells is scarce. Therefore, methods that promote the expansion of these cell populations are essential for their use from the bench to the bed side. Notably, a number of cell lines for rodent spermatogonia, spermatocytes and Sertoli cells have been developed, and significantly we have successfully established a human spermatogonial stem cell line with an unlimited proliferation potential and no tumor formation. This newly developed cell line could provide an abundant source of cells for uncovering molecular mechanisms underlying human spermatogenesis and for their utilization in the field of reproductive and regenerative medicine. In this review, we discuss the methods for establishing spermatogonial, spermatocyte and Sertoli cell lines using various kinds of approaches, including spontaneity, transgenic animals with oncogenes, simian virus 40 (SV40) large T antigen, the gene coding for a temperature-sensitive mutant of p53, telomerase reverse gene (Tert), and the specific promoter-based selection strategy. We further highlight the essential applications of these cell lines in basic research and translation medicine. PMID:27069011

  9. Detection of cytoplasmic and surface membrane markers in cells of some human hematopoietic cell lines.

    PubMed

    Koníková, E; Babusíková, O; Kusenda, J; Glasová, M

    1992-01-01

    The cells of some human leukemia-lymphoma T cell lines (JURKAT, MOLT4), B cell lines (DAUDI, U-266) and of myeloid U-937 cell line were characterized for their surface membrane and cytoplasmic marker profiles. The usefulness of some fixation and permeabilization methods of cell membrane for detection of cytoplasmic markers by flow cytometry was studied. The methods of cell fixation in suspension were found to be more sensitive than the methods of cell fixation in smears. With the very short buffered formaldehyde-acetone (BFA) fixation used in this study an optimal penetration of the monoclonal antibodies (MoAbs) through the plasma membrane and specific binding to the appropriate structures were achieved. CD22 antigen was detected in cytoplasm but not on membrane of DAUDI cells. In another B cell line, U-266, CD22 antigen was present both in cell membrane and cytoplasm. The marker corresponding to anti-CD19 MoAb was detected in cytoplasm but was absent on membrane of U-266 cells. Furthermore, the antigen estimated by anti-CD3 MoAb could be detected intracellularly in cells of both T cell lines tested, while it was absent on cell membrane of these cells. The phenotypic study of U-937 cells showed that the majority of cells expressed myeloid associated antigens. In our study the CD14 marker detected on cell surface membrane of U-937 cells was missing in their cytoplasm. The surface antigens remained intact after BFA fixation enabling a simultaneous detection of membrane and cytoplasmic markers in double immunofluorescence studies. Through this combination of markers minor cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for a rapid and quantitative immunodiagnosis. PMID:1491722

  10. 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

  11. Transcriptional profiling and assessment of cell lines as in vitro models for mantle cell lymphoma.

    PubMed

    Ek, Sara; Ortega, Eva; Borrebaeck, Carl A K

    2005-02-01

    Mantle cell lymphoma (MCL) is an aggressive malignancy and new treatment modalities must be established to increase patient survival time. In the search for new therapeutic targets, reliable and well-characterised in vitro models are essential. In this study, we have characterised three MCL cell lines (SP53, Granta 519 and NCEB1) in comparison with primary tumours from MCL, follicular lymphomas (FL), a FL cell line (RL), a Burkitt lymphoma cell line (RAJI) and five different B cell populations from healthy individuals. Expression profiling was used to determine the relative expression of >12000 transcripts in these samples, and flow cytometry analysis was performed to establish a phenotypic signature for each of the cell lines. In addition, the cell lines were sequenced, and the frequency of somatic mutations and immunoglobulin (Ig) variable heavy chain (VH) usage were determined. We show by hierarchical clustering that the cell lines retain a genetic signature similar to primary MCL, which readily separated the MCL samples from the other lymphoma cell lines and the FL tumours. Furthermore, the MCL cell lines showed differences in the frequency of VH somatic mutations (0-2.1%). The increased number of mutations in NCEB1, compared to the other MCL cell lines, was in agreement with a decreased expression of CD31, CD44, CXCR5, CCR7 and CCR6. Taken together, our data show that the cell lines are clearly derived from MCL tumours and expressed similar genetic and phenotypic signatures compared to primary tumours, which confirmed their usefulness as in vitro models. PMID:15607370

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

    PubMed

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

    1989-07-01

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

  13. A Novel Cell Line from Spontaneously Immortalized Murine Microglia

    PubMed Central

    Kulas, Joshua; Combs, Colin K.

    2014-01-01

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

  14. 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...

  15. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    PubMed

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

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

    PubMed

    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

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

    EPA Science Inventory

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

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

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

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

  19. 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...

  20. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

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

    Methylation of Arsenite by Some Mammalian Cell Lines.

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

  1. Cryopreservation of specialized chicken lines using cultured primordial germ cells

    PubMed Central

    Nandi, S.; Whyte, J.; Taylor, L.; Sherman, A.; Nair, V.; Kaiser, P.; McGrew, M. J.

    2016-01-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells (PGCs). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- (MHC-) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  2. USING NEUROBLASTOMA CELL LINES TO EXAMINE ORGANOPHOSPHATE NEUROTOXICITY

    EPA Science Inventory

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

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

    PubMed

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

    1997-06-01

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

  4. 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. PMID:25050633

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

    PubMed

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

    2015-12-01

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

  6. Simultaneous measurement of NK cell cytotoxicity against two target cell lines labelled with fluorescent lanthanide chelates.

    PubMed

    Lövgren, J; Blomberg, K

    1994-07-12

    We describe a cytotoxicity assay which permits the simultaneous measurement of natural killer cell activity against two different cell lines. The target cell lines are labelled either with a fluorescent europium chelate or with a fluorescent terbium chelate and cell death is quantified by measuring the chelate release. K-562, Molt4 and Daudi cell lines have been used as targets. The release of the two chelates from the target cells can be detected with the help of time resolved fluorometry. As the measurements are made after background fluorescence has decayed no additional steps are needed to correct for the background from the medium. The assay procedure used for measurement of cytotoxicity against two target cell lines is very similar to the widely used 51Cr release assay. PMID:8034979

  7. Metal mutagenesis in transgenic Chinese hamster cell lines.

    PubMed Central

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

    1994-01-01

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

  8. 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

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

    PubMed

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

    2016-01-01

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

  10. Characteristics of taurine transport in cultured renal epithelial cell lines: asymmetric polarity of proximal and distal cell lines.

    PubMed

    Jones, D P; Miller, L A; Budreau, A; Chesney, R W

    1992-01-01

    Taurine transport was determined in two continuous, renal epithelial cell lines: LLC-PK1 derived from the proximal tubule of the pig, and the Madin-Darby canine kidney cell (MDCK) from the distal tubule of the dog. In LLC-PK1, taurine transport is maximal at the apical surface, whereas in MDCK cells, transport is greatest at the basolateral surface. Transport is highly dependent on both sodium and chloride in the external medium, and is specific for beta-amino acids. The apical and basolateral surfaces of both cell lines show an adaptive response to extracellular taurine concentration, but only the basolateral surface of the MDCK cell responds to hyperosomolality by increased taurine accumulation. Thus, differential control of the beta-amino acid transport system by substrate and external tonicity exists. The role of the beta-amino acid transport system may differ according to the origin of the cell: in the proximal renal tubular cell, net transepithelial reabsorption of filtered taurine increases the body pool. By contrast, taurine accumulation by distal tubular cells may form a mechanism of cell volume regulation in response to osmotic stress. PMID:1509959

  11. Off-line test of the KISS gas cell

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  12. Growth inhibition by tyrosine kinase inhibitors in mesothelioma cell lines.

    PubMed

    Nutt, Joyce E; O'Toole, Kieran; Gonzalez, David; Lunec, John

    2009-06-01

    Clinical outcome following chemotherapy for malignant pleural mesothelioma is poor and improvements are needed. This preclinical study investigates the effect of five tyrosine kinase inhibitors (PTK787, ZD6474, ZD1839, SU6668 and SU11248) on the growth of three mesothelioma cell lines (NCI H226, NCI H28 and MSTO 211H), the presence of growth factor receptors and inhibition of their downstream signalling pathways. GI50 values were determined: ZD6474 and SU11248, mainly VEGFR2 inhibitors, gave the lowest GI50 across all cell lines (3.5-6.9 microM) whereas ZD1839 gave a GI50 in this range only in H28 cells. All cell lines were positive for EGFR, but only H226 cells were positive for VEGFR2 by Western blotting. ZD6474 and ZD1839 inhibited EGF-induced phosphorylation of EGFR, AKT and ERK, whereas VEGF-induced phosphorylation of VEGFR2 was completely inhibited with 0.1 microM SU11248. VEGFR2 was detected in tumour samples by immunohistochemistry. VEGFR2 tyrosine kinase inhibitors warrant further investigation in mesothelioma. PMID:19318229

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

    PubMed

    Kowarz, Eric; Löscher, Denise; Marschalek, Rolf

    2015-04-01

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

  14. BRITER: A BMP Responsive Osteoblast Reporter Cell Line

    PubMed Central

    Bandyopadhyay, Amitabha

    2012-01-01

    Background BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. Methodology/Principal Findings We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line), responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. Conclusion As the dynamic range of the assay (for BMP responsiveness) is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance. PMID:22611465

  15. 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

  16. CD40 expression in Wehi-164 cell line

    PubMed Central

    Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

    2010-01-01

    CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body’s defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system. PMID:20496113

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

    PubMed Central

    Piskareva, Olga; Muniyappa, Mohan

    2007-01-01

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

  18. 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.

  19. Inhibitory Effects of Purple Sweet Potato Leaf Extract on the Proliferation and Lipogenesis of the 3T3-L1 Preadipocytes.

    PubMed

    Lee, Shou-Lun; Lee, Hsien-Kuang; Chin, Ting-Yu; Tu, Ssu-Chieh; Kuo, Ming-Hsun; Kao, Ming-Ching; Wu, Yang-Chang

    2015-01-01

    Purple sweet potato leaves (PSPLs) are healthy vegetable that is rich in anti-oxidants. A solution of boiling water extract of PSPL (PSPLE) is believed to be able to prevent obesity and metabolic syndrome in the countryside of Taiwan, but its efficacy has not yet been verified. The purpose of this study was to investigate the possible anti-adipogenesis effect of PSPLE in vitro. PSPLE was used to treat the 3T3-L1 cells, and the effects on cell proliferation and adipogenesis were investigated. The results showed that PSPLE caused a dose-dependent decrease in the cell proliferation of 3T3-L1 preadipocytes, but did not alter the cell viability. In addition, PSPLE induced ERK inactivation in the 3T3-L1 preadipocytes. Furthermore, pre-treatment of confluent 3T3-L1 cells with PSPLE led to reduced lipid accumulation in differentiated 3T3-L1 cells. The inhibition of lipogenesis could result from the PSPLE-induced down-regulation of the expression of the C/EBPα and SREBP-1 transcription factors during 3T3-L1 adipocyte differentiation. These results suggest that PSPLE not only inhibits cell proliferation at an early stage but also inhibits adipogenesis at a later stage of the differentiation program. PMID:26205968

  20. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

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

    2014-06-01

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

  1. Transgenic cell lines for detection of animal viruses.

    PubMed Central

    Olivo, P D

    1996-01-01

    Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses. PMID:8809463

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2015-04-01

    Niclosamide, used as an antihelminthic, has demonstrated some properties of anticancer effects. However, its role in osteosarcoma remains to be determined. The aim of this study was to determine the effect of niclosamide on human osteosarcoma cell lines. The human MG-63 and U2OS osteosarcoma cell lines were treated with different concentrations of niclosamide. The cell inhibitory rate was calculated by CCK-8 assay. Cell cycle was detected by flow cytometry. Cell apoptosis was determined by Hoechst 33324 staining, flow cytometry and fluorescence microscope, respectively. The expression of bcl-2, bax and pro-caspase-3 were measured by western blotting. Niclosamide exerted an inhibitory effect on the two cell lines in a time- and dose-dependent manner. Niclosamide was found to induce the arrest of S and G2/M phase in U2OS cells and G2/M in MG-63 cells. Moreover, niclosamide induced apoptosis in MG-63 and U2OS cells. The bax/bcl-2 ratio increased while the expression of pro‑caspase-3 decreased significantly in the two cell lines. The results indicated that niclosamide inhibits proliferation, and induces apoptosis and cell cycle arrest in human osteosarcoma cell lines. PMID:25634333

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    1990-01-01

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

  8. 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.

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

    PubMed

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

    2015-06-01

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

  10. Interaction of a mouse macrophage cell line with homologous erythrocytes.

    PubMed

    Singer, J A; Walker, W S; Morrison, M

    1982-06-01

    The interaction of the IC-21 murine macrophage cell line and homologous red blood cells (RBC) was assessed in the absence of exogenous opsonins. These results were used to evaluate this system as a potential model for macrophage-mediated clearance of old or damaged RBC. The binding and ingestion of density-separated and unseparated RBC by IC-21 cells were quantitated in assays that involved both 51Cr-labeled RBC and direct microscopy. The number of unseparated RBC that bound to IC-21 macrophages depended on the number of RBC added. Macrophages phagocytized an appreciable proportion of RBC within 3 hours with the ratio of RBC:macrophage of 10, a point at which the RBC-binding was not rate limiting. The mouse RBC were separated into dense- and less-dense fractions which are presumably enriched for old and young cells, respectively. When these RBC fractions were incubated with the IC-21 macrophage, significantly more of these dense cells were phagocytized. These results show that IC-21 macrophage cell line is a useful model for defining the processes whereby aged or damaged RBC are recognized and removed from circulation by macrophages. PMID:7120230

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1999-12-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-08-01

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

  15. 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

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

    PubMed

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

    2015-01-01

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

  17. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  18. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    PubMed Central

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

    2014-01-01

    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. PMID:24728108

  19. Cell line profiling to improve monoclonal antibody production.

    PubMed

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

    2014-04-01

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

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

    PubMed

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

    2007-12-01

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

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

    PubMed

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

    2009-08-01

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

  2. Establishment of human cell lines showing circadian rhythms of bioluminescence.

    PubMed

    Yoshikawa, Aki; Shimada, Hiroko; Numazawa, Kahori; Sasaki, Tsukasa; Ikeda, Masaaki; Kawashima, Minae; Kato, Nobumasa; Tokunaga, Katsushi; Ebisawa, Takashi

    2008-11-28

    We have established human retinal pigment epithelial cell lines stably expressing the luciferase gene, driven by the human Bmal1 promoter, to obtain human-derived cells that show circadian rhythms of bioluminescence after dexamethasone treatment. The average circadian period of bioluminescence for the obtained clones was 24.07+/-0.48 h. Lithium (10 mM) in the medium significantly lengthened the circadian period of bioluminescence, which is consistent with previous reports, while 2 mM or 5 mM lithium had no effect. This is the first report on the establishment of human-derived cell lines that proliferate infinitely and show circadian rhythms of bioluminescence, and also the first to investigate the effects of low-dose lithium on the circadian rhythms of human-derived cells in vitro. The established cells will be useful for various in vitro studies of human circadian rhythms and for the development of new therapies for human disorders related to circadian rhythm disturbances. PMID:18809466

  3. 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. PMID:25701818

  4. Hepatitis C virus infection of cholangiocarcinoma cell lines

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1989-07-01

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

  6. 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. PMID:22511037

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  9. MicroRNA profiles in various hepatocellular carcinoma cell lines

    PubMed Central

    Morishita, Asahiro; Iwama, Hisakazu; Fujihara, Shintaro; Sakamoto, Teppei; Fujita, Koji; Tani, Joji; Miyoshi, Hisaaki; Yoneyama, Hirohito; Himoto, Takashi; Masaki, Tsutomu

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortality worldwide. Although surgery is considered the most effective treatment for patients with HCC, its indication is restricted by limited criteria and a high relapse rate following surgery; therefore, systemic chemotherapy is required for patients with advanced-stage HCC to prolong their survival. MicroRNAs (miRNAs) are endogenous non-coding RNAs of 18–22 nucleotides in length. It has been reported that aberrant expression of miRNAs is a feature shared by various types of human cancer. Previous studies have indicated that the modulation of non-coding RNAs, particularly miRNAs, may be a valuable therapeutic target for HCC. The aim of the present study was to elucidate the miRNA profiles associated with differentiation and hepatitis B virus (HBV) infection observed in HCC cell lines. The human Alex, Hep3B, HepG2, HuH1, HuH7, JHH1, JHH2, JHH5, JHH6, HLE, HLF and Li-7 HCC cell lines were used for an miRNA array. Replicate data were analyzed following their classification into: i) Poorly- and well-differentiated human HCC cells and ii) HBV-positive and -negative human HCC cells. Out of the 1,719 miRNAs, 4 were found to be significantly upregulated and 52 significantly downregulated in the poorly-differentiated cells, as compared with the well-differentiated cells. Conversely, in the HBV-positive cells 125 miRNAs were found to be upregulated and 2 downregulated, as compared with the HBV-negative cells. Unsupervised hierarchical clustering analysis with Pearson's correlation revealed that the miRNA expression levels were clustered both together and separately in each group. In conclusion, miRNA profile characterization based on various parameters may be a novel approach to determine the etiology of HCC.

  10. Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

    PubMed Central

    Carlier, Géraldine; Maugein, Alicia; Cordier, Corinne; Pechberty, Séverine; Garfa-Traoré, Meriem; Martin, Patrick; Scharfmann, Raphaël; Albagli, Olivier

    2014-01-01

    Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation. PMID:25259951

  11. Responding to hypoxia: lessons from a model cell line.

    PubMed

    Seta, K A; Spicer, Z; Yuan, Y; Lu, G; Millhorn, D E

    2002-08-20

    Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. It is, therefore, not surprising that sophisticated mechanisms have evolved that allow cells to adapt to hypoxia. "Oxygen-sensing" is a special phenotype that functions to detect changes in oxygen tension and to transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in various organisms. Oxygen-sensing cells can be segregated into two distinct cell types: those that functionally depolarize (excitable) and those that do not functionally depolarize (nonexcitable) in response to reduced oxygen. Theoretically, excitable cells have all the same signaling capabilities as the nonexcitable cells, but the nonexcitable cells cannot have all the signaling capabilities as excitable cells. A number of signaling pathways have been identified that regulate gene expression during hypoxia. These include the Ca2+-calmodulin pathway, the 3'-5' adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, the p42 and p44 mitogen-activated protein kinase [(MAPK); also known as the extracellular signal-related kinase (ERK) for ERK1 and ERK2] pathway, the stress-activated protein kinase (SAPK; also known as p38 kinase) pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. In this review, we describe hypoxia-induced signaling in the model O2-sensing rat pheochromocytoma (PC12) cell line, the current level of understanding of the major signaling events that are activated by reduced O2, and how these signaling events lead to altered gene expression in both excitable and nonexcitable oxygen-sensing cells. PMID:12189251

  12. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

    PubMed Central

    Gudjonsson, Thorarinn; Villadsen, René; Nielsen, Helga Lind; Rønnov-Jessen, Lone; Bissell, Mina J.; Petersen, Ole William

    2002-01-01

    The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by

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

    PubMed

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

    2009-12-01

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

  14. Early attempts at production of prawn cell lines.

    PubMed

    Owens, L; Smith, J

    1999-01-01

    This report describes some unsuccessful attempts to produce continuous cell lines from penaeid prawn tissues in the late 1980s. This information is presented so that others might save time by not repeating the unsuccessful measures that were attempted. The osmolarity of Penaeus monodon haemolymph was measured at 687 mOsmol/kg (N = 10). Of the media tested, the best medium for cell growth and maintenance was shown to be double strength L-15, supplemented with 10% foetal bovine serum, and 10% prawn muscle extract at 28 degrees C ( approximately 675.5 mOsmol/kg). Prawn muscle extract was made by homogenizing 30 g of prawn muscle in 50/50 ratio of distilled water/autoclaved seawater, clarified stepwise by centrifugation at 2k, 14k, 14k xg for 30 minutes each. The resultant supernatant was heat-inactivated on occasions with no improvement in growth. Preconditioned medium, cholesterol, galactose and trehalose supplements and the use of Cell-Tak did not improve growth conditions, and haemolymph extracts were detrimental to the cells. In addition it was shown that Nunc 25 cm(2) plastic culture flasks were better than Linbro and both were better than glass as substrates. The fate of 101 individual primary cell cultures, established from penaeid prawns, was as follows. Fifteen of the cultures succumbed to bacterial contamination, five became contaminated with fungi, four with thauastrochytrids, four succumbed to presumptive viral autocultures and two to ciliate contamination. Cell cultures derived from heart tissue could be maintained for a mean of 12.7 days (sd 9.7d), those derived from the epidermis 15.6 days (sd 9.0d), ovarian tissue 10 days (sd 2d), lymphoid organ 6.8 days (sd 0.4d), nerve cord and hepatopancreas 2 days. The most persistent cell cultures -- those derived from the heart explants -- contained dividing cells at 40 days, and epidermis cells were still dividing at 30 days. The longest lasting, non- proliferating, but viable, cell cultures were those of

  15. Cytotoxic Activity of New Acetoxycoumarin Derivatives in Cancer Cell Lines

    PubMed Central

    Musa, Musiliyu A.; Badisa, Veera L. D.; Latinwo, Lekan M.; Cooperwood, John; Sinclair, Andre; Abdullah, Ahkinyala

    2012-01-01

    Background Coumarin and their derivatives are important and useful compounds with diverse pharmacological properties. In the present study, we evaluated the in vitro cytotoxic activity of new acetoxycoumarin derivatives: 4-(7-methoxy-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (1), 4-(1-methyl-3-oxo-3H-benzo[f]chromen-2-yl)phenyl acetate (2), 4-(6-propionamido-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (3), 4-(7-acetoxy-2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (4), 4-(2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (5), 4-(6-bromo-2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (6), 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (7), 4-(6,8-dibromo-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (8) against A549 human lung cancer, CRL 1548 rat liver cancer and CRL 1439 normal rat liver cells. Materials and Methods The cytotoxic activity was evaluated by crystal violet dye-binding assay. The effect of compounds 5 and 7 on different phases of the cell cycle was determined using flow cytometry. Results In the A549 lung cancer cell line, the 50% lethal dose (LD50) values for compounds 1–4, 6 and 8 were found to be >100 μM while those for 5 and 7 were 89.3 and 48.1 μM, respectively after 48 h treatment. In the CRL 1548 liver cancer cell line, only compound 7 showed toxicity, with an LD50 of 45.1 μM. Compounds 5 and 7 caused different cell phase arrest in lung and liver cancer cell lines. Conclusion The results indicate that 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (7) had the highest cytotoxic activity in all of the examined cell lines. PMID:21737617

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  17. 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. PMID:26358937

  18. Assessment of Cell Line Models of Primary Human Cells by Raman Spectral Phenotyping

    PubMed Central

    Swain, Robin J.; Kemp, Sarah J.; Goldstraw, Peter; Tetley, Teresa D.; Stevens, Molly M.

    2010-01-01

    Abstract Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures. PMID:20409492

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

    PubMed Central

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

    2016-01-01

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

  20. Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers

    PubMed Central

    Qiu, Zhixin; Zou, Keke; Zhuang, Liping; Qin, Jianjie; Li, Hong; Li, Chao; Zhang, Zhengtao; Chen, Xiaotao; Cen, Jin; Meng, Zhiqiang; Zhang, Haibin; Li, Yixue; Hui, Lijian

    2016-01-01

    Hepatocellular carcinoma (HCC) cell lines are useful in vitro models for the study of primary HCCs. Because cell lines acquire additional mutations in culture, it is important to understand to what extent HCC cell lines retain the genetic landscapes of primary HCCs. Most HCC cell lines were established during the last century, precluding comparison between cell lines and primary cancers. In this study, 9 Chinese HCC cell lines with matched patient-derived cells at low passages (PDCs) were established in the defined culture condition. Whole genome analyses of 4 HCC cell lines showed that genomic mutation landscapes, including mutations, copy number alterations (CNAs) and HBV integrations, were highly stable during cell line establishment. Importantly, genetic alterations in cancer drivers and druggable genes were reserved in cell lines. HCC cell lines also retained gene expression patterns of primary HCCs during in vitro culture. Finally, sequential analysis of HCC cell lines and PDCs at different passages revealed their comparable and stable genomic and transcriptomic levels if maintained within proper passages. These results show that HCC cell lines largely retain the genomic and transcriptomic landscapes of primary HCCs, thus laying the rationale for testing HCC cell lines as preclinical models in precision medicine. PMID:27273737

  1. Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers.

    PubMed

    Qiu, Zhixin; Zou, Keke; Zhuang, Liping; Qin, Jianjie; Li, Hong; Li, Chao; Zhang, Zhengtao; Chen, Xiaotao; Cen, Jin; Meng, Zhiqiang; Zhang, Haibin; Li, Yixue; Hui, Lijian

    2016-01-01

    Hepatocellular carcinoma (HCC) cell lines are useful in vitro models for the study of primary HCCs. Because cell lines acquire additional mutations in culture, it is important to understand to what extent HCC cell lines retain the genetic landscapes of primary HCCs. Most HCC cell lines were established during the last century, precluding comparison between cell lines and primary cancers. In this study, 9 Chinese HCC cell lines with matched patient-derived cells at low passages (PDCs) were established in the defined culture condition. Whole genome analyses of 4 HCC cell lines showed that genomic mutation landscapes, including mutations, copy number alterations (CNAs) and HBV integrations, were highly stable during cell line establishment. Importantly, genetic alterations in cancer drivers and druggable genes were reserved in cell lines. HCC cell lines also retained gene expression patterns of primary HCCs during in vitro culture. Finally, sequential analysis of HCC cell lines and PDCs at different passages revealed their comparable and stable genomic and transcriptomic levels if maintained within proper passages. These results show that HCC cell lines largely retain the genomic and transcriptomic landscapes of primary HCCs, thus laying the rationale for testing HCC cell lines as preclinical models in precision medicine. PMID:27273737

  2. Dynamic DNA methylation across diverse human cell lines and tissues

    PubMed Central

    Varley, Katherine E.; Gertz, Jason; Bowling, Kevin M.; Parker, Stephanie L.; Reddy, Timothy E.; Pauli-Behn, Florencia; Cross, Marie K.; Williams, Brian A.; Stamatoyannopoulos, John A.; Crawford, Gregory E.; Absher, Devin M.; Wold, Barbara J.; Myers, Richard M.

    2013-01-01

    As studies of DNA methylation increase in scope, it has become evident that methylation has a complex relationship with gene expression, plays an important role in defining cell types, and is disrupted in many diseases. We describe large-scale single-base resolution DNA methylation profiling on a diverse collection of 82 human cell lines and tissues using reduced representation bisulfite sequencing (RRBS). Analysis integrating RNA-seq and ChIP-seq data illuminates the functional role of this dynamic mark. Loci that are hypermethylated across cancer types are enriched for sites bound by NANOG in embryonic stem cells, which supports and expands the model of a stem/progenitor cell signature in cancer. CpGs that are hypomethylated across cancer types are concentrated in megabase-scale domains that occur near the telomeres and centromeres of chromosomes, are depleted of genes, and are enriched for cancer-specific EZH2 binding and H3K27me3 (repressive chromatin). In noncancer samples, there are cell-type specific methylation signatures preserved in primary cell lines and tissues as well as methylation differences induced by cell culture. The relationship between methylation and expression is context-dependent, and we find that CpG-rich enhancers bound by EP300 in the bodies of expressed genes are unmethylated despite the dense gene-body methylation surrounding them. Non-CpG cytosine methylation occurs in human somatic tissue, is particularly prevalent in brain tissue, and is reproducible across many individuals. This study provides an atlas of DNA methylation across diverse and well-characterized samples and enables new discoveries about DNA methylation and its role in gene regulation and disease. PMID:23325432

  3. 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

  4. 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

    2016-08-01

    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. PMID:27267063

  5. 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.

  6. 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.

  7. Interactions of Streptococcus iniae with phagocytic cell line.

    PubMed

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

    2015-04-01

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

  8. 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

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

    PubMed

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

    2016-04-01

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

  10. 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.

  11. 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

  12. Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells

    PubMed Central

    Choi, Su Mi; Liu, Hua; Chaudhari, Pooja; Kim, Yonghak; Cheng, Linzhao; Feng, Jian; Sharkis, Saul

    2011-01-01

    EBV-immortalized B lymphocyte cell lines have been widely banked for studying a variety of diseases, including rare genetic disorders. These cell lines represent an important resource for disease modeling with the induced pluripotent stem cell (iPSC) technology. Here we report the generation of iPSCs from EBV-immortalized B-cell lines derived from multiple inherited disease patients via a nonviral method. The reprogramming method for the EBV cell lines involves a distinct protocol compared with that of patient fibroblasts. The B-cell line–derived iPSCs expressed pluripotency markers, retained the inherited mutation and the parental V(D)J rearrangement profile, and differentiated into all 3 germ layer cell types. There was no integration of the reprogramming-related transgenes or the EBV-associated genes in these iPSCs. The ability to reprogram the widely banked patient B-cell lines will offer an unprecedented opportunity to generate human disease models and provide novel drug therapies. PMID:21628406

  13. Photodynamic effect of hypericin in primary cultures of human umbilical endothelial cells and glioma cell lines.

    PubMed

    Stupáková, Viktória; Varinská, Lenka; Mirossay, Andrej; Sarisský, Marek; Mojzis, Ján; Dankovcík, Róbert; Urdzík, Peter; Ostró, Alexander; Mirossay, Ladislav

    2009-06-01

    Hypericin is the most powerful naturally occurring photosensitizer and as such there is renaissant interest in the potentials of this compound for anticancer photodynamic therapy (PDT). The purpose of this study was to investigate the hypericin-mediated photodynamic therapy effects on normal human umbilical endothelial cells (HUVECs) in comparison with cancer human glioma cell lines U-87 MG and U-373 MG, in in vitro conditions. The data suggest that endothelial cells as well as glioma cell lines are sensitive only to photoactivated hypericin. The inhibitory effects of photoactivated hypericin did not differ in endothelial compared with tumor cells in cytotoxicity MTT and DNA fragmentation assays. However, an important difference in sensitivity was found between the above mentioned cell types in migration and metalloproteinases inhibition assays performed as cell function tests. The findings in both function tests were supported by the high sensitivity of endothelial cells in an additional angiogenesis test of tubular formation in vitro. PMID:19173218

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

    PubMed

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

    2016-01-01

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

  15. 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...

  16. 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 ...

  17. Study of oleanolic acid on the estrodiol production and the fat production of mouse preadipocyte 3T3-L1 in vitro.

    PubMed

    Wan, Qian; Lu, Hua; Liu, Xia; Yie, Shangmian; Xiang, Junbei; Yao, Zouying

    2015-01-01

    The women during the menopause period have an increased tendency for the obesity, which represents the more fat production than during the premenopausal period. Although this is not beneficial overall, it could provide a compensatory source for the estrogen production for the menopausal women. So it would be meaningful to find an agent that could inhibit the fat production while does not disturb the total estrogen production by fat tissues. In the present study, the effect of oleanolic acid (OA) on the fat production and the total estrogen production of the differentiating mouse preadipocyte 3T3-L1 as well as the mechanisms behind those effects were preliminarily investigated. The cell line 3T3-L1 was chosen as the model cell because it is usually used for the research about the obesity. During the induced differentiation of 3T3-L1 cells, cells were intervened continuously with OA. The fat production was determined with the oil red staining assay and the total estrogen production was measured with the ELISA assay. Finally, the expression patterns for important genes of the fat production and the estrogen production were studied, respectively with the real-time fluorescence quantitative PCR (qPCR). The results showed that for the differentiating 3T3-L1 cells, OA could significantly inhibit the fat production and did not disturb the total estrogen production significantly. In the mechanism studies, OA was found to significantly down-regulate ACC, the key gene for fat synthesis, which could explain the inhibitory effect of OA on the fat production; OA was also found to significantly up-regulate CYP11A1, CYP17, CYP19, the key genes for the estrogen synthesis and significantly down-regulate CYP1A1, the key gene for the estrogen decomposition, which preliminarily explained the lack of the effect of OA on the total estrogen production. In conclusion, OA was found able to inhibit the fat production while maintaining the total estrogen level and the mechanisms for the above

  18. Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis.

    PubMed

    Zhao, Yunxue; Li, Jing; Wang, Jingjian; Xing, Yanli; Geng, Meiyu

    2007-06-01

    Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo - the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, beta-1,6-GlcNAc-branching, alpha-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(beta-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis. PMID:17650582

  19. 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.

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

    PubMed

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

    2016-01-22

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

  1. Chloride channels in the small intestinal cell line IEC-18.

    PubMed

    Basavappa, Srisaila; Vulapalli, Sreesatya Raju; Zhang, Hui; Yule, David; Coon, Steven; Sundaram, Uma

    2005-01-01

    Small intestinal crypt cells play a critical role in modulating Cl- secretion during digestion. The types of Cl- channels mediating Cl- secretion in the small intestine was investigated using the intestinal epithelial cell line, IEC-18, which was derived from rat small intestine crypt cells. In initial radioisotope efflux studies, exposure to forskolin, ionomycin or a decrease in extracellular osmolarity significantly increased 36Cl efflux as compared to control cells. Whole cell patch clamp techniques were subsequently used to examine in more detail the swelling-, Ca2+-, and cAMP-activated Cl- conductance. Decreasing the extracellular osmolarity from 290 to 200 mOsm activated a large outwardly rectifying Cl- current that was voltage-independent and had an anion selectivity of I- > Cl-. Increasing cytosolic Ca2+ by ionomycin activated whole cell Cl- currents, which were also outwardly rectifying but were voltage-dependent. The increase in intracellular Ca2+ levels with ionomycin was confirmed with fura-2 loaded IEC-18 cells. A third type of whole cell Cl- current was observed after increases in intracellular cAMP induced by forskolin. These cAMP-activated Cl- currents have properties consistent with cystic fibrosis transmembrane regulator (CFTR) Cl- channels, as the currents were blocked by glibenclamide or NPPB but insensitive to DIDS. In addition, the current-voltage relationship was linear and had an anion selectivity of Cl- > I-. Confocal immunofluorescence studies and Western blots with two different anti-CFTR antibodies confirmed the expression of CFTR. These results suggest that small intestinal crypt cells express multiple types of Cl- channels, which may all contribute to net Cl- secretion. PMID:15389550

  2. Cycle reset in a melanoma cell line caused by cooling.

    PubMed

    Dewey, D L

    1987-11-01

    When cells in culture are released from G0 into cycle by diluting into fresh medium there is a delay of many hours before they re-enter the cycle and start DNA synthesis. A mouse melanoma cell line designated HP2 has been used to investigate the effects of non-standard temperatures between the time of plating and DNA synthesis. When the cells were incubated in a 5% CO2 box at 8 degrees C for periods during the G0-G1 transition there was an extra delay before the start of S, approximately equal to the time that the cells were held at 8 degrees C and independent of the time when the cold pulse was administered. When the cells were cooled to 25 degrees C the delay was longer than the time for which the cells had been kept at 25 degrees C, and this extra delay was also dependent on the point in G0-G1 when the cells were cooled, as though the cells could be reset to an earlier time by this treatment. It is suggested that a labile substance required for progression is destroyed faster than it is made at 25 degrees C but at 8 degrees C the rate of destruction is very low. Another phenomenon noted during these cooling experiments was that the peak height of the S phase profile, as measured by frequent pulse-thymidine incorporation experiments, was substantially higher for cells which had been cooled at a later stage in the G0-G1 transition, even though the overall times at 37 degrees C and at the colder temperature were identical. By varying the temperature of the cold pulse it was possible to separate the change in the peak height and the delay as separate entities. PMID:3502929

  3. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

    PubMed Central

    Taylor, AW; Dixit, S; Yu, J

    2015-01-01

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

  4. Responses in mantle cell lymphoma cells to SNS-032 depend on the biological context of each cell line.

    PubMed

    Chen, Rong; Chubb, Sherri; Cheng, Tiewei; Hawtin, Rachael E; Gandhi, Varsha; Plunkett, William

    2010-08-15

    SNS-032 is a potent inhibitor of cyclin-dependent kinases (Cdk) 2, 7, and 9 that regulate the cell cycle and transcription. Our studies in indolent primary chronic lymphocytic leukemia cells showed that SNS-032 inhibited transcription, diminished the antiapoptotic protein Mcl-1, and induced apoptosis. The present study focuses on evaluating this compound in four proliferating mantle cell lymphoma lines (Jeko-1, Granta 519, Mino, and SP-53). Consistent with its action against Cdk9 and Cdk7, SNS-032 inhibited the phosphorylation of RNA pol II in all four lines and blocked RNA synthesis. The transcripts and protein levels of short-lived proteins decreased, including cyclin D1 and Mcl-1. Cell growth was inhibited in a concentration-dependent manner in all lines. Apoptosis was induced in JeKo-1, Mino, and SP-53 cells without disrupting cell cycle distribution. However, apoptosis was limited in Granta cells; rather, there was a significant reduction of clonogenic survival. Small interfering RNA was used to specifically knock down Mcl-1 and cyclin D1 in JeKo-1 and Granta cells. Knocking down Mcl-1 induced significant apoptosis in Jeko-1 cells but not Granta cells. Reducing cyclin D1, rather than Mcl-1, was associated with loss of clonogenic survival in Granta cells. Thus, these results indicated that mantle cell lymphoma cell lines have distinct mechanisms sustaining their survival, and the mechanism of action of SNS-032 is dependent on the biological context of an individual line. PMID:20663900

  5. cis9, trans11-Conjugated Linoleic Acid Differentiates Mouse 3T3-L1 Preadipocytes into Mature Small Adipocytes through Induction of Peroxisome Proliferator-activated Receptor γ.

    PubMed

    Sakuma, Satoru; Nishioka, Yuki; Imanishi, Ryohta; Nishikawa, Kenji; Sakamoto, Hirotada; Fujisawa, Junji; Wada, Koichiro; Kamisaki, Yoshinori; Fujimoto, Yohko

    2010-09-01

    Dietary conjugated linoleic acid (CLA) has been reported to exhibit a number of therapeutic effects in animal models and patients, such as anti-hypertensive, anti-hyperlipidemic, anti-arteriosclerotic, anti-carcinogenic, and anti-diabetic effects. However, the underlying mechanism is not well-characterized. In the present study, the effects of cis(c)9, trans(t)11-CLA on the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes were examined. Treatment with c9, t11-CLA in the presence of insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (differentiation cocktail) significantly stimulated the accumulation of triacylglycerol. The microscopic observation of cells stained by Oil Red O demonstrated that c9, t11-CLA increases the amount and proportion of small mature adipocytes secreting adiponectin, a benign adipocytokine, when compared to the differentiation cocktail alone. Furthermore, c9, t11-CLA increased bioactive peroxisome proliferator-activated receptor γ (PPARγ) levels in a nuclear extract of 3T3-L1 cells, suggesting the enhancing effect of this fatty acid on the nuclear transmission of PPARγ, a master regulator of adipocyte differentiation, in 3T3-L1 cells. These results suggest that the therapeutic effects of c9, t11-CLA on lifestyle-related diseases are partially due to the enhanced formation of small adipocytes from preadipocytes via PPARγ stimulation. PMID:20838573

  6. 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

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

    PubMed

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

    2010-02-24

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

  8. Shotgun Lipidomic Profiling of the NCI60 Cell Line Panel Using Rapid Evaporative Ionization Mass Spectrometry.

    PubMed

    Strittmatter, Nicole; Lovrics, Anna; Sessler, Judit; McKenzie, James S; Bodai, Zsolt; Doria, M Luisa; Kucsma, Nora; Szakacs, Gergely; Takats, Zoltan

    2016-08-01

    Rapid evaporative ionization mass spectrometry (REIMS) was used for the rapid mass spectrometric profiling of cancer cell lines. Spectral reproducibility was assessed for three different cell lines, and the extent of interclass differences and intraclass variance was found to allow the identification of these cell lines based on the REIMS data. Subsequently, the NCI60 cell line panel was subjected to REIMS analysis, and the resulting data set was investigated for its distinction of individual cell lines and different tissue types of origin. Information content of REIMS spectral profiles of cell lines were found to be similar to those obtained from mammalian tissues although pronounced differences in relative lipid intensity were observed. Ultimately, REIMS was shown to detect changes in lipid content of cell lines due to mycoplasma infection. The data show that REIMS is an attractive means to study cell lines involving minimal sample preparation and analysis times in the range of seconds. PMID:27377867

  9. Investigation of Freeze-Linings in Aluminum Production Cells

    NASA Astrophysics Data System (ADS)

    Fallah-Mehrjardi, Ata; Hayes, Peter C.; Jak, Evgueni

    2014-08-01

    The molten cryolite bath creates chemically a very aggressive environment in the Hall-Héroult cell, and thus, the formation of a protective solid layer (freeze-lining) on the cell wall is essential for the operation of the present cell designs. To provide further information on the formation of the freeze-lining deposit in this system, laboratory-based studies were undertaken using an air-cooled probe technique The effects of process conditions, i.e., time, bath agitation, and superheat on the microstructures, morphologies of the phases, and the phase assemblages adjacent to the deposit/bath interface were investigated. A detailed microstructural analysis of the steady-state deposits shows that a dense sealing primary-phase layer of cryolite solid solution was formed at the interface of the bath deposit for the process conditions examined. The formation of sealing primary-phase layer at the bath/deposit interface explicitly indicates that the deposit/liquid bath interface temperature is equal to that of the liquidus of the bulk bath. The experimentally investigated liquidus temperature and subliquidus equilibria differ significantly from those previously reported.

  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. PMID:25673530

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

    PubMed Central

    Mehta, Rajvi H.

    2014-01-01

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

  12. Development and Characterization of Six New Human Papillary Thyroid Carcinoma Cell Lines

    PubMed Central

    Henderson, Ying C.; Ahn, Soon-Hyun; Ryu, Junsun; Chen, Yunyun; Williams, Michelle D.; El-Naggar, Adel K.; Gagea, Mihai; Schweppe, Rebecca E.; Haugen, Bryan R.; Lai, Stephen Y.

    2015-01-01

    Context: Cell lines are a widely used tool in cancer research. However, despite the relatively high incidence of papillary thyroid carcinoma (PTC), there are only four PTC cell lines available for international research audience. Objective: The objective of this study was to establish and characterize new PTC cell lines that represent primary tumor biology. Surgical specimens were obtained to generate PTC cell lines. Short tandem repeat profiling was used to confirm the uniqueness of the cell lines against databases of known cell lines and mutations were assessed using Sequenom. The expression of thyroid-specific genes was examined using real-time PCR. Tumorigenicity was determined using an orthotopic thyroid xenograft tumor mouse model. Results: Six PTC cell lines (five conventional PTCs and one follicular variant of PTC) were generated and found to be unique when compared by short tandem repeat profiling against databases of all existing cell lines. The five conventional PTC cell lines carry the BRAF V600E mutation and the follicular variant of PTC cell line had an NRAS mutation. Five of the six cell lines had a mutation in the promoter of the human telomerase reverse transcriptase gene. None of the cell lines have RET/PTC rearrangements. Three cell lines were tumorigenic in the orthotopic thyroid xenograft tumor mouse model. Conclusions: These five characterized conventional PTC cell lines and the unique follicular variant of PTC cell line should be valuable reagents for thyroid cancer research. The three tumorigenic cell lines can be used for in vivo testing of targeted therapeutic and novel agents. PMID:25427145

  13. Role of glutathione in the intrinsic radioresistance of cell lines from a mouse squamous cell carcinoma

    SciTech Connect

    Miura, M.; Sasaki, T. )

    1991-05-01

    The role of glutathione (GSH) in determining the intrinsic cellular radioresistance under aerobic conditions was studied with the parent cell line MSCC and its radioresistant subclone R1 isolated from a mouse squamous cell carcinoma. The mean inactivation doses (D) of the survival curves were 2.1 and 4.0 Gy for exponentially growing MSCC and R1 cells, respectively. The corresponding GSH content was 22.6 and 13.4 nmol/10(6) cells. There was no significant difference in either the distribution of GSH between nucleus and cytoplasm or the turnover rate of GSH between the two cell lines. Thus it appeared that the radioresistance of R1 cells resulted from mechanisms unrelated to GSH. However, R1 cells became progressively more radiosensitive with a decrease of the GSH content with buthionine sulfoximine (BSO) treatment until about 20 h, and the radiosensitivity showed little change thereafter. The MSCC cells showed little change in the radiosensitivity with the same treatment. In fact, dose-survival curves showed that the enhancement ratio of D with the 24-h BSO treatment was 1.1 for MSCC and 1.4 for R1 cells, although the GSH content was reduced to 1 to 2% of the untreated level for both cell lines. There was no significant difference in the activities of GSH S-transferase and GSH reductase between MSCC and R1 cells before and after BSO treatment, or between BSO-treated and untreated cells of the same cell lines. Although the exact mechanisms of GSH-related radioresistance of R1 cells are unclear, these results suggest that there may exist GSH-related mechanisms in addition to radical scavenging which determine the intrinsic cellular radioresistance under aerobic conditions.

  14. 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.

  15. Transcriptional profiling in an MPNST-derived cell line and normal human Schwann cells

    PubMed Central

    LEE, PHILIP R.; COHEN, JONATHAN E.; TENDI, ELISABETTA A.; FARRER, ROBERT; DE VRIES, GEORGE H.; BECKER, KEVIN G.; FIELDS, R. DOUGLAS

    2005-01-01

    cDNA microarrays were utilized to identify abnormally expressed genes in a malignant peripheral nerve sheath tumor (MPNST)-derived cell line, T265, by comparing the mRNA abundance profiles with that of normal human Schwann cells (nhSCs). The findings characterize the molecular phenotype of this important cell-line model of MPNSTs, and elucidate the contribution of Schwann cells in MPNSTs. In total, 4608 cDNA sequences were screened and hybridizations replicated on custom cDNA microarrays. In order to verify the microarray data, a large selection of differentially expressed mRNA transcripts were subjected to semi-quantitative reverse transcription PCR (LightCycler). Western blotting was performed to investigate a selection of genes and signal transduction pathways, as a further validation of the microarray data. The data generated from multiple microarray screens, semi-quantitative RT–PCR and Western blotting are in broad agreement. This study represents a comprehensive gene-expression analysis of an MPNST-derived cell line and the first comprehensive global mRNA profile of nhSCs in culture. This study has identified ~900 genes that are expressed abnormally in the T265 cell line and detected many genes not previously reported to be expressed in nhSCs. The results provide crucial information on the T265 cells that is essential for investigation using this cell line in experimental studies in neurofibromatosis type I (NF1), and important information on normal human Schwann cells that is applicable to a wide range of studies on Schwann cells in cell culture. PMID:16429615

  16. 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.

  17. 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. PMID:24026291

  18. Light can rescue auxin-dependent synchrony of cell division in a tobacco cell line

    PubMed Central

    Qiao, Fei; Petrášek, Jan; Nick, Peter

    2010-01-01

    Pattern formation in plants has to cope with ambient variability and therefore must integrate environmental cues such as light. Synchrony of cell divisions was previously observed in cell files of tobacco suspension cultures, which represents a simple case of pattern formation. To develop cellular approaches for light-dependent patterning, light-responsive tobacco cell lines were screened from the cell line Nicotiana tabacum L. cv. Virginia Bright Italia 0 (VBI-0). The light responsive and auxin-autonomous cell line VBI-3 was isolated. As in the progenitor line VBI-0, cell divisions are synchronized in VBI-3 during exponential growth phase. This synchrony can be inhibited by 1-N-naphthylphthalamic acid, an auxin transport inhibitor, and this process was accompanied by the disassembly of actin filaments. However, the synchrony could be rescued when the cells were cultured under white light or with exogenous indolyl-3-acetic acid. The rescue was most efficient for continuous far-red light followed by continuous blue light, whereas continuous red light was least effective. These findings are discussed in the context of phytochrome-induced auxin biosynthesis and auxin-dependent synchrony of cell division. PMID:19884227

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

    PubMed

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

    2013-06-01

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

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

    PubMed

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

    2014-02-28

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

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

    NASA Astrophysics Data System (ADS)

    Dulbecco, Renato; Bologna, Mauro; Unger, Michael

    1980-03-01

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

  2. Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

    PubMed Central

    2011-01-01

    Background There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. Results The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. Conclusion This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target. PMID:21982118

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

    PubMed

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

    2012-01-15

    Bendamustine is clinically useful in mantle-cell lymphoma (MCL). Its favorable toxicity profile in-vivo favors its combination with other cytotoxic drugs. Cytarabine is a key drug in the treatment of younger patients with MCL. The current study investigated the in-vitro cytotoxic effect of bendamustine and cytarabine, alone or combined, on two MCL cell lines representing the classic and blastoid variant of the lymphoma subtype (JEKO-1 and GRANTA-519). Cell lines were exposed to each drug alone, or simultaneously and consecutively to both drugs, for different time schedules. Apoptosis was measured by flow cytometry. Mitochondrial damage, cell proliferation/metabolic activity, and cell cycle analysis were also assessed. The synergistic, additive, or antagonistic effects of the drugs were calculated with the combination index (CI) method. Bendamustine and cytarabine alone exhibited relevant cytotoxic activity on both cell lines. Both drugs induced cell cycle arrest in S phase. Drug combinations were associated with significantly higher cytotoxic effects than each drug alone. Among the combination schedules, the consecutive incubation of bendamustine followed by cytarabine was associated with the lower CI, being 10-100-fold lower than with simultaneous incubations. The cytotoxic effect of the consecutive combination was prominent on both cell lines, indicating a very strong and highly significant synergy in inducing apoptosis. Similar results were obtained measuring mitochondrial damage or the decline of the metabolic activity in all cell lines. The strong synergistic effect of bendamustine and cytarabine on MCL cells provides a rationale for developing schedules combining these agents in the treatment of MCL. PMID:22036761

  4. Getting personal: Endogenous adenosine receptor signaling in lymphoblastoid cell lines.

    PubMed

    Hillger, J M; Diehl, C; van Spronsen, E; Boomsma, D I; Slagboom, P E; Heitman, L H; IJzerman, A P

    2016-09-01

    Genetic differences between individuals that affect drug action form a challenge in drug therapy. Many drugs target G protein-coupled receptors (GPCRs), and a number of receptor variants have been noted to impact drug efficacy. This, however, has never been addressed in a systematic way, and, hence, we studied real-life genetic variation of receptor function in personalized cell lines. As a showcase we studied adenosine A2A receptor (A2AR) signaling in lymphoblastoid cell lines (LCLs) derived from a family of four from the Netherlands Twin Register (NTR), using a non-invasive label-free cellular assay. The potency of a partial agonist differed significantly for one individual. Genotype comparison revealed differences in two intron SNPs including rs2236624, which has been associated with caffeine-induced sleep disorders. While further validation is needed to confirm genotype-specific effects, this set-up clearly demonstrated that LCLs are a suitable model system to study genetic influences on A2AR response in particular and GPCR responses in general. PMID:27297283

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

    PubMed Central

    Agris, P F

    1975-01-01

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

  6. Origin of the U87MG glioma cell line: Good news and bad news.

    PubMed

    Allen, Marie; Bjerke, Mia; Edlund, Hanna; Nelander, Sven; Westermark, Bengt

    2016-08-31

    Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin. PMID:27582061

  7. 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

  8. '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.

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

    PubMed Central

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

    2010-01-01

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

  10. Resistance to telomerase inhibition by human squamous cell carcinoma cell lines.

    PubMed

    Bojovic, Bojana; Crowe, David L

    2011-04-01

    Telomeres are nucleoprotein structures at the ends of chromosomes that are composed of a repetitive G rich sequence and telomeric binding proteins. Telomeres prevent the degradation of chromosomal ends and protect against inappropriate recombination. Telomere attrition involves a tumor suppressor pathway that limits the replication of premalignant cells. The loss of telomeric DNA with each round of replication leads to growth arrest accompanied by senescence or apoptosis. Many tumor cells activate the telomerase gene to bypass senescence. Telomerase is a multisubunit ribonucleoprotein that uses an RNA template to catalyze the addition of telomeric DNA to chromosomal ends. Overexpression of the TERT subunit leads to telomere lengthening and extension of the replicative lifespan. Dominant-negative telomerase has been shown to inhibit telomerase activity in many tumor cell lines, and this is associated with telomere shortening and apoptosis. Additionally, pharmacological telomerase inhibitors have been developed which lead to progressive telomere shortening and programmed cell death. In this study, we report a series of human squamous cell carcinoma cell lines that have high telomerase activity and short telomeres. Dominant-negative telomerase expression and pharmacological telomerase inhibition failed to completely inhibit enzymatic activity which was accompanied by the lack of telomere shortening. These cells continued to proliferate and demonstrated fewer responsive genes when treated with a pharmacological telomerase inhibitor. We concluded that some human squamous cell carcinoma cell lines are resistant to telomerase inhibition. PMID:21305252

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

    PubMed

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

    2015-08-01

    Resistance to chemotherapy leading to poor outcome and survival remains a challenge for developing strategies for therapeutic interventions in all types of cancer, including head and neck cancer. In vitro chemoresistant cell line models are an indispensable resource towards delineating the mechanisms involved in drug resistance/response and for the development of novel drugs. Current treatment for head and neck cancer includes chemotherapy with cisplatin, docetaxel and 5-fluorouracil (5-FU) and the response rates to these drugs in patients is 60-80%. The present study aimed to generate head and neck cancer triple drug-resistant cell lines in an effort towards elucidating the mechanisms underlying chemoresistance and providing a resourceful tool for drug design. Using two head and neck squamous cell carcinoma cell lines, Hep-2 (larynx) and CAL-27 (oral cavity), the present study sequentially exposed these cells to increasing concentrations of the combination of docetaxel, cisplatin and 5-FU (TPF) to generate triple drug-resistant cells, termed Hep-2 TPF resistant (TPFR) and CAL-27 TPFR. The effect of the drug treatments on the cell viability, apoptosis, cell cycle and the expression of genes associated with multidrug resistance were analyzed in the parental cells and drug-resistant counterparts. PMID:25962396

  12. 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

  13. Secretion of mucus proteinase inhibitor and elafin by Clara cell and type II pneumocyte cell lines.

    PubMed

    Sallenave, J M; Silva, A; Marsden, M E; Ryle, A P

    1993-02-01

    The regulation of proteinases secreted by neutrophils is very important for the prevention of tissue injury. We recently described the isolation of elafin from bronchial secretions, a new elastase-specific inhibitor that is also found in the skin of patients with psoriasis. In this study, we investigated the secretion of elafin and mucus proteinase inhibitor (MPI), another inhibitor showing sequence similarity with elafin, in two lung carcinoma cell lines, NCI-H322 and A549, which have features of Clara cells and type II alveolar cells, respectively. The results presented show that the two inhibitors are produced when the cells are cultured either in serum-free or in serum-containing media. MPI was detected immunologically as a unique molecule of M(r) 14 kD, in accordance with previous studies. Conversely, one or two elafin-immunoreactive species were detected depending on the cell line: a 12- to 14-kD species was observed in the A549 cell line, regardless of the culture conditions, whereas in the NCI-H322 cell line we detected a 6-kD species in serum-containing (10% fetal calf serum) conditions and a 12- to 14-kD species in serum-free conditions. The 12- to 14-kD molecule probably represents an active precursor of elafin. Whether the cleavage of the 12- to 14-kD precursor giving rise to the elafin molecule is of any physiologic significance is not known. In showing for the first time that MPI and elafin (and its precursor) are secreted by the A549 cell line, this report implicates the type II alveolar cell in the defense of the peripheral lung against the neutrophil elastase secreted during inflammation. PMID:8427705

  14. 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

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

    PubMed Central

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

    2016-01-01

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

  16. Mechanisms of lymphocyte adhesion to endothelial cells: studies using a LFA-1-deficient cell line.

    PubMed Central

    Haskard, D O; Strobel, S; Thornhill, M; Pitzalis, C; Levinsky, R J

    1989-01-01

    In order to investigate the role of lymphocyte function-associated antigen 1 (LFA-1) in lymphocyte adhesion to endothelial cells (EC), we have studied the adhesion of a LFA-1-deficient lymphoblastoid cell line, ICH-KM, which has < 10% of the cell surface LFA-1 expressed on a normal lymphoblastoid cell line, ICH-BJ. The adhesion of ICH-KM cells to unstimulated EC was 49.9 +/- 8.6% (mean +/- SD) that of ICH-BJ cells. Moreover, phorbol ester-stimulated ICH-KM cells showed a considerably weaker increase in adhesion to unstimulated EC compared with ICH-BJ cells (mean +/- SD increase in percentage adhesion, 3.8 +/- 2.3 compared with 18.5 +/- 8.0; P<0.025). In contrast, there was no significant difference between the enhanced adhesion of ICH-KM cells and ICH-BJ cells to interleukin-1 (IL-1)-stimulated EC. Thus ICH-KM cells showed a 22.7 +/- 11.0 (mean +/- SD) increase in percentage adhesion to IL-1-stimulated EC compared with the 24.8 +/- 8.5 increase in percentage adhesion of ICH-BJ cells. Anti-LFA-1 monoclonal antibodies had no effect on the enhanced adhesion of ICH-KM and ICH-BJ cells to IL-1-stimulated EC but abolished the differences in adhesion between the two cell lines. The study therefore indicates that although a major part of unstimulated and phorbol ester-stimulated lymphocyte-EC adhesion is dependent upon LFA-1, the enhanced adhesion due to stimulation of EC with IL-1 is not dependent upon this molecule. The data therefore supports the existence of cytokine-inducible LFA-1-independent adhesion molecules for lymphocytes on EC. PMID:15493272

  17. A rapid and sensitive fluorometric microassay for determining cell mediated cytotoxicity to adherent growing cell lines.

    PubMed

    Krüger-Krasagakes, S; Garbe, C; Kossman, P; Orfanos, C E

    1992-11-25

    In order to measure cell mediated cytotoxicity to adherent growing cell lines in vitro more rapidly and conveniently, a fluorometric microassay was developed and results were compared with those obtained by the 51Cr release assay. The fluorometric method is based on the hydrolysis of the fluorochrome 4-methylumbelliferyl heptanoate (MUH) by intracellular esterases of viable cells. Melanoma cell monolayers were incubated with lymphokine activated killer (LAK) cells for 4 h at various effector: target (E:T) cell ratios (E:T = 16, 8, 4, 2:1). Thereafter surviving adherent melanoma cells were stained with MUH for 30 min and fluorescence was measured directly in a 96 well plate reader. For the calculation of LAK cell cytotoxicity fluorescence values were corrected for the number of nonspecifically detached tumor cells during the washes and the number of nonspecifically adherent LAK cells. Using identical target and effector cell preparations both assays showed a nearly proportional increase of percentage cytotoxicity with rising numbers of lymphocytes. Compared with the 51Cr release assay, however, higher cytotoxicity values were obtained with the fluorometric MUH microassay: 57% with MUH versus 26% with 51Cr and 39% versus 14% for cell lines StML-11 and SKMel-28, respectively (E:T ratio = 16:1). The higher cytotoxicity rates obtained with the fluorometric MUH microassay were not due to the additional 30 min staining with MUH or due to nonspecific hydrolysis of MUH by extracellular esterases released from damaged cells, as could be shown by a series of experiments. In conclusion, a simple and rapid fluorometric microassay has been developed showing reliable reproducibility and a higher sensitivity compared with the 51Cr release assay for the determination of cellular cytotoxicity to adherent growing cell lines, avoiding hazardous radioactive labels. PMID:1431156

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2013-07-01

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

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

    PubMed Central

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2007-10-01

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

  2. Gene expression profiling analysis of osteosarcoma cell lines

    PubMed Central

    SUN, LU; LI, JIE; YAN, BING

    2015-01-01

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

  3. Gene expression profiling analysis of osteosarcoma cell lines.

    PubMed

    Sun, Lu; Li, Jie; Yan, Bing

    2015-09-01

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

  4. Continuous cell lines from the common white, Pieris rapae crucivora Boisduval.

    PubMed

    Mitsuhashi, Jun; Hayasaka, Shouji; Imanishi, Shigeo

    2003-01-01

    Three continuous cell lines, NIAS-PRC-819A, NIAS-PRC-819B, and NIAS-PRC-819C, were established from the pupal ovaries of the common white, Pieris rapae crucivora Boisduval (Insecta, Lepidoptera, Pieridae). The primary culture was initiated as explant cultures with ovariole fragments in MGM-464 medium supplemented with 20% fetal bovine serum at 25 degrees C. About 6 mo after the culture was set up, the first subculture was prepared. Thereafter, cells were subcultured with decreasing passage intervals, resulting in a cell population that multiplied continuously. The karyotypes of these cell lines were similar to each other, and the majority of the cells showed about 100 microchromosomes. The population-doubling times of these cell lines were 3 to 7 d. The cell lines were susceptible to a microsporidia, Nosema bombycis. Immunodiffusion experiments proved that these cell lines derived from the common white and not from other cell lines by contamination. PMID:14505438

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

    PubMed Central

    Sandberg, Rickard; Ernberg, Ingemar

    2005-01-01

    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. PMID:15671165

  6. 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...

  7. 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.

  8. 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

  9. 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...

  10. 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

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

    DOE PAGESBeta

    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 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

  12. Comparison of antibody molecules produced from two cell lines with contrasting productivities and aggregate contents.

    PubMed

    Ishii, Yoichi; Imamoto, Yasufumi; Yamamoto, Rie; Tsukahara, Masayoshi; Wakamatsu, Kaori

    2015-01-01

    Cell culture processes that produce therapeutic antibodies with high productivity (titer) and low aggregate content reduce the risk of adverse effects and expense to patients. To elucidate the mechanism of aggregate formation, we compared trastuzumab samples produced from two contrasting cell lines: cell line A, which exhibits high titer and low aggregate content, and cell line B, which exhibits low titer and high aggregate content. Cell line B produced significantly fewer (approximately 1/3) antibodies compared with cell line A and contained higher (approximately 3-fold) percentages of aggregates. The aggregates of antibodies found in the protein A-purified samples of cell line B were associated mostly with noncovalent interactions. Cell line B exhibited a low content of monomers/dimers of light chains in the medium and within cells. Because light chains are essential for the correct folding of heavy chains and secretion of mature antibodies, the characteristics of cell line B may be attributed to low levels of light chain production. In addition, protein A-purified antibodies from cell line B (but not those from cell line A) contained fragments that are expected to expose the hydrophobic CH3 domain, which may serve as nuclei for aggregation. PMID:25501618

  13. 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

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

    PubMed

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

    2010-06-01

    Two adherent cell lines, BAEC and HeLa, and non-adherent Jurkat, were treated with snake venom metalloproteinase BaP1 to determine whether cytotoxicity, previously reported for this toxin, could be mediated by the process of anoikis. It was observed that there was no correlation between the ability of this toxin to induce loss of adherence, and the cytotoxic effect, since concentrations that do not induce loss of adherence (3-6 microg/mL), were able to trigger 50% of cytotoxicity in BAEC. In the case of HeLa, where toxicity was very low (less than 20% at maximun concentrations and times of exposure), significant detachment and no toxicity was observed at concentrations of 1.5 microg/mL, showing also no correlation between both events. We also observed differences between BAEC toxicity measured by XTT reduction and DNA fragmentation determined by flow cytometry (as an indicator of apoptosis), since concentrations that induce 100% of cytotoxicity barely showed any DNA fragmentation (12% at 24h), suggesting that if apoptosis was involved, DNA damage is still not present, although chromatin condensation, another indicator of apoptosis, is observed in 40% of the cells. Inhibition of BAEC cytotoxicity by caspase inhibitors indicate that apoptosis is playing a role in this process, but other mechanisms of cell death could be participating also. Another way to determine whether the mechanism of cell death was related to anoikis was using a non-adherent cell line, which should show substrate independence. We determined by TUNEL that at 50 microg/ml BaP1 triggered 50% of apoptosis at 96 h, an effect that was seen earlier, suggesting also that if this toxin was inducing apoptosis in a non-adherent cell line, the mechanism could not be related to loss of attachment. Cell cycle arrest in S phase was also observed in Jurkat cells, an effect that could be leading to apoptosis. In conclusion, since there was no correlation between cell detachment and cytotoxicity (and apoptosis

  15. Detection of antigens specific for B-lymphoid cultured cell lines with human alloantisera.

    PubMed

    Mann, D L; Abelson, L; Harris, S; Amos, D B

    1975-07-01

    Human sera were tested for cytotoxicity to pairs of long-term tissue-cultured cell lines. Each pair had been derived from the same individual and one of the pairs possessed the characteristics of either "T" or "B" cells. The alloantisera used were HL-A-typing reagents or sera obtained from Amish multiparas. Selected cytotoxicity was found against the B-cell lines by direct testing. Cytotoxicity was abolished by absorption with B-cell line but not by absorption with the T-cell lines. The results suggest that a group of allotypic antigens may be expressed exculsively on human B cells. PMID:1080182

  16. 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.

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

    PubMed

    Torabi, Sheida; Mo, Huanbiao

    2016-03-01

    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

  18. Effects of feeder cells (human cancer cell lines) on the development of mouse embryos by co-culture.

    PubMed

    Ishiwata, I; Tokieda, Y; Ishiwata, C; Okane, N; Iguchi, M; Sato, K; Ishikawa, H

    1997-12-01

    In order to establish the best co-culture system on embryogenesis such as egg fertilization, egg cleavage, blastocyst formation, hatching and implantation etc., several kinds of cell lines as a feeder cell and mouse fertilized eggs (zygotes) were co-cultured in the organ culture dish, and embryotrophic effects of feeder cells were investigated. Best feeder cell on the embryogenesis was SKG-II cell line derived from squamous cell carcinoma of human uterine cervix which was chosen from 10 of the human tumor cell lines. Furthermore, in order to isolate and determinate embryotrophic factors produced by feeder cells, we established a SKG-II SF subline which was grown in serum free medium derived from SKG-II cell line. The SKG-II SF cell line secreted an epidermal growth factor (EGF) into the medium. Also, cleavaged egg produced and secreted interleukin (IL)-1 alpha into the medium. PMID:9573483

  19. Mantle cell lymphoma cell lines show no evident immunoglobulin heavy chain stereotypy but frequent light chain stereotypy.

    PubMed

    Pighi, Chiara; Barbi, Stefano; Bertolaso, Anna; Zamò, Alberto

    2013-08-01

    Mantle cell lymphoma shows a peculiar immunogenetic profile, but the functional consequences of this fact are unknown. We have determined the precise sequences of rearranged heavy and light chain genes in several mantle cell lymphoma cell lines and investigated the presence of heavy and light chain stereotypy. These cell lines use IGHV and IGLV genes that are known to be preferentially rearranged in mantle cell lymphoma, but we found no evidence of heavy chain stereotypy. In contrast, one cell line (Mino) showed a nearly identical light chain complementarity-determining region 3 when compared to the only published light chain cluster. Two cell line couples (Jeko-1/UPN-2 and JVM-2/JVM-13) showed a highly similar light chain that satisfied the criteria for stereotypy. Our data show that mantle cell lymphoma cell lines resemble the IGHV and IGLV usage of mantle cell lymphoma, and foster the hypothesis that light chain stereotypy might be under-recognized. PMID:23245212

  20. Mutations and polymorphisms in mitochondrial DNA in head and neck cancer cell lines

    PubMed Central

    Allegra, E; Garozzo, A; Lombardo, N; De Clemente, M; Carey, TE

    2006-01-01

    Summary Changes in mitochondrial DNA have been reported in cancer cells. Since little information exists regarding mt DNA mutations in head and neck, the present study focused on ten head and neck cancer cell lines in the attempt to detect alterations in the ND4 gene sequence. DNA was extracted from 10 head and neck squamous cell carcinoma lines from 9 patients. MtDNA sequences were compared in normal and tumour cell line DNA. In ten head and neck squamous cell carcinoma cell lines, 8 somatic mutations and 5 polymorphisms of the mitochondrial gene for ND4 were found. All 5 polymorphisms were silent. Of the 8 somatic mutations, 3 altered the amino acid sequence suggesting a possible effect on enzyme function. The mitochondrial mutations and polymorphisms found demonstrated that these can serve as clonal markers for individual cell lines and demonstrate that the mitochondrial genome remains stable in the cell lines during in vitro culture. PMID:18236634

  1. Transcription factor binding predicts histone modifications in human cell lines

    PubMed Central

    Benveniste, Dan; Sonntag, Hans-Joachim; Sanguinetti, Guido; Sproul, Duncan

    2014-01-01

    Gene expression in higher organisms is thought to be regulated by a complex network of transcription factor binding and chromatin modifications, yet the relative importance of these two factors remains a matter of debate. Here, we show that a computational approach allows surprisingly accurate prediction of histone modifications solely from knowledge of transcription factor binding both at promoters and at potential distal regulatory elements. This accuracy significantly and substantially exceeds what could be achieved by using DNA sequence as an input feature. Remarkably, we show that transcription factor binding enables strikingly accurate predictions across different cell lines. Analysis of the relative importance of specific transcription factors as predictors of specific histone marks recapitulated known interactions between transcription factors and histone modifiers. Our results demonstrate that reported associations between histone marks and gene expression may be indirect effects caused by interactions between transcription factors and histone-modifying complexes. PMID:25187560

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

    SciTech Connect

    Blattmann, Claudia; Oertel, Susanne; Ehemann, Volker

    2010-09-01

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

  3. Cadmium and mercury toxicity in a human fetal hepatic cell line (WRL-68 cells).

    PubMed

    Bucio, L; Souza, V; Albores, A; Sierra, A; Chávez, E; Cárabez, A; Gutiérrez-Ruiz, M C

    1995-09-18

    The toxic effects of cadmium (Cd) and mercury (Hg), as chloride salts, were studied using an hepatic human fetal cell line (WRL-68 cells). From viability curves and the proliferative capacity of the cell in the presence of the metal, three different cell treatments were chosen, (1) 0.5 microM of the metal chloride for 24 h (acute low dose treatment), (2) 0.5 microM of the metal chloride for 7 days (chronic treatment), and (3) 5 microM of the metal chloride for 24 h (acute high dose treatment). WRL-68 cells grown in the presence of Cd exhibited the same proliferative curve as control cells, whereas in the case of Hg, the cells increased their proliferative capacity. Both metals produced ultrastructural alterations in different degrees, mainly observed as mitochondrial and RER structural changes, depending of the treatment and concentration of the metal used. Cytotoxicity was assessed by measuring the release of lactate dehydrogenase from the cells. Acutely high dose-treated cells showed the highest value for this parameter, and Cd-treated cells presented higher lactate dehydrogenase release than the Hg-treated ones. Cell damage was also measured by alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activities. Acute high dose Cd treatment caused the highest value of enzymatic release. Lipid peroxidation was significantly different with respect to control cells in chronic and acute high dose treatments with both metals. Metallothionein (MT) induction in response to Hg treatment was not detected. However, a dramatic induction of this protein occurred in Cd-treated cells. WRL-68 cells differentially respond to Cd and Hg making this hepatic fetal human cell line a useful tool in investigating the mechanism of toxicity of these heavy metals. PMID:7482568

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

    PubMed

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

    2015-11-01

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

  5. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines.

    PubMed

    Joseph, Immanual; Tressler, Robert; Bassett, Ekaterina; Harley, Calvin; Buseman, Christen M; Pattamatta, Preeti; Wright, Woodring E; Shay, Jerry W; Go, Ning F

    2010-11-15

    Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus, inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study, we investigated the effects of imetelstat (GRN163L), a potent telomerase inhibitor, on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally, imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat, but not control oligonucleotides, also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after <4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice, concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat, suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy. PMID:21062983

  6. Biological characteristics of a novel giant cell tumor cell line derived from spine.

    PubMed

    Zhou, Zhenhua; Li, Yan; Xu, Leqin; Wang, Xudong; Chen, Su; Yang, Cheng; Xiao, Jianru

    2016-07-01

    Giant cell tumor of bone(GCTB) is a special bone tumor for it consists of various cell types, and its biological characteristics is different from common benign or malignant neoplasm. In the present study, we report the biological features of a primary Asian GCTB cell line named GCTB28. We analyzed extensive properties of the GCTB28 cells including morphological observations, growth, cell cycle, karyotype, proliferation, proteins expression, surface biomarker verification, and tumorigenicity in nude mice. We found that the stromal cells of GCTB were endowed with self-renewal capacity and played dominant roles in GCTB development. Moreover, we confirmed that GCTB cells can be CD33(-)CD14(-) phenotype which was not in accord with previous study. This study provides an in vitro model system to investigate pathogenic mechanisms and molecular characteristics of GCTB and also provides a useful tool for researching the therapeutic targeting of GCTB. PMID:26801673

  7. Establishment and Characterization of 7 Novel Hepatocellular Carcinoma Cell Lines from Patient-Derived Tumor Xenografts

    PubMed Central

    Hu, Gang; Xie, Fubo; Ouyang, Kedong; Tang, Xuzhen; Wang, Minjun; Wen, Danyi; Zhu, Yizhun; Qin, Xiaoran

    2014-01-01

    Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening. PMID:24416385

  8. Development of a cell line from the American eel brain expressing endothelial cell properties.

    PubMed

    Bloch, Sophia R; Vo, Nguyen T K; Walsh, Sarah K; Chen, Cici; Lee, Lucy E J; Hodson, Peter V; Bols, Niels C

    2016-04-01

    A cell line (eelB) was developed from the outgrowth of adherent cells from brain explants of the American eel, Anguilla rostrata (Lesueur). EelB cells have been grown routinely in L-15 with 10% fetal bovine serum (FBS), undergone over 100 passages, and cryopreserved successfully. The cells from late-passage cultures (>45) were polygonal, formed capillary-like structures (CLS) on Matrigel, and stained immunocytochemically for von Willebrand factor (vWF) and for three tight junction proteins, zonula occludens-1 (ZO-1), claudin 3, and claudin 5. These results suggest that eelB is an endothelial cell line, one of the few from fish and the first from the brain. Despite this, eelB did not respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the induction of CYP1A protein. The cells from early-passage cultures (<20) had more varied shapes and did not form CLS on Matrigel. Only cells from early-passage cultures formed in suspension three-dimensional aggregates that had some cells expressing alkaline phosphatase and nestin. These cells are thought to be neural stem cells and the aggregates neurospheres. The emergence of endothelial-like cells upon the continued subcultivation of cells from early-passage cultures that had neural stem cells has been described previously for mammals, but this is a first for teleosts. Remarkably, cells from all passage levels were stained strongly for senescence-associated β-galactosidase (SA β-Gal) activity. PMID:26714751

  9. 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.

  10. Macrophage cell lines derived from major histocompatibility complex II-negative mice

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

  11. BST-1, a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth.

    PubMed Central

    Kaisho, T; Ishikawa, J; Oritani, K; Inazawa, J; Tomizawa, H; Muraoka, O; Ochi, T; Hirano, T

    1994-01-01

    Bone marrow stromal cells are essential for B-lymphocyte development. However, how stromal cells regulate B lymphopoiesis is not clear. In this paper, we report the molecular cloning of a stromal cell line-derived glycosyl-phosphatidylinositol-anchored molecule, BST-1, that facilitates pre-B-cell growth. The deduced amino acid sequence of BST-1 exhibited 33% identity with CD38. BST-1 was expressed in a wide range of tissues and in umbilical vein endothelial cells, whereas it was scarcely expressed in a variety of hematopoietic cell lines. The gene for BST-1 was assigned to chromosome 14q32.3, where immunoglobulin heavy-chain genes are clustered. BST-1 expression was enhanced in rheumatoid arthritis patient-derived bone marrow stromal cell lines that were previously shown to have an enhanced ability to support the growth of a pre-B-cell line as compared with stromal cell lines derived from healthy donors. Images PMID:8202488

  12. Drug-Resistant Urothelial Cancer Cell Lines Display Diverse Sensitivity Profiles to Potential Second-Line Therapeutics.

    PubMed

    Vallo, Stefan; Michaelis, Martin; Rothweiler, Florian; Bartsch, Georg; Gust, Kilian M; Limbart, Dominik M; Rödel, Franz; Wezel, Felix; Haferkamp, Axel; Cinatl, Jindrich

    2015-06-01

    Combination chemotherapy with gemcitabine and cisplatin in patients with metastatic urothelial cancer of the bladder frequently results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance could help to identify candidate treatments for an efficient second-line therapy. Six cisplatin- and six gemcitabine-resistant cell lines were established. Cell viability assays were performed to evaluate the sensitivity to 16 different chemotherapeutic substances. The activity of the drug transporter ATP-binding cassette transporter, subfamily B, member 1 (ABCB1, a critical mediator of multidrug resistance in cancer) was evaluated using fluorescent ABCB1 substrates. For functional assessment, cells overexpressing ABCB1 were generated by transduction with a lentiviral vector encoding for ABCB1, while zosuquidar was used for selective inhibition. In this study, 8 of 12 gemcitabine- or cisplatin-resistant cell lines were cross-resistant to carboplatin, 5 to pemetrexed, 4 to methotrexate, 3 to oxaliplatin, 5-fluorouracil, and paclitaxel, and 2 to cabazitaxel, larotaxel, docetaxel, topotecan, doxorubicin, and mitomycin c, and 1 of 12 cell lines was cross-resistant to vinflunine and vinblastine. In one cell line with acquired resistance to gemcitabine (TCC-SUP(r)GEMCI(20)), cross-resistance seemed to be mediated by ABCB1 expression. Our model identified the vinca alkaloids vinblastine and vinflunine, in Europe an already approved second-line therapeutic for metastatic bladder cancer, as the most effective compounds in urothelial cancer cells with acquired resistance to gemcitabine or cisplatin. These results demonstrate that this in vitro model can reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of metastatic bladder cancer. PMID:26055179

  13. Characterization of novel low passage primary and metastatic colorectal cancer cell lines

    PubMed Central

    Boot, Arnoud; van Eendenburg, Jaap; Crobach, Stijn; Ruano, Dina; Speetjens, Frank; Calame, Jan; Oosting, Jan; Morreau, Hans; van Wezel, Tom

    2016-01-01

    In vitro models are essential to understanding the molecular characteristics of colorectal cancer (CRC) and the testing of therapies for CRC. Many efforts to establish and characterize primary CRC cell lines have been published, most describing a small number of novel cell lines. However, there remains a lack of a large panel of uniformly established and characterized cell lines. To this end we established 20 novel CRC cell lines, of which six were derived from liver metastases. Genetic, genomic and transcriptomic profiling was performed in order to characterize these new cell lines. All data are made publically available upon publication. By combining mutation profiles with CNA and gene expression profiles, we generated an overall profile of the alterations in the major CRC-related signaling pathways. The combination of mutation profiles with genome, transcriptome and methylome data means that these low passage cell lines are among the best characterized of all CRC cell lines. This will allow researchers to select model cell lines appropriate to specific experiments, facilitating the optimal use of these cell lines as in vitro models for CRC. All cell lines are available for further research. PMID:26894854

  14. 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.

  15. The endocannabinoid system in the human granulosa cell line KGN.

    PubMed

    Ernst, Jana; Grabiec, Urszula; Greither, Thomas; Fischer, Bernd; Dehghani, Faramarz

    2016-03-01

    Ovarian steroidogenesis is embedded in a sensitive network of regulatory mechanisms crucial for human fertility. The endocannabinoid system (ECS) represents an intrinsic modulating system involved in the regulation of endocrine functions. In the present study we characterized the ECS in the human granulosa cell line KGN and its impact on gonadotropin sensitivity and steroid hormone synthesis under basal and FSH-stimulated conditions. Expression studies were performed and estradiol was measured. CB1, CB2, DAGL, FAAH, GPR55, MAGL, NAPE-PLD and TRPV1 were expressed without FSH-dependent effects. Treatment with selective cannabinoid receptor agonists reduced basal but not FSH-stimulated estradiol and CYP19. Progesterone was not altered by ECS manipulation. CB1 agonist changed the expression of miRNAs associated with granulosa cell function, e.g. miR-23a, miR-24, miR-181a and miR-320a. Present data indicate a modulating role of the intrinsic ovarian ECS in the regulation of estradiol synthesis. PMID:26773729

  16. 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. PMID:27241584

  17. More similar than different: Host cell protein production using three null CHO cell lines.

    PubMed

    Yuk, Inn H; Nishihara, Julie; Walker, Donald; Huang, Eric; Gunawan, Feny; Subramanian, Jayashree; Pynn, Abigail F J; Yu, X Christopher; Zhu-Shimoni, Judith; Vanderlaan, Martin; Krawitz, Denise C

    2015-10-01

    To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a

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

    PubMed

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

    2012-08-01

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

  19. Evaluation of effect of triterpenes and limonoids on cell growth, cell cycle and apoptosis in human tumor cell line.

    PubMed

    Cazal, Cristiane M; Choosang, Kantima; Severino, Vanessa Gisele P; Soares, Marcio S; Sarria, Andre Lucio F; Fernandes, Joao B; Silva, Maria Fatima G F; Vieira, Paulo Cezar; Pakkong, Pannee; Almeida, Gabriela M; Vasconcelos, M Helena; Nascimento, Maria S J; Pinto, Madalena M M

    2010-12-01

    Six triterpenes and eight limonoids were evaluated for their capacity to inhibit the growth of three human tumour cell lines, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and melanoma (A375-C5). The mechanisms involved in the observed cell growth arrest of the four most potent compounds were carried out by studying their effect in cell cycle profile and programmed cell death. The results showed that one triterpene (odoratol) and two limonoids (gedunin and cedrelone) caused cell cycle arrest while only the limonoids gedunin and cedrelone were found to be very potent inducers of apoptosis. PMID:21269253

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

    SciTech Connect

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

    2006-11-10

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

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

    PubMed

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

    2007-08-01

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

  2. 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

  3. 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.

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

    SciTech Connect

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

    2013-11-15

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

  5. Cell and membrane lipid analysis by proton magnetic resonance spectroscopy in five breast cancer cell lines.

    PubMed

    Le Moyec, L; Tatoud, R; Eugène, M; Gauvillé, C; Primot, I; Charlemagne, D; Calvo, F

    1992-10-01

    The lipid composition of five human breast cancer cell lines (MCF-7, T47D, ZR-75-1, SKBR3 and MDA-MB231) was assessed by proton magnetic resonance spectroscopy (MRS) in whole cells and membrane-enriched fractions. The proportions of the three main lipid resonances in 1D spectra were different for each cell line. These resonances included mobile methyl and methylene functions from fatty acids of triglycerides and phospholipids and N-trimethyl from choline of phospholipids. T47D and ZR-75-1 cells presented a high methylene/methyl ratio (6.02 +/- 0.35 and 6.28 +/- 0.90). This ratio was significantly lower for SKBR3, MCF-7 and MDA-MB231 cells (2.76 +/- 0.22, 2.27 +/- 0.57 and 1.39 +/- 0.39). The N-trimethyl/methyl ratio was high for MDA-MB231 and SKBR3 cells (1.38 +/- 0.54 and 0.86 +/- 0.32), but lower for MCF-7, T47D and ZR-75-1 cells (0.49 +/- 0.11, 0.16 +/- 0.07 and 0.07 +/- 0.03). 2D COSY spectra confirmed these different proportions in mobile lipids. From 1D spectra obtained on membrane preparations, T47D and ZR-75-1 were the only cell lines to retain a signal from mobile methylene functions. These differences might be related to the heterogeneity found for several parameters of these cells (tumorigenicity, growth rate, hormone receptors); an extended number of cases from fresh samples might enable clinical correlations. PMID:1329906

  6. 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.

  7. 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

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

    PubMed

    Liu, Guangfu; Xu, Yipeng; Yu, Xiaoping

    2015-03-01

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

  9. [Establishment and characterization of cell lines derived from nude mice transplanted squamous cell carcinoma of uterine cervix].

    PubMed

    Imanishi, K; Kato, Y; Karasaki, S; Yoshida, R; Kawana, T; Mizuno, M

    1990-06-01

    Two human cell lines, KIMI-1 and -2 were established from nude mice transplanted tumor originated from a human squamous cell carcinoma of the uterine cervix. These two cell lines have different shapes, chromosome numbers and tumor markers, respectively. PMID:2085478

  10. Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro.

    PubMed

    Matsumoto, Asako; Harada, Hidemitsu; Saito, Masahiro; Taniguchi, Akiyoshi

    2011-01-01

    Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions. PMID:21082283

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. 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. ...

  13. Synthesis and biological evaluation of Fotemustine analogues on human melanoma cell lines.

    PubMed

    Winum, Jean Yves; Bouissière, Jean Luc; Passagne, Isabelle; Evrard, Alexandre; Montero, Véronique; Cuq, Pierre; Montero, Jean Louis

    2003-03-01

    Two new analogues of Fotemustine have been synthesized and tested on two melanoma cell lines. Compounds 4 and 8 proved to be more potent than the reference compound on A375 cell line which express the MGMT enzyme involved in the chemoresistance of tumoral cells. PMID:12667699

  14. 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

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

    PubMed

    Genzel, Yvonne

    2015-05-01

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

  16. Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation

    PubMed Central

    Pryzhkova, Marina V; Peters, Ann; Zambidis, Elias T

    2012-01-01

    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 insitu 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. PMID:20541472

  17. The Influence of Clinical and Demographic Risk Factors on the Establishment of Head and Neck Squamous Cell Carcinoma Cell Lines

    PubMed Central

    White, Jason S.; Weissfeld, Joel L.; Ragin, Camille C. R.; Rossie, Karen M.; Martin, Christa Lese; Shuster, Michele; Ishwad, Chandramohan S.; Law, John C.; Myers, Eugene N.; Johnson, Jonas T.; Gollin, Susanne M.

    2007-01-01

    The purpose of this study was to generate stable cell cultures from head and neck squamous cell carcinomas (HNSCC), and retrospectively analyze the factors associated with successful cell line establishment. Fifty-two HNSCC cell lines were isolated from a series of 199 tumors collected between 1992 and 1997 at the University of Pittsburgh Medical Center. Cell lines were characterized at the molecular and cellular level to determine the features associated with cell line formation. Successful cell line formation was dependent on multiple factors, including gene amplification involving chromosomal band 11q13, local and/or regional involvement of lymph nodes, and alcohol usage. The establishment of HNSCC cell lines enriches the resources available for cancer research. Our findings indicate that generation of stable cell lines from HNSCC is biased towards tumors with a poor prognosis. Our 52 stable lines comprise one of the largest series of HNSCC cell lines in the literature, with complete demographic, histopathologic, clinical, and survival data. PMID:17112776

  18. Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line.

    PubMed

    Lee, Suk Kyoo; Lee, Gyun Min

    2003-06-30

    Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins. PMID:12701155

  19. Establishment and characterization of a cell line from the Chinese soft-shelled turtle Pelodiscus sinensis.

    PubMed

    Guo, Haijie; Xia, Zhaonan; Tang, Wei; Mao, Zhijuan; Qian, Guoying; Wang, Caisheng

    2016-06-01

    The establishment and partial characterization of Pelodiscus sinensis continuous cell line is described here. A novel P. sinensis fibroblast cell line, designated PSF, was established from heart tissue by the semi-digestion explant culture technique. Since its initiation in July 2013, the cell line has been subcultured at 30°C in minimal essential medium (MEM) containing 15% (v/v) fetal bovine serum for more than 50 passages. The growth curve of the cell line revealed the population doubling time was 51.1 h. Karyotyping analysis indicated the modal chromosome number was 66, and no microbial contamination was detected. The PSF cell line produced significant fluorescent signals after transfection with plasmid pEGFP-C3. Analysis of mitochondrial cytochrome D-loop sequences revealed 96% identity among other Chinese turtle subspecies. Several cell line characterizations included morphological analysis and immunocytochemistry, which revealed the origin of the PSF cell line was fibroblast-like cells. Measurement of the isoenzymes lactic dehydrogenase and malic dehydrogenase showed no cross-contamination of this cell line with other species. This newly established cell line will be a valuable tool for transgenic and genetic manipulation studies and will act as an efficient instrument for studies of the viral diseases of the soft-shelled turtle. PMID:27059326

  20. Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours

    PubMed Central

    Ince, Tan A.; Sousa, Aurea D.; Jones, Michelle A.; Harrell, J. Chuck; Agoston, Elin S.; Krohn, Marit; Selfors, Laura M.; Liu, Wenbin; Chen, Ken; Yong, Mao; Buchwald, Peter; Wang, Bin; Hale, Katherine S.; Cohick, Evan; Sergent, Petra; Witt, Abigail; Kozhekbaeva, Zhanna; Gao, Sizhen; Agoston, Agoston T.; Merritt, Melissa A.; Foster, Rosemary; Rueda, Bo R.; Crum, Christopher P.; Brugge, Joan S.; Mills, Gordon B.

    2015-01-01

    Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy. PMID:26080861

  1. Standard melanoma-associated markers do not identify the MM127 metastatic melanoma cell line

    PubMed Central

    Haridas, Parvathi; McGovern, Jacqui A.; Kashyap, Abhishek S.; McElwain, D. L. Sean; Simpson, Matthew J.

    2016-01-01

    Reliable identification of different melanoma cell lines is important for many aspects of melanoma research. Common markers used to identify melanoma cell lines include: S100; HMB-45; and Melan-A. We explore the expression of these three markers in four different melanoma cell lines: WM35; WM793; SK-MEL-28; and MM127. The expression of these markers is examined at both the mRNA and protein level. Our results show that the metastatic cell line, MM127, cannot be detected using any of the commonly used melanoma-associated markers. This implies that it would be very difficult to identify this particular cell line in a heterogeneous sample, and as a result this cell line should be used with care. PMID:27087056

  2. Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours.

    PubMed

    Ince, Tan A; Sousa, Aurea D; Jones, Michelle A; Harrell, J Chuck; Agoston, Elin S; Krohn, Marit; Selfors, Laura M; Liu, Wenbin; Chen, Ken; Yong, Mao; Buchwald, Peter; Wang, Bin; Hale, Katherine S; Cohick, Evan; Sergent, Petra; Witt, Abigail; Kozhekbaeva, Zhanna; Gao, Sizhen; Agoston, Agoston T; Merritt, Melissa A; Foster, Rosemary; Rueda, Bo R; Crum, Christopher P; Brugge, Joan S; Mills, Gordon B

    2015-01-01

    Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy. PMID:26080861

  3. Standard melanoma-associated markers do not identify the MM127 metastatic melanoma cell line.

    PubMed

    Haridas, Parvathi; McGovern, Jacqui A; Kashyap, Abhishek S; McElwain, D L Sean; Simpson, Matthew J

    2016-01-01

    Reliable identification of different melanoma cell lines is important for many aspects of melanoma research. Common markers used to identify melanoma cell lines include: S100; HMB-45; and Melan-A. We explore the expression of these three markers in four different melanoma cell lines: WM35; WM793; SK-MEL-28; and MM127. The expression of these markers is examined at both the mRNA and protein level. Our results show that the metastatic cell line, MM127, cannot be detected using any of the commonly used melanoma-associated markers. This implies that it would be very difficult to identify this particular cell line in a heterogeneous sample, and as a result this cell line should be used with care. PMID:27087056

  4. 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.

  5. Targeted Inhibition of CD133+ Cells in Oral Cancer Cell Lines

    PubMed Central

    Damek-Poprawa, M.; Volgina, A.; Korostoff, J.; Sollecito, T.P.; Brose, M.S.; O’Malley, B.W.; Akintoye, S.O.; DiRienzo, J.M.

    2011-01-01

    Resistance to treatment and the appearance of secondary tumors in head and neck squamous cell carcinomas (HNSCC) have been attributed to the presence of cells with stem-cell-like properties in the basal layer of the epithelium at the site of the lesion. In this study, we tested the hypothesis that these putative cancer stem cells (CSC) in HNSCC could be specifically targeted and inhibited. We found that 9 of 10 head and neck tumor biopsies contained a subpopulation of cells that expressed CD133, an unusual surface-exposed membrane-spanning glycoprotein associated with CSC. A genetically modified cytolethal distending toxin (Cdt), from the periodontal pathogen Aggregatibacter actinomycetemcomitans, was conjugated to an anti-human CD133 monoclonal antibody (MAb). The Cdt-MAb complex preferentially inhibited the proliferation of CD133+ cells in cultures of established cell lines derived from HNSCC. Inhibition of the CD133+ cells was rate- and dose-dependent. Saturation kinetics indicated that the response to the Cdt-MAb complex was specific. Healthy primary gingival epithelial cells that are native targets of the wild-type Cdt were not affected. Analysis of these data provides a foundation for the future development of new therapies to target CSC in the early treatment of HNSCC. Abbreviations: Cdt, cytolethal distending toxin; CSC, cancer stem cells; HNSCC, head and neck squamous cell carcinoma; MAb, monoclonal antibody. PMID:21220361

  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) rank