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Sample records for 3t3 mouse fibroblasts

  1. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Highlights: • LPA5 inhibits the cell growth and motile activities of 3T3 cells. • LPA5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA5 on the cell motile activities inhibited by LPA1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA5 in 3T3 cells. • LPA signaling via LPA5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA1–LPA6) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA1 and LPA5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA1

  2. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  3. Exogenous lactate interferes with cell-cycle control in BALB/3T3 mouse fibroblasts

    Purpose: Previous studies have shown that exogenous lactate may influence proliferation rates, radiation sensitivity, and postirradiation repair capacity of mammalian cells. In the present study, we addressed the question of potential underlying mechanisms and, therefore, examined effects of exogenous lactate on proliferation rates and cell-cycle distribution in immortal but nontumorigenic mammalian cells. Methods and Materials: Cells were grown at 37 deg. C in an incubator with 5% CO2 and 95% air, in a culture medium supplemented or not with lactate at a 10 mM concentration. Daily, we changed the culture medium and counted cells per dish. On selected days, cell-cycle distribution was determined by flow cytometry. Balb/3T3 mouse fibroblasts were used. Results: During the exponential phase of cell proliferation, mean population doubling time was significantly increased from 17.7 to 19.9 h, due to selective prolongation of G2/M. However, in density-inhibited cultures, exogenous lactate stimulated entry into S and proliferation to a significantly higher saturation density. Conclusions: These findings indicate that exogenous lactate interferes with mechanisms of cell-cycle control at two different points in the cell-cycle, depending on cell density and the resulting absence or presence of inhibition of cell proliferation. Interference with cell-cycle control may underlay the modification by exogenous lactate of radiosensitivity and postirradiation repair capacity in mammalian cells

  4. Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress

    Lambert, Ian Henry; Enghoff, Maria Stine; Brandi, Marie-Luise;

    2015-01-01

    The aim of this project was to analyze the regulation of p53 expression in NIH3T3 fibroblasts under the influence of increasing hyperosmotic stress. Expression of p53 showed a biphasic response pattern in NIH3T3 cells under increasing osmotic stress (337 mOsm to 737 mOsm) with a maximum at 587 m......Osm. Under isotonic conditions p53 expression increased after addition of the proteasome inhibitor MG132 indicating that cellular p53 levels in unperturbed cells is kept low by proteasomal degradation. However, under hypertonic conditions p53 synthesis as well as p53 degradation were significantly reduced...... and it is demonstrated that the increase in p53 expression observed when tonicity is increased from 337 to 587 mOsm reflects that degradation is more inhibited than synthesis, whereas the decrease in p53 expression at higher tonicities reflects that synthesis is more inhibited than degradation. The...

  5. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

    Friis, Martin Barfred; Vorum, Katrine Gribel; Lambert, Ian Henry

    2008-01-01

    Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein...... the Ca2+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid...... (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the...

  6. Cytotoxicity of MEIC chemicals Nos. 11-30 in 3T3 mouse fibroblasts with and without microsomal activation

    Rasmussen, Eva

    1999-01-01

    The cytotoxicity of MEIC chemicals Nos, 11-30 was evaluated by determination of neutral red uptake in Balb/c 3T3 mouse fibroblasts with and without the addition of a microsomal activation mixture. The use of microsomes significantly decreased the cytotoxicity of malathion, 2,4-dichlorophenoxyacetic...... acid, propranolol, thioridazine, lithium sulfate, copper sulfate and thallium sulfate, whereas the cytotoxicity of 1,1,1-trichloroethylene, phenol, nicotine, and paraquat was significantly increased by use of the microsomal activation mixture. These cytotoxicity data are in line with observations in...... other studies on microsomal modulation of the cytotoxicity of the test substances. Moderate to good correlations were found between the cytotoxicity data and rodent lethality data, and the addition of microsomes slightly improved the in vitro/in vivo concordance. The evidence to support the relevance of...

  7. Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts

    Hansen, Daniel Bloch; Friis, Martin Barfred; Hoffmann, Else Kay;

    2012-01-01

    The present work was initiated to investigate regulation of the taurine transporter TauT by reactive oxygen species (ROS) and the tonicity-responsive enhancer binding protein (TonEBP) in NIH3T3 mouse fibroblasts during acute and long-term (4 h) exposure to low-sodium/hypo-osmotic stress. Taurine...... influx is reduced following reduction in osmolarity, keeping the extracellular Na(+) concentration constant. TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions. In contrast, TonEBP activity and TauT transcription are...... ROS under hypo-osmotic, low-sodium conditions, whereas the TauT mRNA level is unaffected. Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics. Thus, swelling-induced ROS production could account for the reduced taurine uptake under low...

  8. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  9. Casein kinase 2 regulates the active uptake of the organic osmolyte taurine in NIH3T3 mouse fibroblasts

    Jacobsen, Jack H; Clement, Christian A; Friis, Martin B;

    2008-01-01

    T to ER but has no detectable effect on TauT protein expression. On the other hand, CK2 inhibition increases the affinity of TauT towards Na(+ )and reduces the Na(+)/taurine stoichiometry for active taurine uptake. It is suggested that CK2 controls the cellular taurine uptake in unperturbated NIH3T3......Inhibition of the constitutively active casein kinase 2 (CK2) with 2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidasole stimulates the Na(+)-dependent taurine influx via the taurine transporter TauT in NIH3T3 cells. CK2 inhibition reduces the TauT mRNA level and increases the localization of Tau...... cells, i.e., inhibition of CK2 increases the affinity of TauT towards Na(+) and hence Na(+)-dependent taurine uptake....

  10. Pharmaco-Phylogenetic Investigation of Methyl Gallate Isolated from Acacia nilotica (L.) Delile and Its Cytotoxic Effect on NIH3T3 Mouse Fibroblast.

    Mishra, Rohit K; Ramakrishna, M; Mishra, Vani; Pathak, Ashutosh; Rajesh, S; Sharma, Shivesh; Pandey, Avinash C; Nageswara Rao, G; Dikshit, Anupam

    2016-01-01

    Present exploration deals with the therapeutic perspective of methyl gallate isolated from the leaf extract of Acacia nilotica (L.) Delile in contrast to food-borne bacterial pathogen's viz., Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus with their evolutionary succession. The extract was subjected to phytochemical analysis and isolated compound was identified as methyl gallate using UV-vis, IR and NMR spectra. It was found most potent against K. pneumoniae with its minimum inhibition concentration (MIC) of 0.32 mg/ml and minimum bactericidal concentration (MBC) at 0.62 mg/ml. The correlation of MIC values with an evolutionary succession assists the relationship between their genetic and toxic properties. The cytotoxic pursuit of methyl gallate was additionally assessed over NIH3T3 mouse fibroblast by Neutral red (NR) uptake, MTT cell proliferation assay and did not disclose any relevant influence on cell viability as well as cell proliferation. As such, the methyl gallate extracted from the leaf of A. nilotica holds massive antibacterial aptitude and hands out towards a new paradigm for food and pharmaceutical industries. PMID:26813302

  11. Neoplastic transformation and tumorigenesis associated with overexpression of imup-1 and imup-2 genes in cultured NIH/3T3 mouse fibroblasts

    Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation

  12. Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles

    The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but not to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (γ-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on cells. -- Highlights: ► Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ► Ludox CL-X silica NPs are cytotoxic to both cell lines. ► In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ► Cell cycle analysis shows

  13. Alteration of proteoglycan metabolism during the differentiation of 3T3- L1 fibroblasts into adipocytes

    1991-01-01

    3T3-L1 fibroblasts were induced to differentiate to 3T3-L1 adipocytes by dexamethasone, isobutyl-methylxanthine, and insulin. To study how differentiation affects extracellular matrix production, the accumulation of proteoglycans was studied by labeling the 3T3-L1 cells with [35S]sulphate for 24 h. The labeled proteoglycans were isolated from the medium and cell layer extracts by anion-exchange chromatography. They were then taken to gel filtration chromatography on Superose 6 before or after...

  14. Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model

    Meixensberger Jürgen; Gebhardt Rolf; Hengstler Jan; Hermes Matthias; Geiger Kathrin D; Fuchs Beate; Zemitzsch Nadine; Renner Christof; Gaunitz Frank

    2010-01-01

    Abstract Background It was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy. Results A mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth fa...

  15. 成纤维细胞系3T3细胞来源exosome对小鼠乳腺癌细胞增殖能力的影响%Effect of exosome Extracted from Fibroblast Cell Line 3T3 on Proliferation of Mouse Breast Cancer Cells

    王雅琴; 陈智

    2016-01-01

    目的 观察小鼠成纤维细胞系3T3来源的外泌小体(exosome)对小鼠乳腺癌细胞4T1增殖能力的影响,并探索其中可能的机制.方法 PureExo Exosome提取试剂盒提取3T3细胞上清液中的exosome,按照不同浓度及时间作用于4T1细胞,CCK8法检测4T1细胞的增殖能力,BrdU/PI双掺入法测定细胞DNA合成及细胞周期;免疫印迹法(Western blot)及荧光定量实时PCR(qPCR)检测人表皮生长因子受体2(epidermal growth factor receptor-2,EGFR2,也称HER2)及下游PI3K/AKT信号转导通路相关蛋白的变化.利用HER2单克隆抗体靶向药物赫赛汀(Herceptin),观察exosome是否影响4T1细胞对于Herceptin敏感度.结果 exosome处理组OD450吸光度值显著高于对照组(P<0.05),细胞增殖及细胞周期进程加快.Western blot及qPCR实验提示随着exosome浓度的增加,HER2表达逐渐升高,AKT磷酸化水平增加.而同时给予exosome可明显增加4T1细胞对Herceptin的敏感度.结论 小鼠成纤维细胞系3T3来源exosome可促进小鼠乳腺癌细胞4T1增殖及周期进程,并且可能通过HER2激活其下游PI3K/AKT信号通路发挥上述作用.

  16. Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts

    Friis, Martin B; Friborg, Christel R; Schneider, Linda;

    2005-01-01

    Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687...... mosmol l(-1)) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced...... in cells expressing a constitutively active G protein, Rac (RacV12A3 cell), indicating that Rac acts upstream to caspase-3 activation. The stress-activated protein kinase, p38, was significantly activated by phosphorylation within 30 min after induction of osmotic shrinkage, the phosphorylation being...

  17. Mitogenic stimuli and phosphatidylinositol (PI) turnover in cultured 3T3 fibroblasts

    The hydrolysis of PI and polyphosphoinositides by phopholipase C is an early and rapid response to cell activation by a variety of neurotransmitters, hormones, growth factors and pharmacological agonists. The authors have examined the role of PI turnover and the generation of second messengers (diacylglycerol and inositol trisphosphate) in the mitogenic response of cultured Balb/c and Swiss 3T3 cells to polypeptide growth factors. Cells were prelabelled with 3H inositol for 18-20 hours, washed and suspended in Herpes + Li+ buffer, and stimulated with platelet-derived growth factor (PDGF), vasopressin, insulin, and other growth factors. PI turnover was measured as the increase in total inositol phosphate (IP) production. IP1, IP2, and IP3 were characterized by sequential elution from a Dowex column. Partially purified PDGF produced a 2-4 fold stimulation of total IP production. This was seen as early as 30 seconds after stimulation and increased for up to 1-2 hours. Balb/c cells were more sensitive than Swiss cells to the mitogenic and PI effects of PDGF. Other mitogenic stimuli had differential effects on PI turnover. Vasopressin (4-400 ng/ml) markedly stimulated PI turnover (3-6 fold) in Swiss, but not Balb/c cells. Insulin (100 ng/ml - 10 μg/ml) increased total IP to a greater degree in Balb/c cells. Epidermal growth factor (10 ng/ml - 10 μg/ml) had no effect on PI turnover and fibroblast growth factor (10 ng/ml - 10 μg/ml) only stimulated at the higher concentrations in Swiss cells. Thrombin (1U/ml - 10 U/ml) produced a 1.5 - 2 fold stimulation in Balb/c cells. Thus, various polypeptide growth factors have differential effects on PI turnover depending on their mitogenic potential and the effector cell type

  18. Sustained calcium influx activated by basic fibroblast growth factor in Balb-c 3T3 fibroblasts.

    Munaron, L; Distasi, C; Carabelli, V; Baccino, F M; Bonelli, G; Lovisolo, D

    1995-05-01

    1. We have investigated the ionic events elicited in Balb-c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2-100 ng ml-1) caused the appearance of an inward current, as observed in whole-cell patch-clamp experiments at a holding potential of -50 mV, that could last for tens of minutes and had a peak density of 4.6 +/- 2.6 pA pF-1. The reversal potential was 18.8 +/- 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low-affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre-incubated with tyrphostin or 5'-methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high-affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365-A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura-2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell-attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high-K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb-c 3T3 cells bFGF induces an inward current that

  19. A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis

    Yue-juan QIN; Zhen-lin ZHANG; Lu-yang YU; Jin-wei HE; Ya-nan HOU; Tian-jin LIU; Jia-cai WU; Song-hua WU; Li-he GUO

    2006-01-01

    Aim: To construct an A20 expression vector under the control of mouse osteocalcin promoter (OC-A20), and investigate osteoblastic MC3T3-E1 cell line, which stably overexpresses A20 protein prevented tumor necrosis factor (TNF)-alpha-induced apoptosis. Methods: OC-A20 vector was constructed by fusing a fragment of the mouse osteocalcin gene-2 promoter with human A20 complementary DNA. Then the mouse MC3T3-E1 cell line, stably transfected by A20, was established. The expression of A20 mRNA and A20 protein in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To determine the specificity of A20 expression in osteoblast, the mouse osteoblastic MC3T3-E1 cell line and mouse embryo fibro-blast NIH3T3 cell line were transiently transfected with OC-A20. The anti-apoptotic role of A20 in MC3T3-E1 cells was determined by Flow cytometric analysis (FACS), terminal dUTP nick endo-labeling (TUNEL) and DNA gel electrophoresis analysis (DNA Ladder), respectively. Results: Weak A20 expression was found in MC3T3-El cells with the primers of mouse A20. A20 mRNA and A20 protein expression were identified in MC3T3-E1 cells transfected with OC-A20 using RT-PCR and Western blot analysis. Only A20 mRNA expression was found in MC3T3-E1 cell after MC3T3-E1 cells and NIH3T3 cells were transient transfected with OC-A20. A decrease obviously occurred in the rate of apoptosis in the OC-A20 group compared with the empty vector (pcDNA3) group by FACS (P<0.001). A significant increase in TUNEL positive staining was found in the pcDNA group compared with OC-A20 group (P<0.001). Simultaneously, similar effects were demonstrated in DNA gel electrophoresis analysis. Conclusion: We constructed an osteoblast-specific expression vector that expressed A20 protein in MC3T3-E1 cells and confirmed that A20 protects osteoblast against TNF-alpha-induced apoptosis.

  20. Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells.

    Smith, J. B.

    1983-01-01

    Vanadyl sulfate and sodium orthovanadate in the concentration range between 5 and 50 microM are shown to be mitogenic for quiescent cultures of Swiss mouse 3T3 and 3T6 cells. The compounds caused a striking shift in the dose-response for the effect of serum on [3H]thymidine incorporation and DNA synthesis. In the absence of serum the effect of vanadium was greatly potentiated by insulin. Vanadium ions produced no more than additive increases in [3H]thymidine incorporation when combined with e...

  1. Collagen gel containing 3T3 fibroblasts (dermal equivalent for raft culture)

    sprotocols

    2014-01-01

    Author: Matt Lewis ### Ingredients for 6 x collagen matrices in a 6-well plate 1. Roughly 3x10e6 J2-3T3s (a fully confluent T75?) - 1.5mL 10x reconstitution buffer - 1.5mL 10x DMEM - 12mL rat tail type 1 collagen (>3.8mg/mL) - 10N NaOH - Glacial acetic acid (in case) ### Method 1. Pre-chill pipettes, keep collagen on ice - *The collagen solidifies above 8ºC* - Mix 1.5mL of 10x DMEM with 1.5mL of 10x reconstitution buffer, keep on ice. Count J2-3T3s...

  2. Changes in laser-induced fluorescence responses of 3T3 fibroblasts to repetitive thermal stress

    Beuthan, J.; Dressler, C.; Zabarylo, U.; Minet, O.

    2009-04-01

    The combined experimental use of laser-induced autofluorescence of cellular metabolites and methodological fundamentals of systems biology will provide access to biological thermal stress analysis on a sub cellular level. A test setup incorporating a pulsed nitrogen laser was realized with which autofluorescence of the coenzyme NADH could be measured in living 3T3 cells. The cells were subjected to different temperature stress at repetitive time intervals. When subjected to a simple mathematical analysis, the NADH concentration change measured through autofluorescence in biological cells exhibited approximate concentration-equivalent balance curves. These results add up to the fundamental know-how about the dosimetry of thermally therapeutic methods.

  3. Macerated-Pineapple Core Crude Extract-derived Bromelain Has Low Cytotoxic Effect in NIH-3T3 Fibroblast

    Dewi Liliany Margaretta

    2015-08-01

    Full Text Available BACKGROUND: Bromelain is a sulfhydryl proteolytic enzyme that can hydrolyze protein, protease or peptide. Bromelain can be found in pineapple stem, fruit and core. Bromelain is composed of 212 amino acid residues with cysteine-25 forming a polypeptide chain that can hydrolyze peptide bonds by H2O. In medicine, bromelain has been developed as antibiotic, cancer drug, anti-inflammatory agent and immunomodulator. In dentistry, bromelain has potential to reduce plaque formation on the teeth and to irrigate root canal. METHODS: Pineapple core was dried for 3 days to get simplicia. Then simplicia was extracted with water solvent for 24 hours. After that, the macerated-pineapple core crude extract-derived bromelain (PCB was separated by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE followed by Coomassie Brilliant Blue (CBB staining to ensure the presence of bromelain. In cytotoxic test, NIH-3T3 fibroblast cultures were treated with extracts in various concentrations to for 24 or 48 hours. Number of fibroblasts was calculated using 3-(4,5-dimethylthiazol-2- yl-2,5-Diphenyltetrazolium bromide (MTT assay. RESULTS: Pineapple core extraction using maceration method produced relative high yield (concentration: 1.5424 g/mL of bromelain, which was confirmed by CBB staining results with the molecular weight of 33 kDa. Based on cytotoxic test results of PCB on NIH-3T3 fibroblasts, 24-hours-incubation LD50 was 95.7 g/L, while 48-hours-incubation LD50 was 51.1 g/L. CONCLUSIONS: PCB has low cytotoxic effect in NIH-3T3 fibroblasts. KEYWORDS: bromelain, pineapple, extract, cytotoxic, MTT.

  4. Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

    Foster, C.M.; Shafer, J.A.; Rozsa, F.W.; Wang, X.; Lewis, S.D.; Renken, D.A.; Natale, J.E.; Schwartz, J.; Carter-Su, C.

    1988-01-12

    Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. /sup 125/I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained in M/sub r/ 134,000 /sup 125/I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of fibroblast form. O-Phosphotyrosine prevented /sup 125/I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with (/sup 32/P)P/sub i/, GH was shown to stimulate formation of a /sup 32/P-labeled protein which bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114,000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. These observations provide strong evidence that binding of GH to its receptor stimulates phosphorylation of tyrosyl residues in the GH receptor.

  5. Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

    Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. 125I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained in M/sub r/ 134,000 125I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of fibroblast form. O-Phosphotyrosine prevented 125I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with [32P]P/sub i/, GH was shown to stimulate formation of a 32P-labeled protein which bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114,000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. These observations provide strong evidence that binding of GH to its receptor stimulates phosphorylation of tyrosyl residues in the GH receptor

  6. Evidence that downregulation of hexose transport limits intracellular glucose in 3T3-L1 fibroblasts

    Measurements of initial glucose entry rate and intracellular glucose concentration in cultured cells are difficult because of rapid transport relative to intracellular volume and a substantial extracellular space from which glucose cannot be completely removed by quick exchanges of medium. In 3T3-L1 cells, we obtained good estimates of initial entry of [14C]methylglucose and D-[14C]glucose with (1) L-[3H]glucose as an extracellular marker together with the [14C]glucose or [14C]methylglucose in the substrate mixture, (2) sampling times as short as 2 s, (3) ice-cold phloretin-containing medium to stop uptake and rinse away the extracellular label, and (4) nonlinear regression of time courses. Methylglucose equilibrated in two phases--the first with a half-time of 1.7 s and the second with a half-time of 23 s; it eventually equilibrated in an intracellular space of 8 microliters/mg protein. Entry of glucose remained almost linear for 10 s, making its transport kinetics easier to study (Km = 5.7 mM, Vmax = 590 nmol.s-1.ml-1 cell water). Steady-state intracellular glucose concentration was 75-90% of extracellular glucose concentration. Cells grown in a high-glucose medium (24 mM) exhibited a 67% reduction of glucose-transport activity and a 50% reduction of steady-state ratio of intracellular glucose to extracellular glucose

  7. A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts

    Magdalon, Juliana

    2011-11-24

    Abstract Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

  8. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity

  9. Effects of cytochalasin B on the uptake of ascorbic acid and glucose by 3T3 fibroblasts: Mechanism of impaired ascorbate transport in diabetes

    Hyperglycemia and/or hypoinsulinemia have been found to inhibit L-ascorbic acid cellular transport. The resultant decrease in intracellular ascorbic acid may de-inhibit aryl sulfatase B and increase degradation of sulfated glycosaminoglycans (sGAG). This could lead to a degeneration of the extracellular matrix and result in increased intimal permeability, the initiating event in atherosclerosis. The present studies show that the glucose transport inhibitor cytochalasin B blocked the uptake of 3H-2-deoxy-D-glucose by mouse 3T3 fibroblasts. Cytochalasin B also blocked the uptake of 14C-L-ascorbic acid. The results of these studies further support the hypothesis that glucose and ascorbate share a common transport system. This may have important implications concerning the vascular pathology associated with diabetes mellitus

  10. Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts

    The metabolic actions of insulin and anti-insulin receptor monoclonal antibodies were compared with their effects on insulin receptor phosphorylation in mouse NIH 3T3 fibroblasts transfected with human insulin receptor cDNA. In serum-starved NIH 3T3 HIR3.5 cells, uptake of 2-deoxy-[3H]glucose was stimulated up to 2-fold after 30 min with insulin, with a half-maximal effect at 0.1 nM insulin. Incorporation of [3H]thymidine was stimulated ∼ 12-fold after a 16-hr preincubation with insulin, with a half-maximal effect at 2 nM insulin. Phosphorylation of insulin receptor β-subunit in cells prelabeled with [32P]phosphate was increased 10- to 20-fold within 5 min of adding insulin. Monoclonal antibodies reacting with four different epitopes on the insulin receptor mimicked the effect of insulin on 2-deoxyglucose uptake. These antibodies also stimulated thymidine incorporation, although the maximum stimulation was only ∼ 30% that of insulin. It is concluded that the insulin-like metabolic effects of antibodies involve a mechanism of receptor activation that is independent of autophosphorylation and hence that receptor autophosphorylation is not an essential step in triggering at least some events in the insulin signaling pathway

  11. Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model

    Meixensberger Jürgen

    2010-01-01

    Full Text Available Abstract Background It was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy. Results A mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu, were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 μl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p Conclusion As a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.

  12. Morphological changes of nuclear and chromatin architecture after microwave electromagnetic field exposure in 3T3 fibroblast cell cultures

    It is already demonstrated in the literature that electromagnetic fields, particularly the microwave irradiation could be a powerful weapon against human tumors , but also against human body itself, depending on the wave parameters and irradiation time. The effects of microwave electromagnetic fields on living systems were studied in detail all over the world and, furthermore, the potential of intracellular damages by cytoskeleton, nuclear, chromatin and DNA alterations were carefully evaluated. In this study, the authors emphasize the morphological changes of nucleus and chromatin in fibroblast cell line 3T3 after microwave exposure with progressive increasing powers and times of irradiation. It was used a pulsed wave with 915 MHz frequency, with forward power ranging between 3 - 10 W, emitted by a helical microwave antenna placed into the cell culture medium, close to the cell monolayer. The authors tried to define certain severity stages of nuclear material alterations following different wave intensities and to compare these effects with other cytoplasmic organelle alterations. It was found that the nuclear material is the most sensitive intracellular structure in microwave electromagnetic field exposure. Also the authors tried to establish a well-defined protocol of irradiation with microwave electromagnetic fields in order to destroy the microtubule system of cytoskeleton in different types of cellular lines, in vitro. The cytoskeleton structure was evaluated by immunofluorescence methods. In non-muscle cells the cytoskeleton stability is achieved by interaction between microtubule system and actin filaments. Microtubule depolymerization by microwave exposure produces a secondary instability of cytoskeleton, the actin filaments coupling and cell contractility. The increasing of fibroblast contractility allows a more efficient treatment of the wounds with low spontaneous healing. Electromagnetic therapy could be an alternative therapy in plastic surgery

  13. Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes

    R. Kuonen

    2013-01-01

    Full Text Available Viscum album L. lipophilic extract (VALE contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10 μg/mL VALE and 1 μg/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo.

  14. Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

    Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup;

    2008-01-01

    immunofluorescence microscopy, Western blot analysis and alkali comet assays to show that phosphorylation of ATM in NIH3T3 fibroblasts occurs prior to apoptotic DNA fragmentation, nuclease degradation and phosphorylation of histone H2A.X in cells treated with low levels of either staurosporine (STS) or tumor...

  15. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  16. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 106 receptors per cell. The cell line with the highest 125I-insulin binding (NIH 3T3 HIR3.5) had 6 x 106 receptors with a K/sub d/ of 10-9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 107 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  17. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-08-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the ..cap alpha.. and ..beta.. subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  18. Effect of two homeopathic remedies at different degrees of dilutions on the wound closure of 3T3 fibroblasts in in vitro scratch assay

    Reinhard Saller

    2012-09-01

    Full Text Available Background: Since ancient times, preparations from traditional medicinal plants e.g. Arnica montana, Calendula officinalis or Hypericum perforatum have been used for different wound healing purposes. The aim of this study was to investigate the efficacy of the commercial low dilution homeopathic remedy Similasan® Arnica plus Spray, a preparation of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2 and medium diluted SIM WuS (Petroleum 15x, Arnica montana 15x, Calcium fluoratum 12x, Calendula officinalis 12x, Hepar sulfuris 12x and Mercurius solubilis 15x; 1101-4, on the wound healing in cultured NIH 3T3 fibroblasts. Both remedies were from Similasan AG (Jonen, Switzerland and prepared according the German Homoeopathic Pharmacopoeia (GHP following descriptions 4a for arnica, 3a for marigold and St. John’s wort, 2a for comfrey, 5a for petroleum, and 6 for calcium fluoride, hepar sulfuris and mercurius solubilis. Materials and Methods: Cell proliferation, migration and wound closure promoting effect of the preparations (0712-2, 1101- 4 and their succussed solvents (0712-1, 1101-3 were investigated on mouse NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined wound area. All assays were performed in three independent controlled experiments. In some experiments diluted unsuccussed alcohol (0712-3 was also investigated. Results: Preparations (0712-1, (0712-2, (0712-3, (1101-3 and (1101-4 were investigated at decimal dilution steps from 1x to 4x. Cell viabilty was not affected by any of the substances and (0712-1 and (0712-2 showed no stimulating effect on cell proliferation. Preparation (0712-2 exerted a stimulating effect on fibroblast migration (31.7% vs 15% with succussed solvent (0712-1 at 1

  19. Direct Comparison of the Spread Area, Contractility, and Migration of balb/c 3T3 Fibroblasts Adhered to Fibronectin- and RGD-Modified Substrata

    Rajagopalan, Padmavathy; Marganski, William A.; Brown, Xin Q; Wong, Joyce Y.

    2004-01-01

    Native proteins are often substituted by short peptide sequences. These peptides can recapitulate key, but not all biofunctional properties of the native proteins. Here, we quantify the similarities and differences in spread area, contractile activity, and migration speed for balb/c 3T3 fibroblasts adhered to fibronectin- (FN) and Arg-Gly-Asp (RGD)-modified substrata of varying surface density. In both cases spread area has a biphasic dependence on surface ligand density (σ) with a maximum at...

  20. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating

  1. Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells

    Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 μM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is ≅ 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself

  2. A homeopathic remedy from arnica, marigold, St. John’s wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts

    Hostanska Katarina

    2012-07-01

    Full Text Available Abstract Background Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. Methods We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712–2, its succussed hydroalcoholic solvent (0712–1 and unsuccussed solvent (0712–3 on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined “wound field”. All assays were performed in three independent controlled experiments. Results None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712–2 exerted a stimulating effect on fibroblast migration (31.9% vs 14.7% with succussed solvent (0712–1 at 1:100 dilutions (p  0.05. Preparation (0712–2 at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p  Conclusion Results of this study showed that the low potency homeopathic remedy (0712–2 exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.

  3. Direct Comparison of the Spread Area, Contractility, and Migration of balb/c 3T3 Fibroblasts Adhered to Fibronectin- and RGD-Modified Substrata

    Rajagopalan, Padmavathy; Marganski, William A.; Brown, Xin Q.; Wong, Joyce Y.

    2004-01-01

    Native proteins are often substituted by short peptide sequences. These peptides can recapitulate key, but not all biofunctional properties of the native proteins. Here, we quantify the similarities and differences in spread area, contractile activity, and migration speed for balb/c 3T3 fibroblasts adhered to fibronectin- (FN) and Arg-Gly-Asp (RGD)-modified substrata of varying surface density. In both cases spread area has a biphasic dependence on surface ligand density (σ) with a maximum at σ ∼ 200 molecules/μm2, whereas the total traction force increases and reaches a plateau as a function of σ. In addition to these qualitative similarities, there are significant quantitative differences between fibroblasts adhered to FN and RGD. For example, fibroblasts on FN have a spread area that is on average greater by ∼200 μm2 over a ∼40-fold change in σ. In addition, fibroblasts on FN exert ∼3–5 times more total force, which reaches a maximum at a value of σ ∼5 times less than for cells adhered to RGD. The data also indicate that the differences in traction are not simply a function of the degree of spreading. In fact, fibroblasts on FN (σ ∼2000 μm−2) and RGD (σ ∼ 200 μm−2) have both similar spread area (∼600 μm2) and migration speed (∼11 μm/h), yet the total force production is five times higher on FN than RGD (∼0.05 dyn compared to ∼0.01 dyn). Thus, the specific interactions between fibroblasts and FN molecules must inherently allow for higher traction force generation in comparison to the interactions between fibroblasts and RGD. PMID:15454473

  4. Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells

    Lambert, Ian Henry

    2007-01-01

    Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H(2)O(2) and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A(2) (PLA(2......)) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca(2+)-independent (iPLA(2))/secretory PLA(2) (sPLA(2)) plus 5-LO...... activity and modulation by ROS. Vanadate and H(2)O(2) stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell...

  5. ToF-SIMS depth profiling of cells: z-correction, 3D imaging, and sputter rate of individual NIH/3T3 fibroblasts.

    Robinson, Michael A; Graham, Daniel J; Castner, David G

    2012-06-01

    Proper display of three-dimensional time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging data of complex, nonflat samples requires a correction of the data in the z-direction. Inaccuracies in displaying three-dimensional ToF-SIMS data arise from projecting data from a nonflat surface onto a 2D image plane, as well as possible variations in the sputter rate of the sample being probed. The current study builds on previous studies by creating software written in Matlab, the ZCorrectorGUI (available at http://mvsa.nb.uw.edu/), to apply the z-correction to entire 3D data sets. Three-dimensional image data sets were acquired from NIH/3T3 fibroblasts by collecting ToF-SIMS images, using a dual beam approach (25 keV Bi(3)(+) for analysis cycles and 20 keV C(60)(2+) for sputter cycles). The entire data cube was then corrected by using the new ZCorrectorGUI software, producing accurate chemical information from single cells in 3D. For the first time, a three-dimensional corrected view of a lipid-rich subcellular region, possibly the nuclear membrane, is presented. Additionally, the key assumption of a constant sputter rate throughout the data acquisition was tested by using ToF-SIMS and atomic force microscopy (AFM) analysis of the same cells. For the dried NIH/3T3 fibroblasts examined in this study, the sputter rate was found to not change appreciably in x, y, or z, and the cellular material was sputtered at a rate of approximately 10 nm per 1.25 × 10(13) ions C(60)(2+)/cm(2). PMID:22530745

  6. Transformation by viral and cellular oncogenes of a mouse BALB/3T3 cell mutant resistant to transformation by chemical carcinogens.

    Ono, M; Yakushinji, M; Segawa, K.; Kuwano, M

    1988-01-01

    The mouse cell line MO-5 is resistant to transformation by various chemical carcinogens and also by UV irradiation (C. Yasutake, Y. Kuratomi, M. Ono, S. Masumi, and M. Kuwano, Cancer Res. 47:4894-4899, 1987). Northern (RNA) blot analysis showed active expression of ras and myc genes in MO-5 and BALB/3T3 cells. The effect of transfection of various oncogenes on transformation was compared in MO-5 cells and parental BALB/3T3 cells. Activated c-H-ras, c-N-ras, and v-mos gene induced transformati...

  7. Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor β

    A mutant clone (MO-5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF):binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-β efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-β while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-β showed low colony formation capacity in soft agar in the absence of TGF-β. Colonies of MO-5 formed by TGF-β in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-β. Pretreatment of MO-5 with TGF-β induced secretion of TGF-β-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-β-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the transformed phenotype in MO-5 is discussed

  8. [Cytotoxicity study of high gold content Degutan surfaces of various degrees of roughness with fibroblasts (BALB 3T3) and osteoblasts (hFOB 1.19)].

    Altvater, T; Hendrich, C; Nöth, U; Rader, C P; Stach, R; Schütze, N; Eulert, J; Thull, R

    2000-09-01

    The cytotoxicity of Degutan surfaces with different degrees of roughness, and the effect of surface structures on osteoblast proliferation and differentiation, was investigated with standardised cell culture systems. Fibroblast cell lines (BALB/3T3) and osteoblast cell lines (hFOB 1.19) were used. The number and variability of the cells were determined for assessment of proliferation and alkaline phosphatase activity, collagen I and osteocalcin production were used as parameters for differentiation. In the early phase, the largest numbers of cells and greatest proliferation were measured on polished Degutan surfaces. In the late phase, however, larger numbers of cells and a greater degree of proliferation were to be seen on sandblasted and sandblasted/heat-treated Degutan surfaces. No differences were found for collagen I, osteocalcin production or alkaline phosphatase activity. Neither the osteoblasts nor the fibroblasts revealed a toxic effect of Degutan. The results for osteoblast differentiation correlate with recent studies on identical structured titanium surfaces. In view of the immeasurable amount of ion release, Degutan may be considered an ideal model for an inert material surface. PMID:11030093

  9. Increased NIH 3T3 fibroblast functions on cell culture dishes which mimic the nanometer fibers of natural tissues

    Bhardwaj G

    2015-08-01

    Full Text Available Garima Bhardwaj,1 Thomas J Webster1,21Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 2Center of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi ArabiaAbstract: Traditional flat tissue cell culture dishes have consisted of polystyrene treated with plasma gases for growing, subculturing, and studying cell behavior in vitro. However, increasingly it has been observed that mimicking natural tissue properties (such as chemistry, three-dimensional structure, mechanical properties, etc in vitro can lead to a better correlation of in vitro to in vivo cellular functions. The following studies compared traditional NIH 3T3 fibroblasts’ functions on XanoMatrix scaffolds to standard tissue culture polystyrene. Results found significantly greater fibroblast adhesion and proliferation on XanoMatrix cell culture dishes which mimic the nanoscale geometry of natural tissue fibers with true, tortuous fiber beds creating a robust, consistent, and versatile growth platform. In this manner, this study supports that cell culture dishes which mimic features of natural tissues should be continually studied for a wide range of applications in which mimicking natural cellular functions are important.Keywords: nanotechnology, cell culture, fibroblasts

  10. Cocoa tea (Camellia ptilophylla) water extract inhibits adipocyte differentiation in mouse 3T3-L1 preadipocytes.

    Li, Kai Kai; Liu, Chuek Lun; Shiu, Hoi Ting; Wong, Hing Lok; Siu, Wing Sum; Zhang, Cheng; Han, Xiao Qiang; Ye, Chuang Xing; Leung, Ping Chung; Ko, Chun Hay

    2016-01-01

    Cocoa tea (Camellia ptilophylla) is a naturally decaffeinated tea plant. Previously we found that cocoa tea demonstrated a beneficial effect against high-fat diet induced obesity, hepatic steatosis, and hyperlipidemia in mice. The present study aimed to investigate the anti-adipogenic effect of cocoa tea in vitro using preadipocytes 3T3-L1. Adipogenic differentiation was confirmed by Oil Red O stain, qPCR and Western blot. Our results demonstrated that cocoa tea significantly inhibited triglyceride accumulation in mature adipocytes in a dose-dependent manner. Cocoa tea was shown to suppress the expressions of key adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPAR γ) and CCAAT/enhancer binding protein (C/EBP α). The tea extract was subsequently found to reduce the expressions of adipocyte-specific genes such as sterol regulatory element binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS), Acetyl-CoA carboxylase (ACC), fatty acid translocase (FAT) and stearoylcoenzyme A desaturase-1 (SCD-1). In addition, JNK, ERK and p38 phosphorylation were inhibited during cocoa tea inhibition of 3T3-L1 adipogenic differentiation. Taken together, this is the first study that demonstrates cocoa tea has the capacity to suppress adipogenesis in pre-adipocyte 3T3-L1 similar to traditional green tea. PMID:26833256

  11. Results of the L5178Y mouse lymphoma assay and the Balb/3t3 cell in vitro transformation assay for eight phthalate esters.

    Barber, E D; Cifone, M; Rundell, J; Przygoda, R; Astill, B D; Moran, E; Mulholland, A; Robinson, E; Schneider, B

    2000-01-01

    Eight phthalate esters, with alcohol chain lengths of 1-11 carbon atoms and with various degrees of branching, were tested in vitro in the L5178Y mouse lymphoma mammalian cell mutation assay and in the Balb/3T3 cell transformation assay. The tests were performed as part of a voluntary testing agreement between the Chemical Manufacturers Association's Phthalate Esters Panel and the United States Environmental Protection Agency (US EPA). The esters tested were: dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), di-¿n-hexyl, n-octyl, n-decyl¿ phthalate (610P), di-isononyl phthalate (DINP), di-¿heptyl, nonyl, undecyl¿ phthalate (711P), di-isodecyl phthalate (DIDP) and di-undecyl phthalate (DUP). Both DMP and DBP were found to produce significant increases in the mutant frequency in the mouse lymphoma assay in the presence but not in the absence of an Aroclor-induced rat liver activation system (S-9). Ester 610P gave equivocal results in the mouse lymphoma assay in the presence and absence of rat liver S-9. There was no indication of mutagenic potential for any of the other test materials in the mouse lymphoma assay, and none of the test materials increased transformation frequency in the Balb/3T3 cell transformation assay. Aldehyde metabolites of the de-esterified alcohols are postulated to play a role in the positive results for DMP and DBP. PMID:10641018

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

    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

  13. Phenotypic and genotypic characteristics of novel mouse cell line (NIH/3T3-adapted human enterovirus 71 strains (EV71:TLLm and EV71:TLLmv.

    Carla Bianca Luena Victorio

    Full Text Available Since its identification in 1969, Enterovirus 71 (EV71 has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71 is hampered by the virus's inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv, which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1 and viral RNA-dependent RNA polymerase (3D. Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136-150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

  14. 3T3细胞源外泌小体对小鼠结直肠癌细胞CT26增殖的影响%Effect of exosome extracted from 3T3 on proliferation of mouse colorectal cancer cells CT26

    朱栋良; 刘志苏; 王芳元

    2015-01-01

    目的 观察小鼠成纤维细胞株3T3来源的外泌小体(exosome)对小鼠结直肠癌细胞CT26增殖能力的影响,并探讨其机制.方法 通过PureExo exosome提取试剂盒提取3T3细胞培养基上清中的exosome,按照不同浓度及时间作用于CT26细胞,并利用细胞计数试剂盒(CCK-8)检测CT26细胞增殖能力,碘化丙锭(PI)染色法检测细胞周期改变,Western blot及实时定量反转录聚合酶链反应(RT-qPCR)检测CT26中DNA甲基转移酶1(DNMT1)及抑癌基因p16的蛋白及mRNA表达.结果 CCK-8实验中450 nm处吸光度(A)值96 h时,exosome处理组(10 mg/L:1.17±0.04,50 mg/L:1.69±0.03,200 mg/L:2.08 ±0.05),显著高于对照组(Control):0.89 ±0.03,即随着exosome处理浓度的提高,CT26细胞增殖逐渐增快;同时细胞周期进程加快,表现为处于S期及G2/M期的细胞比例增多,处于G0/G1期比例减少.而Western blot提示调控DNA甲基化的重要蛋白DNMT1明显增多,同时伴有其下游蛋白及细胞增殖、周期相关的分子如p16、p21、细胞周期素(Cyclin) D1和CDK6的水平变化,RT-qPCR也验证了上述分子mRNA水平的变化.结论 小鼠成纤维细胞株3T3来源exosome可促进小鼠结直肠癌细胞CT26增殖,加快其细胞周期进程,并且可能通过DNMT1改变其下游分子发挥上述作用.%Objective To investigate the effect of exosome secreted by 3T3 on proliferation of mouse colorectal cancer cells CT26,and explore the potential underlying mechanism.Methods The exosome was obtained and purified by PureExo exosome kit from mouse fibroblast 3T3 cells cultural supernatant,and CT26 cells were cultured and incubated with different doses of exosome for indicated time.Then cell counting kit-8 (CCK-8) assay and propidium iodide (PI) incorporation were performed to measure the cell proliferation and cell cycle of CT26.Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) were used to detect and analyze the potential

  15. The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts.

    Rousseau, Marthe; Pereira-Mouriès, Lucilia; Almeida, Maria José; Milet, Christian; Lopez, Evelyne

    2003-05-01

    Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts and to mineralize in the presence of beta-glycerophosphate and ascorbic acid. Cell proliferation and alkaline phosphatase activity were measured as markers of osteoblast differentiation, and mineralization was analyzed. These studies revealed that WSM stimulates osteoblast differentiation and mineralization by day 6 instead of the 21-day period required for cells grown in normal mineralizing media. We compared the activity of WSM with that of dexamethasone on this cell line. WSM can inhibit alkaline phosphatase (ALP) activity and the activity of dexamethasone on MC3T3-E1 cells. This study shows that nacre WSM could speed up the differentiation and mineralization of this cell line more effectively than dexamethasone. PMID:12781967

  16. Ca2+-mobilizing actions of platelet-derived growth factor differ from those of bombesin and vasopressin in Swiss 3T3 mouse cells

    Addition of the mitogenic peptides bombesin and vasopressin to quiescent Swiss 3T3 mouse cells increased the cytosolic Ca2+ concentration without any measurable delay. In contrast, there was a significant lag period (16 +/- 1.2 s) before platelet-derived growth factor (PDGF) increased cytosolic Ca2+ concentration. This lag was not diminished at high concentrations of either porcine or human PDGF. Similar results were obtained in 3T3 cells loaded with quin-2 or fura-2. The differences in the effects of bombesin, vasopressin, and PDGF on Ca2+ movements were also substantiated by measurements of 45Ca2+ efflux and of cellular 45Ca2+ content. Activation of protein kinase C by phorbol esters inhibited Ca2+ mobilization induced by either bombesin or vasopressin. In contrast, phorbol esters had no effect on PDGF-induced cytosolic Ca2+ concentration increase or acceleration of 45Ca2+ efflux. Finally, bombesin and vasopressin caused a rapid increase in the production of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate, whereas PDGF, even at a saturating concentration, exerted only a small effect. These results indicate that the signal transduction pathway activated by PDGF that lead to Ca2+ mobilization can be distinguished form those utilized by bombesin and vasopressin

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

    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. 31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

    Civan, M M; Williams, S R; Gadian, D G; Rozengurt, E

    1986-01-01

    Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event. PMID:3543375

  19. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-01-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli-type chickpeas, which are

  20. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes.

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-08-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli

  1. Using quantitative PCR to Identify Kinesin-3 Genes that are Upregulated During Growth Arrest in MouseNIH3T3 Cells

    Thorsteinsson, Rikke; Christensen, Søren Tvorup; Pedersen, Lotte Bang

    2009-01-01

    mouse NIH3T3 cells and those that might have cilia-related functions. We employed this method to specifically search for mouse kinesin-3 genes that are upregulated during growth arrest and identified three such genes (Kif13A, Kif13B, and Kif16A). In principle, however, the method can be extended to...

  2. Data from proteomic characterization of the role of Snail1 in murine mesenchymal stem cells and 3T3-L1 fibroblasts differentiation

    A. Peláez-García

    2015-09-01

    Full Text Available The transcription factor (TF Snail1 is a major inducer of the epithelial–mesenchymal transition (EMT during embryonic development and cancer progression. Ectopic expression of Snail in murine mesenchymal stem cells (mMSC abrogated their differentiation to osteoblasts or adipocytes. We used either stable isotopic metabolic labeling (SILAC for 3T3-L1 cells or isobaric labeling with tandem mass tags (TMT for mMSC stably transfected cells with Snail1 or control. We carried out a proteomic analysis on the nuclear fraction since Snail is a nuclear TF that mediates its effects mainly through the regulation of other TFs. Proteomics data have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifiers PXD001529 and PXD002157 (Vizcaino et al., 2014 [1]. Data are associated with a research article published in Molecular and Cellular Proteomics (Pelaez-Garcia et al., 2015 [2].

  3. Recombinant protein of tissue inhibitor of metaIloproteinase-3 induces apoptosis of mouse MC3T3-E1 osteoblasts

    袁凌青

    2006-01-01

    Objective To investigate the action of recombinantprotein of tissue inhibitor of metalloproteinase-3 (TIMP-3) on apoptosis of MC3T3-E1 osteoblasts. Methods Cell survival rate and apoptosis were measured by MTT and ELISA respectively. The expressions of Fas, FasL, Bel-2, Bax, caspase-3 , caspase-8, cytochrome c and phosphorylations of JNK, p38 and extracellular signalregulated kinase (ERK) 1/2 were analysed by Western blotting. Results TIMP-3 reduced survival rate of MC3T3-E1 cells and promoted apoptosis of MC3T3-E1

  4. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity.

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography-Mass Spectrometry (GC-MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC-MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  5. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography–Mass Spectrometry (GC–MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC–MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  6. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3

    Phan, Chia-Wei; David, Pamela; Naidu, Murali; Wong, Kah-Hui; Sabaratnam, Vikineswary

    2013-01-01

    Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (...

  7. Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

    Chan Shih-Hung

    2012-07-01

    Full Text Available Abstract Background Insulin receptor substrate (IRS-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels. Methods and results In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3, aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress

  8. Rho family GTP binding proteins are involved in the regulatory volume decrease process in NIH3T3 mouse fibroblasts

    Pedersen, Stine F; Beisner, Kristine H; Willumsen, Berthe M;

    2002-01-01

    kinase inhibitor Y-27632 and the phosphatidyl-inositol 3 kinase (PI3K) inhibitor wortmannin. The maximal rates of swelling-activated K+ (86 Rb+ as tracer) and taurine ([3H]taurine as tracer) efflux after a 30 % reduction in extracellular osmolarity were increased about twofold in cells with maximal Rho......AV14 expression compared to wild-type cells, but were unaffected by Y-27632. The volume set points for activation of release of both osmolytes appeared to be reduced by RhoAV14 expression. The maximal taurine efflux rate constant was potentiated by the tyrosine phosphatase inhibitor Na(3)VO(4), and...... inhibited by the tyrosine kinase inhibitor genistein. The magnitude of the swelling-activated Cl- current (I(Cl,swell) ) was higher in RhoAV14 than in wild-type cells after a 7.5 % reduction in extracellular osmolarity, but, in contrast to 86Rb+ and [3H]taurine efflux, similar in both strains after a 30...

  9. Differentially expressed genes and signalling pathways are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-b estradiol

    Zhen-Zhen Shang; Xin Li; Hui-Qiang Sun; Guo-Ning Xiao; Cun-Wei Wang; Qi Gong

    2014-01-01

    Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation. In this study, complementary DNA (cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha (a-MEM) cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with 1028 mol?L21 17-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase (ALP) activity;thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes, of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology (GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta (TGF-b)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large

  10. T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA.

    Ray, Durwood B; Merrill, Gerald A; Brenner, Frederic J; Lytle, Laurie S; Lam, Tan; McElhinney, Aaron; Anders, Joel; Rock, Tara Tauber; Lyker, Jennifer Kier; Barcus, Scott; Leslie, Kara Hust; Kramer, Jill M; Rubenstein, Eric M; Pryor Schanz, Karen; Parkhurst, Amy J; Peck, Michelle; Good, Kimberly; Granath, Kristi Lemke; Cifra, Nicole; Detweiler, Jessalee Wantz; Stevens, Laura; Albertson, Richard; Deir, Rachael; Stewart, Elisabeth; Wingard, Katherine; Richardson, Micah Rose; Blizard, Sarah B; Gillespie, Lauren E; Kriley, Charles E; Rzewnicki, Daniel I; Jones, David H

    2016-01-01

    Cancer cells often arise progressively from "normal" to "pre-cancer" to "transformed" to "local metastasis" to "metastatic disease" to "aggressive metastatic disease". Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic "punctuated evolution" (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6-10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7

  11. Roles of Na+/H+ exchange in regulation of p38 mitogen-activated protein kinase activity and cell death after chemical anoxia in NIH3T3 fibroblasts

    Rentsch, Maria L; Ossum, Carlo G; Hoffmann, Else K; Pedersen, Stine F

    2007-01-01

    , p38 mitogen-activated protein kinase (MAPK), ERK1/2, p53, and Akt activity, and cell death, after chemical anoxia in NIH3T3 fibroblasts. The NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (EIPA) (5 muM), as well as removal of extracellular Na(+) [replaced by N-methyl-D: -glucamine (NMDG......(+))], prevented recovery of intracellular pH (pH(i)) during chemical anoxia (10 mM NaN(3) +/- 10 mM glucose), indicating that activation of NHE was the dominating mechanism of pH(i) regulation under these conditions. NHE activation by chemical anoxia was unaffected by inhibitors of p38 MAPK (SB203580) and...... extracellular signal-regulated kinase (ERK) (PD98059). In contrast, chemical anoxia activated p38 MAPK in an NHE-dependent manner, while ERK1/2 activity was unaffected. Anoxia-induced cell death was caspase-3-independent, mildly attenuated by EIPA, potently exacerbated by SB203580, and unaffected by PD98059...

  12. Phosphatidylcholine induces apoptosis of 3T3-L1 adipocytes

    Li Hailan

    2011-12-01

    Full Text Available Abstract Background Phosphatidylcholine (PPC formulation is used for lipolytic injection, even though its mechanism of action is not well understood. Methods The viability of 3T3-L1 pre-adipocytes and differentiated 3T3-L1 cells was measured after treatment of PPC alone, its vehicle sodium deoxycholate (SD, and a PPC formulation. Western blot analysis was performed to examine PPC-induced signaling pathways. Results PPC, SD, and PPC formulation significantly decreased 3T3-L1 cell viability in a concentration-dependent manner. PPC alone was not cytotoxic to CCD-25Sk human fibroblasts at concentrations Conclusions PPC results in apoptosis of 3T3-L1 cells.

  13. Gene expression profile of mouse fibroblasts exposed to a biodegradable iron alloy for stents.

    Purnama, Agung; Hermawan, Hendra; Champetier, Serge; Mantovani, Diego; Couet, Jacques

    2013-11-01

    Iron-based materials could constitute an interesting option for cardiovascular biodegradable stent applications due to their superior ductility compared to their counterparts - magnesium alloys. Since the predicted degradation rate of pure iron is considered slow, manganese (35% w/w), an alloying element for iron, was explored to counteract this problem through the powder metallurgy process (Fe-35 Mn). However, manganese presents a high cytotoxic potential; thus its effect on cells must first be established. Here, we established the gene expression profile of mouse 3T3 fibroblasts exposed to Fe-35 Mn degradation products in order to better understand cell response to potentially cytotoxic degradable metallic material (DMM). Mouse 3T3 cells were exposed to degradation products eluting through tissue culture insert filter (3 μm pore size) containing cytostatic amounts of 3.25 mg ml(-1) of Fe-35 Mn powder, 0.25 mg ml(-1) of pure Mn powder or 5 mg ml(-1) of pure iron powder for 24 h. We then conducted a gene expression profiling study from these cells. Exposure of 3T3 cells to Fe-35 Mn was associated with the up-regulation of 75 genes and down-regulation of 59 genes, while 126 were up-regulated and 76 down-regulated genes in the presence of manganese. No genes were found regulated for the iron powder. When comparing the GEP of 3T3 fibroblasts in the presence of Fe-35 Mn and Mn, 68 up-regulated and 54 down-regulated genes were common. These results were confirmed by quantitative RT-PCR for a subset of these genes. This GEP study could provide clues about the mechanism behind degradation products effects on cells of the Fe-35 Mn alloy and may help in the appraisal of its potential for DMM applications. PMID:23499988

  14. Mouse white adipocytes and 3T3-L1 cells display an anomalous pattern of carnitine palmitoyltransferase (CPT) I isoform expression during differentiation. Inter-tissue and inter-species expression of CPT I and CPT II enzymes.

    Brown, N F; Hill, J K; Esser, V; Kirkland, J L; Corkey, B E; Foster, D W; McGarry, J D

    1997-01-01

    The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPT I) represents the initial and regulated step in the beta-oxidation of fatty acids. It exists in at least two isoforms, denoted L (liver) and M (muscle) types, with very different kinetic properties and sensitivities to malonyl-CoA. Here we have examined the relative expression of the CPT I isoforms in two different models of adipocyte differentiation and in a number of rat tissues. Adipocytes from mice, hamsters and humans were also evaluated. Primary monolayer cultures of undifferentiated rat preadipocytes expressed solely L-CPT I, but significant levels of M-CPT I emerged after only 3 days of differentiation in vitro; in the mature cell M-CPT I predominated. In sharp contrast, the murine 3T3-L1 preadipocyte expressed essentially exclusively L-CPT I, both in the undifferentiated state and throughout the differentiation process in vitro. This was also true of the mature mouse white fat cell. Fully developed adipocytes from the hamster and human behaved similarly to those of the rat. Thus the mouse white fat cell differs fundamentally from those of the other species examined in terms of tis choice of a key regulatory enzyme in fatty acid metabolism. In contrast, brown adipose tissue from all three rodents displayed the same isoform profiles, each expressing overwhelmingly M-CPT I. Northern blot analysis of other rat tissues established L-CPT I as the dominant isoform not only in liver but also in kidney, lung, ovary, spleen, brain, intestine and pancreatic islets. In addition to its primacy in skeletal muscle, heart and fat, M-CPT I was also found to dominate the testis. The same inter-tissue isoform pattern (with the exception of white fat) was found in the mouse. Taken together, the data bring to light an intriguing divergence between white adipocytes of the mouse and other mammalian species. They also raise a cautionary note that should be considered in the choice of animal model used

  15. Piwil2基因表达诱导NIH3T3细胞恶性转化%Malignant transformation of HIN3T3 cells induced by expression of Piwil2 genes

    李跃波; 冯定庆; 凌斌; 程敏

    2013-01-01

    目的 通过建立稳定表达外源Piwil2基因的NIH3T3细胞,初步探讨Piwil2基因对NIH3T3细胞生物学特性的调控作用.方法 通过脂质体介导的方法和G418的筛选,建立稳定表达Piwil2基因的NIH3T3细胞株,之后利用细胞集落形成实验、细胞增殖实验、细胞周期分析和裸鼠成瘤实验,观察Piwil2基因表达对NIH3T3细胞的生物学特性的影响.结果 建立了稳定过表达Piwil2基因的NIH3T3细胞株.与对照组空白载体细胞系相比,过表达Piwil2基因的NIH3T3细胞增殖和集落形成能力增强;细胞周期G0/G1期减少、S期明显升高;接种裸鼠后形成的肿瘤体积显著大于对照组.结论 Piwil2基因在NIH3T3细胞中稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能.%Objective To establish a mouse fibroblastio cell line stably transfected with Piwil2 gene, and use such cell line to investigate tumor development and progression imposed by the ectopic expression of Piwil2 gene. Methods Eukaryotic expression vector pIRES2-EGFP-Piwil2 was transfected into mouse fibroblast cell line NIH3T3 by lipofectamine. Stable transfect-ants were selected by G418. The integration and expression of Piwil2 gene were analyzed by PCR. And then cell proliferation, cycle and apoptosis in experimental analysis, colony formation and tumor growth experiment were used to observe the impact of Piwil2 gene expression of NIH3T3 on biological characteristics. Results NIH3T3 cell line with stably expression of Piwil2 gene was successfully established and confirmed. Compared with control group, the NIH3T3 cell line with high expression of Piwil2 gene growed quickly and had high capability of colony formation. The S phase and M phase increased significantly. Tumor formation in nude mice was significantly greater than in the control group. Conclusion Expression of Piwil2 gene in NIH3T3 cells can induce malignant transformation of mouse fibroblastic cells both in vitro and in

  16. Effects of chemical anoxia on NHE1, p38 MAPK, p53, Akt and ERM proteins in NIH3T3 fibroblasts: evidence for a role of NHE1 upstream of p38 MAPK

    Rentsch, M. L.; Hoffmann, E. K.; Pedersen, Stine Helene Falsig

    Activation of the plasma membrane Na+/H+ exchanger NHE1 contributes importantly to ischemic/anoxic cell damage, yet the mechanisms involved are unclear. In NIH3T3 cells, PCR studies confirmed the expression of NHE1 and -8, yet not NHE2, -3, and -4. Chemical anoxia (10 mM azide, 10 min) was...... associated with a decrease in pHi which was exacerbated by the NHE1 inhibitor EIPA (5 µM). Reperfusion (azide washout) elicited a rapid, EIPA-sensitive alkalinization to 7.60 ± 0.057 (n=6), compared to a starting pHi of 7.49 ± 0.032 (n=6). Cell survival was reduced by prolonged chemical anoxia (to 87% at 3 h...... and 41% at 24 h, MTT assay), an effect counteracted by EIPA at early ( 6 h) time points. Chemical anoxia was furthermore associated with: (i) a rapid ( 10 min) and transient phosphorylation of p38 MAPK, which was abolished by NHE1 inhibitors (EIPA, cariporide, 5 µM); (ii) increased phosphorylation of...

  17. Vasonatrin peptide promotes the synthesis of adiponectin in 3T3-L1 adipocytes of mouse and the underlying mechanism%血管钠肽促进小鼠3T3-L1脂肪细胞合成脂联素及其可能机制

    铁茹; 邢文娟; 陈小丽; 金坚; 张海锋; 于军; 陈宝莹

    2012-01-01

    目的 探讨血管钠肽(VNP)对脂肪因子脂联素生成的影响及其机制.方法 在3T3-L1细胞分化的脂肪细胞中加入不同浓度的VNP,分别用实时定量PCR法和Western blot法检测脂联素的mRNA水平和蛋白表达,放免法测定细胞内cGMP的水平.结果 VNP可显著增加脂联素mRNA水平和蛋白表达,同时提高细胞内cGMP,含量为(38±5)~(265±35)nmol/L,显著高于对照组的(10±2)nmol/L(P<0.01);该效应可用8-Br-cGMP诱导,可被cGMP依赖性蛋白激酶抑制剂KT-5823或钠尿肽受体NPR阻断剂HS-142-1抑制.结论VNP可通过NPR/cGMP/PKG信号通路增加脂肪细胞脂联素的表达.%Objective To identify the roles of vasonatrin peptide (VNP) on adiponectin production and the underlying mechanisms. Methods 3T3-L1 cells were differentiated into adipocytes and exposed to various concentrations of VNP. Quantitative PCR and immunoassays were performed to determine the mRNA levels of adiponectin. Involved signaling pathway was identified by radioimmunoassay to detect the levels of intracellular cGMP[ (38+5) ~ (265 ± 35)nmol/L]. Results VNP markedly enhanced adiponectin mRNA expression as well as protein secretion. In addition, VNP significantly enhanced the intracellular level of cGMP. The effects of VNP were mimicked by 8-Br-cGMP, whereas inhibited by HS-142-1 or KT-5823. Conclusions VNP regulates adiponectin production in adipocytes via a guanylyl cyclase-coupled NPR/cGMP/PKG pathway.

  18. 鼠PVRL-2慢病毒载体的构建及其在3T3-L1细胞中的表达%Potential role of mouse PVRL-2 gene in the fatty acid metabolism

    马静; 刘晓萌; 张传海; 郑宗基; 赵倩伟; 杨鸣琦; 张雷

    2013-01-01

    Excess fat and cholesterol in food such as meat,eggs or milk could lead to hyperlipoidemia in human.Currently,to explore genes expression and their mechanisms associated with lipid metabolism has been a major focus in veterinary science.Growing bodies of evidence indicated that molecular functions of fatty acid metabolism related genes such as ApoE,ApoC1 and Tomm40 were very well characterized; however,function of their chromosomal neighbor such as PVRL-2 gene in the fatty acid metabolism remains unclear.Present study was aim to investigate potential role of mouse PVRL-2 gene in regulation of fatty acid related gene expression using preadipogenic 3T3-L1 cells.The cells were infected by Lentiviral particles which was produced by lentiviral plasmid containing Pvrl2 gene,and RNA were extracted 48h post viral infection.Quantitative real-time PCR analysis confirmed that PVRL-2 overexpressed more than 100 folds upon PVRL-2 virus transformation compared to the control.Notably,the expression of PPARα gene which is a key player in the fatty acid oxidation was strongly induced (4.5 fold increase) post PVRL-2 viral infection,but not other genes that related to the fatty acid metabolism such as CPT1A,FASN,COX7A,PGC1B,ASADM showed similar changes.Furthermore,bioinformatics analyses revealed that Nectin-2,coded by PVRL-2,should be a transmembrane protein with a signal peptide.In conclusion,the present study demonstrated that overexpression of PVRL-2 induce the expression of PPARα,which highlight the potential roles of PVRL-2 gene in fatty acid metabolism.Future studies are needed to determine detailed molecular function of PVRL-2 gene in fatty acid metabolism.%过多的脂肪和胆固醇随着肉蛋奶被人体摄入是导致人类高血脂等各种疾病诱发的原因之一,而探索脂代谢通路相关基因的表达变化及其调控机制已经成为分子生物学技术在兽医学领域中的研究热点.与高血脂有关的ApoE、ApoC1和Tomm40等基因研究较多,

  19. Irradiated Human Dermal Fibroblasts Are as Efficient as Mouse Fibroblasts as a Feeder Layer to Improve Human Epidermal Cell Culture Lifespan

    Lucie Germain

    2013-02-01

    Full Text Available A fibroblast feeder layer is currently the best option for large scale expansion of autologous skin keratinocytes that are to be used for the treatment of severely burned patients. In a clinical context, using a human rather than a mouse feeder layer is desirable to reduce the risk of introducing animal antigens and unknown viruses. This study was designed to evaluate if irradiated human fibroblasts can be used in keratinocyte cultures without affecting their morphological and physiological properties. Keratinocytes were grown either with or without a feeder layer in serum-containing medium. Our results showed that keratinocytes grown either on an irradiated human feeder layer or irradiated 3T3 cells (i3T3 can be cultured for a comparable number of passages. The average epithelial cell size and morphology were also similar. On the other hand, keratinocytes grown without a feeder layer showed heavily bloated cells at early passages and stop proliferating after only a few passages. On the molecular aspect, the expression level of the transcription factor Sp1, a useful marker of keratinocytes lifespan, was maintained and stabilized for a high number of passages in keratinocytes grown with feeder layers whereas Sp1 expression dropped quickly without a feeder layer. Furthermore, gene profiling on microarrays identified potential target genes whose expression is differentially regulated in the absence or presence of an i3T3 feeder layer and which may contribute at preserving the growth characteristics of these cells. Irradiated human dermal fibroblasts therefore provide a good human feeder layer for an effective expansion of keratinocytes in vitro that are to be used for clinical purposes.

  20. Regulation of taurine homeostasis by protein kinase CK2 in mouse fibroblasts

    Hansen, Daniel Bloch; Guerra, Barbara; Jacobsen, Jack Hummeland;

    2011-01-01

    Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume is a...... critical step in cell proliferation, differentiation and induction of apoptosis. In the present study, we use mouse NIH3T3 fibroblasts and Ehrlich Lettré ascites tumour cells with different CK2 expression levels. Taurine uptake via the Na(+) dependent transporter TauT and taurine release are increased and...... reduced, respectively, following pharmacological CK2 inhibition. The effect of CK2 inhibition on TauT involves modulation of transport kinetics, whereas the effect on the taurine release pathway involves reduction in the open-probability of the efflux pathway. Stimulation of PLA(2) activity, exposure to...

  1. Regulation of lipoprotein lipase synthesis in 3T3-L1 adipocytes by interleukin-1

    When fully differentiated 3T3-L1 fatty fibroblasts were exposed to purified, recombinant murine interleukin-1, a dose dependent suppression of lipoprotein lipase activity was observed. The loss of activity reached a maximum of 60-70% of control and appeared to be due to a specific effect on the synthesis of the enzyme as judged by a suppression of the ability to incorporate [35S]methionine into immunoprecipitable lipoprotein lipase. There was no general effect on protein synthesis as determined by radiolabel incorporated into acid precipitable protein, however, after a 17 h exposure of the 3T3-L1 cells to interleukin-1, the synthesis of two proteins (molecular weights, 19,400 and 165,000 daltons) was enhanced several fold. The observed effects on protein synthesis in the adipocytes occur at a concentration of interleukin-1 which is similar to the concentration necessary for the stimulation of [3H]thymidine incorporation into mouse thymocyte DNA. The present study represents the first unequivocal report of the ability of interleukin-1 to regulate protein synthesis in intact cells, specifically adipocytes. Moreover, their results demonstrate the ability of interleukin-1 to regulate metabolism by controlling the synthesis of specific proteins

  2. Verapamil inhibits 3T3-L1 preadipocyte differentiation

    Nan Gu; Shi Liu; Xirong Guo; Li Fei; Xiaoqin Pan; Mei Guo; Ronghua Chen

    2009-01-01

    Objective: To investigate the effect of the calcium channel blocker verapamil on adipocyte differentiation and its mechanism of action. Methods: Preadipocytes from 3T3-L1 strain mouse embryos were cultured and differentiated into matured adipocytes in vitro. Verapamil was added to the culture medium in the concentration of 30 μmol/L on Day 0. Cell differentiation was determined by Oil Red O staining and marker gene mRNA expression was evaluated and compared by RT-PCR. The fluo-3/AM probe and laser scanning confocal microscopy were used to measure intracellular calcium concentrations. Results: ①The differentiation rate of 3T3-L1 preadipocytes exposed to verapamil was lower than that of untreated cells. ②Verapamil promoted the retention of pref-1 gene expression. Lipoprotein lipase expression in the verapamil group was significantly lower than that in the control group on Day 4, Day 6 and Day 8 (P 0.05). Conclusion: In 3T3-L1 preadipocytes verapamil significantly reduced adipocyte differentiation, down-regulated the mRNA expression of three marker genes for adipocytes differentiation, and prolonged the mRNA expression of an inhibitor of differentiation. The inhibitory effect of verapamil on differentiation may involve its role as a blocker of calcium influx in adipocytes.

  3. Anti-adipogenic effect of mulberry leaf ethanol extract in 3T3-L1 adipocytes

    Yang, Soo Jin; Park, Na-Young; LIM, YUNSOOK

    2014-01-01

    BACKGROUND/OBJECTIVES Adipogenesis is part of the cell differentiation process in which undifferentiated fibroblasts (pre-adipocytes) become mature adipocytes with the accumulation of lipid droplets and subsequent cell morphological changes. Several transcription factors and food components have been suggested to be involved in adipogenesis. The aim of this study was to determine whether mulberry leaf ethanol extract (MLEE) affects adipogenesis in 3T3-L1 adipocytes. MATERIALS/METHODS The 3T3-...

  4. Defining the identity of mouse embryonic dermal fibroblasts.

    Budnick, Isadore; Hamburg-Shields, Emily; Chen, Demeng; Torre, Eduardo; Jarrell, Andrew; Akhtar-Zaidi, Batool; Cordovan, Olivia; Spitale, Rob C; Scacheri, Peter; Atit, Radhika P

    2016-08-01

    Embryonic dermal fibroblasts in the skin have the exceptional ability to initiate hair follicle morphogenesis and contribute to scarless wound healing. Activation of the Wnt signaling pathway is critical for dermal fibroblast fate selection and hair follicle induction. In humans, mutations in Wnt pathway components and target genes lead to congenital focal dermal hypoplasias with diminished hair. The gene expression signature of embryonic dermal fibroblasts during differentiation and its dependence on Wnt signaling is unknown. Here we applied Shannon entropy analysis to identify the gene expression signature of mouse embryonic dermal fibroblasts. We used available human DNase-seq and histone modification ChiP-seq data on various cell-types to demonstrate that genes in the fibroblast cell identity signature can be epigenetically repressed in other cell-types. We found a subset of the signature genes whose expression is dependent on Wnt/β-catenin activity in vivo. With our approach, we have defined and validated a statistically derived gene expression signature that may mediate dermal fibroblast identity and function in development and disease. genesis 54:415-430, 2016. © 2016 Wiley Periodicals, Inc. PMID:27265328

  5. Lipid Droplets Characterization in Adipocyte Differentiated 3T3-L1 Cells: Size and Optical Density Distribution

    V. Rizzatti; F. Boschi; Pedrotti, M.; E. Zoico; A. Sbarbati; Zamboni, M.

    2013-01-01

    The 3T3-L1 cell line, derived from 3T3 cells, is widely used in biological research on adipose tissue. 3T3-L1 cells have a fibroblast-like morphology, but, under appropriate conditions, they differentiate into an adipocyte-like phenotype. During the differentiation process, 3T3-L1 cells increase the synthesis of triglycerides and acquire the behavior of adipose cells. In particular, triglycerides accumulate in lipid droplets (LDs) embedded in the cytoplasm. The number and the size distributio...

  6. Control of insulin receptor level in 3T3 cells: effect of insulin-induced down-regulation and dexamethasone-induced up-regulation on rate of receptor inactivation.

    Knutson, V P; Ronnett, G V; Lane, M D

    1982-01-01

    Chronic exposure of 3T3 mouse fibroblasts to insulin or to the glucocorticoid dexamethasone induces down-regulation and up-regulation, respectively, of cell-surface and total cellular insulin binding capacity. Both processes are reversed upon withdrawal of the inducer. Scatchard analysis of insulin binding for receptors in the down- and up-regulated states indicates that the changes in binding capacity result primarily from alterations in insulin receptor level. That these alterations in tota...

  7. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    Hakim-Weber, Robab; Krogsdam, Anne-M; Jørgensen, Claus;

    2011-01-01

    Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate...... this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene...... experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of...

  8. Transcriptional profiling of immortalized and K-ras-transformed mouse fibroblasts upon PKA stimulation by forskolin in low glucose availability.

    Chiaradonna, Ferdinando; Pirola, Yuri; Ricciardiello, Francesca; Palorini, Roberta

    2016-09-01

    Forskolin (FSK) induces activation of protein kinase A (PKA). This activation protects specifically some cancer cells from death induced by glucose starvation. Cell effects upon FSK treatment prompted us to investigate in detail the physiological role of PKA in the activation of pro-survival mechanisms in glucose starvation. In this regard we performed a microarray analysis of normal NIH3T3 and transformed NIH3T3-K-ras mouse fibroblasts cultured at 1 mM glucose and daily treated or not with 10 μM FSK until 72 h of growth, when the samples were collected. The microarray is deposited into Gene Expression Omnibus under Series GSE68266. The microarray data revealed that the activation of PKA regulates the expression of genes involved in metabolic, stress-response and pro-survival processes, like glutamine metabolism, autophagy and unfolded protein response, preventing cancer cell death in glucose starvation. Altogether these findings suggest that PKA activation, by inducing a complex transcriptional program, leads to cancer survival in nutrient stress, a typical feature of developing tumor. These transcriptional data, identifying this important role of PKA, will be useful to identify novel target in cancer therapy. PMID:27486565

  9. RNase-L regulates the stability of mitochondrial DNA-encoded mRNAs in mouse embryo fibroblasts

    Accelerated decrease in the levels of mitochondrial DNA-encoded mRNA (mt-mRNA) occurs in neuronal cells exposed either to the excitatory amino acid, glutamate or to the sodium ionophore, monensin, suggesting a role of mitochondrial RNase(s) on the stability of mt-mRNAs. Here we report that in mouse embryo fibroblasts that are devoid of the interferon-regulated RNase, RNase-L, the monensin-induced decrease in the half-life of mt-mRNA was reduced. In monensin (250 nM)-treated RNase-L+/+ cells the average half-life of mt-mRNA, determined after termination of transcription with actinomycin D, was found to be 3 h, whereas in monensin-treated RNase-L-/- cells the half-life of mt-mRNA was >6 h. In contrast, the stability of nuclear DNA-encoded β-actin mRNA was unaffected. Induction of RNase-L expression in mouse 3T3 fibroblasts further decreased the monensin-induced reduction in mt-mRNA half-life to 1.5 h. The results indicate that the RNase-L-dependent decrease in mtDNA-encoded mRNA transcript levels occurs through a decrease in the half-life of mt-mRNA, and that RNase-L may play a role in the stability of mt-mRNA

  10. Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

    Nicken, Petra; Empl, Michael T; Gerhard, Daniel; Hausmann, Julia; Steinberg, Pablo

    2016-09-01

    High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development. PMID:27427305

  11. Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

    Battey, J F; Way, J M; Corjay, M H; Shapira, H; Kusano, K; Harkins, R.; Wu, J M; Slattery, T; Mann, E.; Feldman, R I

    1991-01-01

    The mammalian bombesin-like peptides gastrin-releasing peptide (GRP) and neuromedin B regulate numerous and varied cell physiologic processes in various cell types and have also been implicated as autocrine growth factors influencing the pathogenesis and progression of human small cell lung carcinomas. We report here the molecular characterization of the bombesin/GRP receptor. Structural analysis of cDNA clones isolated from Swiss 3T3 murine embryonal fibroblasts shows that the GRP receptor i...

  12. Culture of IRM-2 mouse embryonic fibroblasts and biological character

    Objective: To isolate, cultivate the primary mouse embryonic fibroblasts (MEFs)derived from IRM-2 mice and identify their inherent radioresistance character. Methods: MEFs were isolated.MEFs cultured were passed on from generation to generation. MEFs of passage 3 and 5 were assessed by cell morphology, growth curves and cell cycles. Results: Primary MEFs were isolated from 14 to 16 day pregnant IRM-2 mice. MEFs in vitro were a kind of adherent cells with good growth ability prior to passage 6. MEFs of passage 3 and 5 reached logarithmic phase from the third day and peaked at the sixth day. MEFs of passage 3 and 5 were of good ability for proliferation, whose proliferation index were 53% and 46% respectively. Conclusion: MEFs are successfully isolated and cultivated from IRM-2 mice, which will lay a foundation for studying their radioresistance mechanism in further research. (authors)

  13. Human ovarian neoplasm cell CD147 stimulates production and activation of matrix metalloproteinases in co-cultures with mouse fibroblasts

    YANG Hong; ZOU Wei; XIN Xiao-yan

    2005-01-01

    Objective: To investigate the expression of CD147 on human ovarian neoplasm cell lines and its influence on production and activation of matrix metallproteinases(MMPs). Methods: The expression of CD147 on different human ovarian neoplasm cell lines was studied by western blotting. Co-culture was carried out to investigate the stimulative effect of the positive expression CD147 cell HO-8910 on the production of MMPs of fibroblast cell in vitro. Zymography and immune blotting were used to study the production and activity of positive MMPs, at the time, to explore the relation between CD147 and MMPs. Results: CD147 was positively presented in 2 ovarian neoplasm cell lines(HO-8910,3-AO), but in SKOV3, TC-1,NIN3T3 cell was negative. MMP-2 and MMP-9 were detected by HO-8910 cell line, mouse fibroblast cell and co-culture cells; but the expression in co-culture cell is obviously higher than individual cultures of each type alone.CD147 stimulated MMPs in dose-dependent manner. Conclusion: CD147 causes increased production and activation of MMP-2, MMP-9.CD147 is probably a indirect marker of some ovarian cancer cells with invasion and metastasis.

  14. Antiproliferative activity of flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the MCF7, KB, and NIH/3T3 cell lines.

    Nedel, Fernanda; Begnini, Karine; Carvalho, Pedro Henrique de Azambuja; Lund, Rafael Guerra; Beira, Fátima T A; Del Pino, Francisco Augusto B

    2012-11-01

    This study assessed the antiproliferative effect in vitro of the flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the human breast adenocarcinoma (MCF-7), human mouth epidermal carcinoma (KB), and mouse embryonic fibroblast (NIH 3T3) cell lines, using sulforhodamine B (SRB) assay. A cell density of 2×10(4)/well was seeded in 96-well plates, and samples at different concentrations ranging from 10 to 500 mg/mL were tested. The optical density was determined in an ELISA multiplate reader (Thermo Plate TP-Reader). Results demonstrated that the hexane extract presented antiproliferative activity against both the tumor cell lines KB and MCF-7, presenting a GI(50) (MCF-7=13.09 mg/mL), TGI (KB=37.76 mg/mL), and IL(50) (KB=291.07 mg/mL). Also, the hexane extract presented antiproliferative activity toward NIH 3T3 cells GI(50) (183.65 mg/mL), TGI (280.54 mg/mL), and IL(50) (384.59 mg/mL). The results indicate that the flower hexane extract obtained from M. spicata associated with M. rotundifolia presents an antineoplastic activity against KB and MCF-7, although an antiproliferative effect at a high concentration of the extract was observed toward NIH 3T3. PMID:23066647

  15. Expression of the Saccharomyces cerevisiae glycoprotein invertase in mouse fibroblasts: glycosylation, secretion, and enzymatic activity

    Oligosaccharide processing is controlled by host- and protein-dependent factors. To increase our understanding of the relative contribution of those factors the authors studied the glycosylation of yeast invertase expressed in a heterologous system. Invertase synthesized in psi-2 cells (an NIH 3T3-derived packaging line) is secreted efficiently, enzymatically active, and heavily glycosylated. It was estimated that the protein contains 8 or 9 carbohydrate chains. Two classes can be observed, of an approximate size of 100-110 kDa and 115-130 kDa, respectively. The size differences are due to differences in glycosylation. The smaller class contains two high-mannose carbohydrate chains; the remainder is of the complex type, sialylated and most likely tri- or tetraantennary. This profile parallels the situation observed with invertase glycosylation in yeast, where 2 of 9 or 10 chains remain unprocessed. The larger size class of invertase expressed in mouse fibroblasts has a different profile, since it contains probably only complex-type glycans. There are no apparent differences, however, in the size of the protein backbone between the two size classes. When invertase is synthesized in the presence of the mannosidase inhibitor 1-deoxymannojirimycin, processing is blocked completely. The glucosidase inhibitor 1-deoxynojirimycin does not inhibit processing completely. The glycosylation inhibitor tunicamycin prevents secretion of invertase completely when cells are cultured at 370C. At 260C, however, nonglycosylated invertase can be detected in the medium. These data suggest that glycosylation of invertase seems to be essential for the early steps of the secretory pathway but is less critical for later events

  16. Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

    Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation

  17. Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

    Tahereh Talaei-Khozani; Fatemeh Heidari; Tahereh Esmaeilpour; Zahra Vojdani; Zohrah Mostafavi-Pour; Leili Rohani

    2014-01-01

    Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA) and 5-Aza-2-Deoxycytidine (5-aza-dC). The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21...

  18. Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition

    Kedar, Padmini S.; Stefanick, Donna F.; Horton, Julie K.; Wilson, Samuel H.

    2008-01-01

    Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR (Horton et al. (2005) J. Biol. Chem., 280, 15773-15785). Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipit...

  19. Topiramate effects lipolysis in 3T3-L1 adipocytes

    MARTINS, GABRIELA POLTRONIERI CAMPAGNARO; de Souza, Camila Oliveira; MARQUES, SCHEROLIN; LUCIANO, THAIS FERNANDES; DA SILVA PIERI, BRUNO LUIZ; Rosa, José César; da Silva, Adelino Sanchez Ramos; PAULI, JOSÉ RODRIGO; Cintra, Dennys Esper; Ropelle, Eduardo Rochete; Rodrigues, Bruno; DE LIRA, FABIO SANTOS; Souza, Claudio Teodoro de

    2015-01-01

    Studies have shown that topiramate (TPM)-induced weight loss can be dependent on the central nervous system (CNS). However, the direct action of TPM on adipose tissue has not been tested previously. Thus, the present study aimed to examine whether TPM modulates lipolysis in 3T3-L1. The 3T3-L1 cells were incubated in 50 µM TPM for 30 min. The β-adrenergic stimulator, isoproterenol, was used as a positive control. The release of lactate dehydrogenase, non-esterified fatty acid, glycerol and inc...

  20. Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

    Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

    2016-12-01

    Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. PMID:27612743

  1. Mouse embryonic fibroblasts exhibit extensive developmental and phenotypic diversity

    Singhal, Prabhat K.; Sassi, Slim; Lan, Lan; Au, Patrick; Halvorsen, Stefan C.; Fukumura, Dai; Jain, Rakesh K.; Seed, Brian

    2016-01-01

    Analysis of embryonic fibroblasts from GFP reporter mice indicates that the fibroblast cell type harbors a large collection of developmentally and phenotypically heterogeneous subtypes. Some of these cells exhibit multipotency, whereas others do not. Multiparameter flow cytometry analysis shows that a large number of distinct populations of fibroblast-like cells can be found in cultures initiated from different embryonic organs, and cells sorted according to their surface phenotype typically retain their characteristics on continued propagation in culture. Similarly, surface phenotypes of individual cloned fibroblast-like cells exhibit significant variation. The fibroblast cell class appears to contain a very large number of denumerable subtypes. PMID:26699463

  2. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    Hansen Jacob B

    2011-05-01

    Full Text Available Abstract Background Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs, and retinoblastoma gene-deficient MEFs (Rb-/- MEFs. Findings Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1. To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. Conclusion In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte

  3. Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

    Friesel, R; Burgess, W H; Maciag, T

    1989-01-01

    Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130...

  4. Dioxin induces genomic instability in mouse embryonic fibroblasts.

    Merja Korkalainen

    Full Text Available Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI, i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD. Mouse embryonic fibroblasts (C3H10T1/2 were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days. For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay, was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

  5. Instability of endogenous MRP/proliferin transcripts in the nucleus of mouse embryo fibroblasts contrasts with their stability when produced during transient transfections.

    Malyankar, U M; Rittling, S R; Denhardt, D T

    1996-02-01

    The mitogen regulated protein/proliferin (MRP/PLF) gene is transcribed in primary mouse embryo fibroblasts (MEFs), but the pre-mRNA is not properly converted into a stable cytoplasmic mRNA and instead is rapidly degraded, apparently in the nucleus [Malyankar et al. (1994): Proc Natl Acad Sci USA 91:335-359]. In 3T3 cells derived from the MEFs by the standard 3T3 immortalization protocol, stable MRP/PLF mRNA is produced. We show here that the processing of intron sequences is similar in the two cell types and that some of the MRP/PLF transcripts are polyadenylated in the MEFs. We also document the production of stable MRP/PLF mRNA generated by transcription of various plasmid constructs containing different portions of the MRP/PLF3 gene after calcium phosphate-mediated transfection into the MEFs. We conclude that the inability of the MRP/PLF mRNA to accumulate in the MEFs is unlikely to result solely from a single localized sequence in the primary transcript (or the mRNA) that causes it to be subject to rapid breakdown; possibly export of the mRNA from the MEF nucleus is defective or some aspect of the transcriptional process marks the transcript for degradation. PMID:8655630

  6. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  7. Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms

  8. Atomic force microscopy of 3T3 and SW-13 cell lines: An investigation of cell elasticity changes due to fixation

    Mechanical properties of single cells are of increasing interest both from a fundamental cell biological perspective and in the context of disease diagnostics. In this respect, atomic force microscopy (AFM) has become a powerful tool for imaging and assessing mechanical properties of biological samples. However, while these tests are typically carried out on chemically fixed cells, the most important data is that on living cells. The present study applies AFM technique to assess the Young's modulus of two cell lines: mouse embryonic fibroblasts (NIH/3T3) and human epithelial cancer cells (SW-13). Both living cells and those fixed with paraformaldehyde were investigated. This analysis quantifies the difference between Young's modulus for these two conditions and provides a coefficient to relate them. Knowing the relation between Young's modulus of living and fixed cells, allows carrying out and comparing data obtained during steady-state measurements on fixed cells that are more frequently available in the clinical and research settings and simpler to maintain and probe. - Highlights: • The influence of fixation process on NIH/3T3 and SW13 cell elasticity was studied. • The two cell lines have been chosen for their different cytoskeletal structures. • There is a difference between AFM data collected at 37 °C and room temperature. • At RT, the longer the time out of the incubator the softer the cells appear. • A coefficient to relate elasticity between fixed and leaving cells is provided

  9. Matrine inhibits proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB

    WU Yan-an; GAO Chun-fang; WANG Hao; HUANG Chao; KONG Xian-tao

    2001-01-01

    To study the effect of matrine on proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB (PDGF-BB). Methods: Mouse skin fibroblasts were obtained from newborn ⅠCR mice and propagated in vitro. Proliferation of cell was analyzed by mitochondrial reduction of tetrazolium salt MTT and actual cell count. Results: Matrine (50 to 500 μg/ml) caused dose-dependent reduction of serum-stimulated cell growth. Growth inhibition was totally reversed after removal of the drug. Matrine also inhibited PDGF-BB induced cell growth dose-dependently. Conclusion: Matrine exhibits potent anti-proliferation effect on mouse skin fibroblast. This effect appears to be mediated by decrease of PDGF-induced growth. These results suggest that matrine might have preventive and therapeutic implication in skin fibrosis.

  10. Alteration of glycolipids in ras-transfected NIH 3T3 cells

    Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. The authors analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, they found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by 125I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1 as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, they found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. These results indicated that ras oncogenes derived form human tumors are capable of inducing alterations in glycolipid composition

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

    Lai, Jing [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); Liu, Gexiu [Institute of Hematology, School of Medicine, Jinan University, Guangzhou, Guangdong (China); Yan, Guoyao [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); He, Dongmei [Institute of Hematology, School of Medicine, Jinan University, Guangzhou, Guangdong (China); Zhou, Ying [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); Chen, Shengting, E-mail: shengtingchen@sina.cn [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China)

    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.

  12. Lipid droplets characterization in adipocyte differentiated 3T3-L1 cells: size and optical density distribution

    V. Rizzatti

    2013-08-01

    Full Text Available The 3T3-L1 cell line, derived from 3T3 cells, is widely used in biological research on adipose tissue. 3T3-L1 cells have a fibroblast-like morphology, but, under appropriate conditions, they differentiate into an adipocyte-like phenotype. During the differentiation process, 3T3-L1 cells increase the synthesis of triglycerides and acquire the behavior of adipose cells. In particular, triglycerides accumulate in lipid droplets (LDs embedded in the cytoplasm. The number and the size distribution of the LDs is often correlated with obesity and many other pathologies linked with fat accumulation. The integrated optical density (IOD of the LDs is related with the amount of triglycerides in the droplets. The aim of this study is the attempt to characterize the size distribution and the IOD of the LDs in 3T3-L1 differentiated cells. The cells were differentiated into adipocytes for 5 days with a standard procedure, stained with Oil Red O and observed with an optical microscope. The diameter, area, optical density of the LDs were measured. We found an asymmetry of the kernel density distribution of the maximum Feret’s diameter of the LDs with a tail due to very large LDs. More information regarding the birth of the LDs could help in finding the best mathematical model in order to analyze fat accumulation in adipocytes.

  13. Cannabidiol promotes browning in 3T3-L1 adipocytes.

    Parray, Hilal Ahmad; Yun, Jong Won

    2016-05-01

    Recruitment of the brown-like phenotype in white adipocytes (browning) and activation of existing brown adipocytes are currently being investigated as a means to combat obesity. Thus, a wide variety of dietary agents that contribute to browning of white adipocytes have been identified. The present study was designed to investigate the effects of cannabidiol (CBD), a major nonpsychotropic phytocannabinoid of Cannabis sativa, on induction of browning in 3T3-L1 adipocytes. CBD enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cited1, Tmem26, Prdm16, Cidea, Tbx1, Fgf21, and Pgc-1α) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of PPARγ and PI3K. In addition, CBD increased protein expression levels of CPT1, ACSL, SIRT1, and PLIN while down-regulating JNK2, SREBP1, and LPL. These data suggest possible roles for CBD in browning of white adipocytes, augmentation of lipolysis, thermogenesis, and reduction of lipogenesis. In conclusion, the current data suggest that CBD plays dual modulatory roles in the form of inducing the brown-like phenotype as well as promoting lipid metabolism. Thus, CBD may be explored as a potentially promising therapeutic agent for the prevention of obesity. PMID:27067870

  14. Generation of Stable Pluripotent Stem Cells From NOD Mouse Tail-Tip Fibroblasts

    Liu, Jun; Ashton, Michelle P.; Sumer, Huseyin; O’Bryan, Moira K.; Brodnicki, Thomas C.; Verma, Paul J.

    2011-01-01

    OBJECTIVE The NOD mouse strain has been widely used to investigate the pathology and genetic susceptibility for type 1 diabetes. Induced pluripotent stem cells (iPSCs) derived from this unique mouse strain would enable new strategies for investigating type 1 diabetes pathogenesis and potential therapeutic targets. The objective of this study was to determine whether somatic fibroblasts from NOD mice could be reprogrammed to become iPSCs, providing an alternative source of stem cells for the p...

  15. Antagonistic effects of a covalently dimerized insulin derivative on insulin receptors in 3T3-L1 adipocytes.

    Weiland, M; Brandenburg, C; Brandenburg, D.; Joost, H. G.

    1990-01-01

    In the present study we describe the antagonistic effects of the covalently dimerized insulin derivative B29,B29'-suberoyl-insulin on insulin receptors in 3T3-L1 mouse cells. In differentiated 3T3-L1 adipocytes, the derivative fully inhibits binding of 125I-labeled insulin to its receptor with about the same affinity as unlabeled insulin. In contrast, the dimerized derivative only partially (approximately 20%) mimics insulin's effects on glucose transport and DNA synthesis in the absence of i...

  16. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells

    Zachary, I.; Gil, J.; Lehmann, W.; Sinnett-Smith, J.; Rozengurt, E. (Imperial Cancer Research Fund, London (England))

    1991-06-01

    The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of M{sub r} 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of {sup 32}P{sub i}-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C{sub {gamma}}, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.

  17. Protein kinase A suppresses the differentiation of 3T3-L1 preadipocytes

    Fuqiang Li; Dongmei Wang; Yiran Zhou; Bo Zhou; Yanan Yang; Hehua Chen; Jianguo Song

    2008-01-01

    cAMP and protein kinase A (PKA) are widely known as signaling molecules that are important for the induction of adipogenesis. Here we show that a strong increase in the amount of cAMP inhibits the adipogenesis of 3T3-L1 fibroblast cells. Stimulation of PKA activity suppresses adipogenesis and, in contrast, inhibition of PKA activity markedly accelerates the adipogenic process. As adipogenesis progresses, there is a significant increase in the expression level of PKA regulatory subunits and a corresponding decrease in PKA activity. Moreover, treatment of 3T3-L1 cells with epidermal growth factor (EGF) stimulates PKA activity and blocks adipogenesis. Inhibition of PKA activity abolishes this suppressive effect of EGF on adipogenesis. Moreover, activation of PKA induces serine/threonine phosphorylation, reduces tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and the association between PKA and IRS-1. Taken together, our study demonstrates that PKA has a pivotal role in the suppression of adipogenesis. cAMP at high concentrations can suppress adipogenesis through PKA activation. These findings could be important and useful for understanding the mechanisms of adipogenesis and the relevant physiological events.

  18. In vitro studies of the diabetic condition using cultured fibroblasts with focus on wound healing

    Hehenberger, Karin M.

    1997-01-01

    This thesis focuses on the diabetic condition at the cellular level, and how thismay lead to late complications. Defect wound healing in diabetic patients is poorlyunderstood, but impaired granulation is observed clinically. We have therefore decidedto study an in vltro system using cultured fibroblasts. These were derived from biopsiesfrom human diabetic and non-diabetic wounds and uninjured skin, Goto-Kakazaki ratsand Wistar rats. In addition Swiss 3T3 mouse fibroblasts we...

  19. The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development.

    Wetzels, A M; Punt-Van der Zalm, A P; Bastiaans, B A; Janssen, B A; Goverde, H J; Rolland, R

    1992-07-01

    A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified. PMID:1500485

  20. Molecular mechanism of 9-cis-retinoic acid inhibition of adipogenesis in 3T3-L1 cells

    Sagara, Chiaki; Takahashi, Katsuhiko [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan); Kagechika, Hiroyuki [School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo 101-0062 (Japan); Takahashi, Noriko, E-mail: t-noriko@hoshi.ac.jp [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)

    2013-03-29

    Highlights: ► We examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1. ► 9-cis-RA inhibited lipid accumulation in adipogenetically-induced 3T3-L1 cells. ► A RXR pan-antagonist suppressed the inhibitory effects of 9-cis-RA on adipogenesis. ► This antagonist had no effects on RXRα and PPARγ levels in 9-cis-RA-treated cells. ► 9-cis-RA-induced decrease in both RXRα and PPARγ was independent of RXR activation. -- Abstract: Retinoic acid (RA) signaling is mediated by specific nuclear hormone receptors. Here we examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1 cells. 9-cis-RA inhibits the lipid accumulation of adipogenetically induced 3T3-L1 cells. The complex of retinoid X receptor α (RXRα) with peroxisome proliferator-activated receptor γ (PPARγ) is a major transcription factor in the process of adipogenesis, and the levels of these molecules were decreased by 9-cis-RA treatment. A RXR pan-antagonist suppressed 9-cis-RA’s inhibitory effects on adipogenesis, but not on the intracellular levels of both RXRα and PPARγ. These results suggest that 9-cis-RA could inhibit adipogenesis by activating RXR, and decrease both RXR and PPARγs levels in a RXR activation-independent manner.

  1. Induction of pigmentation in mouse fibroblasts by expression of human tyrosinase cDNA

    1989-01-01

    A distinguishing characteristic of cells of the melanocyte lineage is the expression of the melanosomal enzyme tyrosinase that catalyzes the synthesis of the pigment melanin. A tyrosinase cDNA clone, designated BBTY-1, was isolated from a library constructed from the pigmented TA99+/CF21+ melanoma cell line SK-MEL-19. Expression of BBTY-1 in mouse L929 fibroblasts led to synthesis and expression of active tyrosinase, and, unexpectedly, to stable production of melanin. Melanin was synthesized ...

  2. Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes.

    MacDougald, O A; Hwang, C. S.; Fan, H; Lane, M D

    1995-01-01

    A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expresse...

  3. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using [32P]phosphorylase α as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at ∼5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase

  4. Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

    Benamer, Najate [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Fares, Nassim [Laboratoire de Physiologie, Faculte de Medecine, Universite Saint Joseph, Beyrouth (Lebanon); Bois, Patrick [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Faivre, Jean-Francois, E-mail: Jean-Francois.Faivre@univ-poitiers.fr [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France)

    2011-04-29

    Highlights: {yields} In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. {yields} S1P increases cell proliferation through SUR2/Kir6.1 activation. {yields} S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. {yields} S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac

  5. Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

    Powers, S; Fisher, P B; Pollack, R.

    1984-01-01

    We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that ...

  6. Effects of secretive bone morphogenetic protein 2 induced by gene transfection on the biological changes of NIH3T3 cells

    SUN Wei-bin; WANG Juan; LU Chun; TANG Gui-xia

    2005-01-01

    Background Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta superfamily, are powerful regulators of cartilage and bone formation. This study investigated the biological changes of NIH3T3 cells incubated with secretive BMP2 that was induced by gene transfection through transwell. Methods Eukaryonic expression vector (pcDNA3.1-B2) was transfered into NIH3T3 cells with SofastTM,a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expressions of BMP2 were determined by immunohistochemical stain and enzyme-linked immunosorbent assay. NIH3T3 cells were co-cultured with hBMP2 gene transfecting cells through transwell, and the ultrastructure, alkaline phosphatase activity and the expression of osteocalcin (the marker of osteogenetic differentiation) changes were observed. Results There were cytoplasmic and extracellular expressions of BMP2 in transfecting NIH3T3 cells. The ultrastructural changes, the high activity of alkaline phosphatase and the positive stain of osteocalcin suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells. Conclusion Secretive BMP2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.

  7. D-Arg1,D-Phe5,D-Trp7,9,Leu11 substance P, a neuropeptide antagonist, blocks binding, Ca2(+)-mobilizing, and mitogenic effects of endothelin and vasoactive intestinal contractor in mouse 3T3 cells

    Endothelin (ET1) and vasoactive intestinal contractor (VIC) stimulate quiescent Swiss 3T3 cells to resume DNA synthesis acting synergistically with epidermal growth factors (EGF) and other mitogens. The peptide [D-Arg1,D-Phe5,D-Trp7,9,Leu11] substance P has been identified as a broad spectrum neuropeptide antagonist which blocks the binding and biological effects of the Ca2(+)-mobilizing neuropeptides bombesin, vasopressin, and bradykinin. In the present study we show that [D-Arg1,D-Phe5,D-Trp7,9,Leu11] substance P also acts as an ET1/VIC antagonist as judged by the following criteria: (a) inhibition of specific 125I-labelled ET1 binding to a ET1/VIC receptor in a competitive and dose-dependent manner; (b) blocking of the rapid increase in the cytosolic Ca2+ concentration promoted by ET1 or VIC; and (c) inhibition of DNA synthesis stimulated by VIC in the presence of EGF. The inhibitory effects of [D-Arg1,D-Phe5,D-Trp7,9,Leu 11] substance P on Ca2+ mobilization and DNA synthesis were reversed by increasing the concentration of VIC. This is the first time that a peptide structurally unrelated to ET1 or VIC is shown to block the binding and mitogenic effects of peptides of the endothelin family

  8. D-Arg1,D-Phe5,D-Trp7,9,Leu11 substance P, a neuropeptide antagonist, blocks binding, Ca2(+)-mobilizing, and mitogenic effects of endothelin and vasoactive intestinal contractor in mouse 3T3 cells

    Fabregat, I.; Rozengurt, E. (Imperial Cancer Research Fund, London (England))

    1990-10-01

    Endothelin (ET1) and vasoactive intestinal contractor (VIC) stimulate quiescent Swiss 3T3 cells to resume DNA synthesis acting synergistically with epidermal growth factors (EGF) and other mitogens. The peptide (D-Arg1,D-Phe5,D-Trp7,9,Leu11) substance P has been identified as a broad spectrum neuropeptide antagonist which blocks the binding and biological effects of the Ca2(+)-mobilizing neuropeptides bombesin, vasopressin, and bradykinin. In the present study we show that (D-Arg1,D-Phe5,D-Trp7,9,Leu11) substance P also acts as an ET1/VIC antagonist as judged by the following criteria: (a) inhibition of specific 125I-labelled ET1 binding to a ET1/VIC receptor in a competitive and dose-dependent manner; (b) blocking of the rapid increase in the cytosolic Ca2+ concentration promoted by ET1 or VIC; and (c) inhibition of DNA synthesis stimulated by VIC in the presence of EGF. The inhibitory effects of (D-Arg1,D-Phe5,D-Trp7,9,Leu 11) substance P on Ca2+ mobilization and DNA synthesis were reversed by increasing the concentration of VIC. This is the first time that a peptide structurally unrelated to ET1 or VIC is shown to block the binding and mitogenic effects of peptides of the endothelin family.

  9. Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

    Tahereh Talaei-Khozani

    2014-03-01

    Full Text Available Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA and 5-Aza-2-Deoxycytidine (5-aza-dC. The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function.

  10. Effect of Fibroblast Co-culture on In Vitro Maturation and Fertilization of Mouse Preantral Follicles

    Mahmoud Heidari

    2011-01-01

    Full Text Available Background: The aim of this study was to evaluate fibroblast co-culture on in vitro maturation andfertilization of prepubertal mouse preantral follicles.Materials and Methods: The ovaries of 12-14 day old mice were dissected and 120-150 μmintact preantral follicles with one or two layers of granulosa cells, and round oocytes were culturedindividually in α-minimal essential medium (α-MEM supplemented with 5% fetal bovine serum(FBS, 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin, seleniummix, 100 μg/ml penicillin and 50 μg/ml streptomycin as base medium for 12 days. A total number of226 follicules were cultured under two conditions: i base medium as control group (n=113; ii basemedium co-cultured with mouse embryonic fibroblast (MEF (n=113. Follicular diameters, alone,in addition to other factors were analyzed by student’s t-test and chi-square test, respectively.Results: The co-culture group showed significant differences (p<0.05 in growth rate (days 4, 6 and8 of the culture period and survival rate. However, there was no significant difference in antrumformation, ovulation rate and embryonic development of released oocytes. There were significantdifferences (p<0.05 in the estradiol and progesterone secretion at all days between the co-cultureand control groups.Conclusion: Fibroblast co-culture increased survival rate and steroid production of preantralfollicles by promoting granulosa cell proliferation.

  11. Proinflammatory cytokine production and insulin sensitivity regulated by overexpression of resistin in 3T3-L1 adipocytes

    Garvey W Timothy

    2006-07-01

    Full Text Available Abstract Resistin is secreted from adipocytes, and high circulating levels have been associated with obesity and insulin resistance. To investigate whether resistin could exert autocrine effects in adipocytes, we expressed resistin gene in 3T3-L1 fibroblasts using a lentiviral vector, and selected several stably-transduced cell lines under blasticidin selection. We observed that 3T3-L1 adipocytes expressing resistin have a decreased gene expression for related transcriptional factors (CCAAT/enhancer binding protein α(C/EBPα , peroxisome proliferator-activated receptor gamma (PPARγ, and adipocyte lipid binding protein (ALBP/aP2 which is one of target genes for the PPARγ during adipocyte differentiation,. Overexpression of resistin increased the levels of three proinflammatory cytokines, tumor necrosis factor alpha (TNFα, interleukin 6 (IL-6 and monocyte chemoattractant protein-1 (MCP-1, which play important roles for insulin resistance, glucose and lipid metabolisms during adipogenesis. Furthermore, overexpressing resistin in adipocytes inhibits glucose transport 4 (GLUT4 activity and its gene expression, reducing insulin's ability for glucose uptake by 30 %. In conclusion, resistin overexpression in stably transduced 3T3-L1 cells resulted in: 1 Attenuation of programmed gene expression responsible for adipogenesis; 2 Increase in expression of proinflammatory cytokines; 3 Decrease in insulin responsiveness of the glucose transport system. These data suggest a new role for resistin as an autocrine/paracrine factor affecting inflammation and insulin sensitivity in adipose tissue.

  12. Differentiation of the insulin-sensitive glucose transporter in 3T3-L1 adipocytes

    3T3-L1 fibroblasts differentiate in culture to resemble adipocytes both morphologically and biochemically. Insulin-sensitive glucose transport, as measured by 2-deoxy-[1-14C]- glucose uptake in the undifferentiated cell is small (2X). In contrast, the rate of glucose transport in fully differentiated cells is elevated 15-fold over basal in the presence of insulin. To determine if this is due to an increase in the number of transporters/cell or accessibility to the transporters, the number of transporters was measured in subcellular fractions over differentiation using a 3H-cytochalasin B binding assay. The increase in the rate of insulin-sensitive glucose transport directly parallels an increase in the number of transporters which reside in an insulin-responsive intracellular compartment. This observation was confirmed by identifying the transporters by immunoblotting using an antibody generated against the human erythrocyte transporter. The molecular weight of this transporter increases over differentiation from a single band of 40kDa to a heterogeneous triplet of 40, 44 and 48kDa. These data suggest that the transporter undergoes differential processing and that the functional, insulin-responsive transporter may be different from the insulin-insensitive (basal) transporter

  13. ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

    Jang, Min Kyung; Kim, Cho Hee [School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735 (Korea, Republic of); Seong, Je Kyung [Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul 151-742 (Korea, Republic of); Jung, Myeong Ho, E-mail: jung0603@pusan.ac.kr [School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735 (Korea, Republic of)

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Overexpression of ATF3 inhibits adipocyte differentiation in 3T3-L1 cells. Black-Right-Pointing-Pointer Overexpression of ATF3 represses C/EBP{alpha} expression. Black-Right-Pointing-Pointer ATF3 directly binds to mouse C/EBP{alpha} promoter spanning from -1928 to -1907. Black-Right-Pointing-Pointer ATF3 may play a role in hypoxia-mediated inhibition of adipocyte differentiation. -- Abstract: ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBP{alpha} transcript and repressed the activity of the 3.6-kb mouse C/EBP{alpha} promoter, demonstrating that ATF3 downregulates C/EBP{alpha} expression. Transfection studies using mutant constructs containing 5 Prime -deletions in the C/EBP{alpha} promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between -1921 and -1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBP{alpha} promoter spanning from -1928 to -1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBP{alpha} mRNA and repress the promoter activity of the C/EBP{alpha} gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBP{alpha} expression. Collectively, these results demonstrate that ATF3 represses the C/EBP{alpha} gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition

  14. Production of glycosylated physiologically normal human α1-antitrypsin by mouse fibroblasts modified by insertion of a human α1-antitrypsin cDNA using a retroviral vector

    α2-Antitrypsin (α1AT) deficiency is a hereditary disorder characterized by reduced serum levels of α1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment α1AT levels in this disorder with physiologically normal human α1AT, the authors have integrated a full-length normal human α1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the α1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi2 and infected NIH 3T3. The clones produced three mRNA transcripts containing human α1AT sequences, secreted an α1AT molecule recognized by an anti-human α1AT antibody, with the same molecular mass as normal human α1AT and that complexed with and inhibited human neutrophil elastase. The psi2 produced α1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal α1AT purified from human plasma and markedly longer than that of nonglycosylated human α1AT cDNA-directed yeast-produced α1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human α1AT cDNA into non-α1AT-producing cells, resulting in the synthesis and secretion of physiologically normal α1AT

  15. Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers.

    Timothy Fee

    Full Text Available To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL and a blend of PCL and gelatin (PCL+Gel to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes.

  16. Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers

    Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

    2016-01-01

    To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes. PMID:27196306

  17. Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers.

    Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

    2016-01-01

    To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes. PMID:27196306

  18. Block by acetylcholine of mouse muscle nicotinic receptors, stably expressed in fibroblasts

    1995-01-01

    We have measured the concentration and voltage dependence of block by acetylcholine (ACh) of fetal- and adult-type mouse muscle nicotinic receptors, expressed in a fibroblast cell line. Data, obtained at a transmembrane potential of -60 mV and with ACh concentrations of 1 mM and above, are broadly consistent with the occlusion of an open channel with a single ACh+ ion (simple open channel block). The rate of recovery from block is approximately 40,000s-1 and has only a weak voltage dependence...

  19. Fate of the surface protein gp70 during entry of retrovirus into mouse fibroblasts

    The kinetics of the viral surface protein gp70 and the viral core proteins p30 and p15C were followed during retrovirus entry into mouse fibroblasts. All three proteins were internalized, but whereas essentially all the gp70 was degraded, approximately one-third of the core proteins remained stable in the cells. These diverging routes of the different proteins are in agreement with the proposed route, that retrovirus enters the cells by endocytosis followed by a membrane fusion between the virus membrane and the vesicle membrane

  20. Imaging collagen remodeling and sensing transplanted autologous fibroblast metabolism in mouse dermis using multimode nonlinear optical imaging

    Collagen remodeling and transplanted autologous fibroblast metabolic states in mouse dermis after cellular injection are investigated using multimode nonlinear optical imaging. Our findings show that the technique can image the progress of collagen remodeling in mouse dermis. It can also image transplanted autologous fibroblasts in their collagen matrix environment in the dermis, because of metabolic activity. It was also found that the approach can provide two-photon ratiometric redox fluorometry based on autologous fibroblast fluorescence from reduced nicotinamide adenine dinucleotide coenzyme and oxidized flavoproteins for sensing the autologous fibroblast metabolic state. These results show that the multimode nonlinear optical imaging technique may have potential in a clinical setting as an in vivo diagnostic and monitoring system for cellular therapy in plastic surgery

  1. Direct lineage reprogramming of mouse fibroblasts to functional midbrain dopaminergic neuronal progenitors

    Han-Seop Kim

    2014-01-01

    Full Text Available The direct lineage reprogramming of somatic cells to other lineages by defined factors has led to innovative cell-fate-change approaches for providing patient-specific cells. Recent reports have demonstrated that four pluripotency factors (Oct4, Sox2, Klf4, and c-Myc are sufficient to directly reprogram fibroblasts to other specific cells, including induced neural stem cells (iNSCs. Here, we show that mouse fibroblasts can be directly reprogrammed into midbrain dopaminergic neuronal progenitors (DPs by temporal expression of the pluripotency factors and environment containing sonic hedgehog and fibroblast growth factor 8. Within thirteen days, self-renewing and functional induced DPs (iDPs were generated. Interestingly, the inhibition of both Jak and Gsk3β notably enhanced the iDP reprogramming efficiency. We confirmed the functionality of the iDPs by showing that the dopaminergic neurons generated from iDPs express midbrain markers, release dopamine, and show typical electrophysiological profiles. Our results demonstrate that the pluripotency factors-mediated direct reprogramming is an invaluable strategy for supplying functional and proliferating iDPs and may be useful for other neural progenitors required for disease modeling and cell therapies for neurodegenerative disorders.

  2. The loss of PIN1 deregulates cyclin E and sensitizes mouse embryo fibroblasts to genomic instability.

    Yeh, Elizabeth S; Lew, Brian O; Means, Anthony R

    2006-01-01

    During the G0/G1-S phase transition, the timely synthesis and degradation of key regulatory proteins is required for normal cell cycle progression. Two of these proteins, c-Myc and cyclin E, are recognized by the Cdc4 E3 ligase of the Skp1/Cul1/Rbx1 (SCF) complex. SCF(Cdc4) binds to a similar phosphodegron sequence in c-Myc and cyclin E proteins resulting in ubiquitylation and degradation of both proteins via the 26 S proteosome. Since the prolyl isomerase Pin1 binds the c-Myc phosphodegron and participates in regulation of c-Myc turnover, we hypothesized that Pin1 would bind to and regulate cyclin E turnover in a similar manner. Here we show that Pin1 regulates the turnover of cyclin E in mouse embryo fibroblasts. Pin1 binds to the cyclin E-Cdk2 complex in a manner that depends on Ser384 of cyclin E, which is phosphorylated by Cdk2. The absence of Pin1 results in an increased steady-state level of cyclin E and stalling of the cells in the G1/S phase of the cell cycle. The cellular changes that result from the loss of Pin1 predispose Pin1 null mouse embryo fibroblasts to undergo more rapid genomic instability when immortalized by conditional inactivation of p53 and sensitizes these cells to more aggressive Ras-dependent transformation and tumorigenesis. PMID:16223725

  3. Generation and characterisation of Friedreich ataxia YG8R mouse fibroblast and neural stem cell models.

    Chiranjeevi Sandi

    Full Text Available BACKGROUND: Friedreich ataxia (FRDA is an autosomal recessive neurodegenerative disease caused by GAA repeat expansion in the first intron of the FXN gene, which encodes frataxin, an essential mitochondrial protein. To further characterise the molecular abnormalities associated with FRDA pathogenesis and to hasten drug screening, the development and use of animal and cellular models is considered essential. Studies of lower organisms have already contributed to understanding FRDA disease pathology, but mammalian cells are more related to FRDA patient cells in physiological terms. METHODOLOGY/PRINCIPAL FINDINGS: We have generated fibroblast cells and neural stem cells (NSCs from control Y47R mice (9 GAA repeats and GAA repeat expansion YG8R mice (190+120 GAA repeats. We then differentiated the NSCs in to neurons, oligodendrocytes and astrocytes as confirmed by immunocytochemical analysis of cell specific markers. The three YG8R mouse cell types (fibroblasts, NSCs and differentiated NSCs exhibit GAA repeat stability, together with reduced expression of frataxin and reduced aconitase activity compared to control Y47R cells. Furthermore, YG8R cells also show increased sensitivity to oxidative stress and downregulation of Pgc-1α and antioxidant gene expression levels, especially Sod2. We also analysed various DNA mismatch repair (MMR gene expression levels and found that YG8R cells displayed significant reduction in expression of several MMR genes, which may contribute to the GAA repeat stability. CONCLUSIONS/SIGNIFICANCE: We describe the first fibroblast and NSC models from YG8R FRDA mice and we confirm that the NSCs can be differentiated into neurons and glia. These novel FRDA mouse cell models, which exhibit a FRDA-like cellular and molecular phenotype, will be valuable resources to further study FRDA molecular pathogenesis. They will also provide very useful tools for preclinical testing of frataxin-increasing compounds for FRDA drug therapy

  4. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

    Ahmed Kamel El-Sayed

    2014-11-01

    Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

  5. Temperature induced modulation of lipid oxidation and lipid accumulation in palmitate-mediated 3T3-L1 adipocytes and 3T3-L1 adipocytes.

    Lin, Xiaofen; Li, Yi; Leung, Polly Hangmei; Li, Jiashen; Hu, Junyan; Liu, Xuan; Li, Zhi

    2016-05-01

    Human skin temperature can vary widely depending on anatomical location and ambient temperature. It is also known that local changes in skin and subcutaneous temperature can affect fat metabolism. This study aimed to explore the potential effects of surrounding thermal environment on fat by investigating cell viability, lipid oxidation, and lipid accumulation in 3T3-L1 adipocytes and palmitate-treated adipocytes after 4h incubation. No significant differences of viability in 3T3-L1 adipocytes were detected under different temperature conditions. Despite no significant increase being observed under warm temperature (39°C) conditions, a similarly significant suppression of intracellular reactive oxygen species (ROS) and lipid peroxidation were found in 3T3-L1 adipocytes and palmitate-treated adipocytes under 4h exposure to cooler temperatures of 31-33°C (Psize of lipid droplets in 3T3-L1 adipocytes (Padipocytes. In the palmitate-induced adiposity model, although excessive ROS and lipid peroxidation has been attenuated by temperature decrease (Psize (P>0.05) and remedy the palmitate damage induced cell death (Padipocytes. PMID:27157327

  6. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

    Zhong, Xing; Xiu, Ling-ling; Wei, Guo-hong; Liu, Yuan-Yuan; Su, Lei; Cao, Xiao-pei; Li, Yan-Bing; Xiao, Hai-peng

    2011-01-01

    Aim: To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects. Methods: MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 w...

  7. Neuronal and astrocyte dysfunction diverges from embryonic fibroblasts in the Ndufs4fky/fky mouse

    Matthew J. Bird

    2014-11-01

    Full Text Available Mitochondrial dysfunction causes a range of early-onset neurological diseases and contributes to neurodegenerative conditions. The mechanisms of neurological damage however are poorly understood, as accessing relevant tissue from patients is difficult, and appropriate models are limited. Hence, we assessed mitochondrial function in neurologically relevant primary cell lines from a CI (complex I deficient Ndufs4 KO (knockout mouse (Ndufs4fky/fky modelling aspects of the mitochondrial disease LS (Leigh syndrome, as well as MEFs (mouse embryonic fibroblasts. Although CI structure and function were compromised in all Ndufs4fky/fky cell types, the mitochondrial membrane potential was selectively impaired in the MEFs, correlating with decreased CI-dependent ATP synthesis. In addition, increased ROS (reactive oxygen species generation and altered sensitivity to cell death were only observed in Ndufs4fky/fky primary MEFs. In contrast, Ndufs4fky/fky primary isocortical neurons and primary isocortical astrocytes displayed only impaired ATP generation without mitochondrial membrane potential changes. Therefore the neurological dysfunction in the Ndufs4fky/fky mouse may partly originate from a more severe ATP depletion in neurons and astrocytes, even at the expense of maintaining the mitochondrial membrane potential. This may provide protection from cell death, but would ultimately compromise cell functionality in neurons and astrocytes. Furthermore, RET (reverse electron transfer from complex II to CI appears more prominent in neurons than MEFs or astrocytes, and is attenuated in Ndufs4fky/fky cells.

  8. EGF raises cytosolic Ca2+ in A431 and Swiss 3T3 cells by a dual mechanism

    The changes in Ca2+ homeostasis and phosphoinositide hydrolysis induced by EGF were studied in human epidermoid carcinoma A431 cells both when attached to a substratum and after detachment and suspension. The cytosolic Ca2+ concentration was measured by the conventional fluorimetric technique, using the specific probe, quin2, as well as by a new microscopic technique in which single cells are investigated after loading with another probe, fura-2. EGF applied in the complete, Ca2+-containing medium caused a rapid rise in the cytosolic 45Ca2+ concentration, that remained elevated for several minutes. In Ca2+-free, EGTA-containing medium, part of this response persisted, as revealed by quin2 results in suspended cells and microscopic results with fura-2. These results, as well as additional microscopic fura-2 results in Swiss 3T3 fibroblasts, demonstrate that the Ca2+ signal elicited by EGF is due to two components: redistribution from an intracellular store and stimulated influx across the plasmalemma. This latter process was not detected in 3T3 cells treated with either PDGF or bombesin. It is therefore suggested that the 45Ca2+ influx effect of EGF is under the control of a separate, as yet unidentified mechanism

  9. Inhibition and recovery of the replication of depurinated parvovirus DNA in mouse fibroblasts

    Vos, J.M.; Avalosse, B.; Su, Z.Z.; Rommelaere, J.

    1984-01-01

    Apurinic sites were introduced in the single-stranded DNA of parvovirus minute-virus-of-mice (MVM) and their effect on viral DNA synthesis was measured in mouse fibroblasts. Approximately one apurinic site per viral genome, is sufficient to block its replication in untreated cells. The exposure of host cells to a sublethal dose of UV-light 15 hours prior to virus infection, enhances their ability to support the replication of depurinated MVM. Cell preirradiation induces the apparent overcome of 10-15% of viral DNA replication blocks. These results indicate that apurinic sites prevent mammalian cells from replicating single-stranded DNA unless a recovery process is activated by cell UV-irradiation.

  10. Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors

    Yang, Ruifeng; Zheng, Ying; Li, Ling; Liu, Shujing; Burrows, Michelle; Wei, Zhi; Nace, Arben; Herlyn, Meenhard; Cui, Rutao; Guo, Wei; Cotsarelis, George; Xu, Xiaowei

    2015-01-01

    Direct reprogramming provides a fundamentally new approach for the generation of patient-specific cells. Here, by screening a pool of candidate transcription factors, we identify that a combination of three factors, MITF, SOX10 and PAX3, directly converts mouse and human fibroblasts to functional melanocytes. Induced melanocytes (iMels) activate melanocyte-specific networks, express components of pigment production and delivery system, and produce melanosomes. Human iMels properly integrate into the dermal-epidermal junction, and produce and deliver melanin pigment to surrounding keratinocytes in a 3D organotypic skin reconstruct. Human iMels generate pigmented epidermis and hair follicles in skin reconstitution assays in vivo. The generation of iMels has important implications for studies of melanocyte lineage commitment, pigmentation disorders and cell replacement therapies. PMID:25510211

  11. Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.

    Yang, Ruifeng; Zheng, Ying; Li, Ling; Liu, Shujing; Burrows, Michelle; Wei, Zhi; Nace, Arben; Herlyn, Meenhard; Cui, Rutao; Guo, Wei; Cotsarelis, George; Xu, Xiaowei

    2014-01-01

    Direct reprogramming provides a fundamentally new approach for the generation of patient-specific cells. Here, by screening a pool of candidate transcription factors, we identify that a combination of the three factors, MITF, SOX10 and PAX3, directly converts mouse and human fibroblasts to functional melanocytes. Induced melanocytes (iMels) activate melanocyte-specific networks, express components of pigment production and delivery system and produce melanosomes. Human iMels properly integrate into the dermal-epidermal junction and produce and deliver melanin pigment to surrounding keratinocytes in a 3D organotypic skin reconstruct. Human iMels generate pigmented epidermis and hair follicles in skin reconstitution assays in vivo. The generation of iMels has important implications for studies of melanocyte lineage commitment, pigmentation disorders and cell replacement therapies. PMID:25510211

  12. The mitochondrial function was impaired in APP knockout mouse embryo fibroblast cells

    SHENG BaiYang; NIU Ying; ZHOU Hui; YAN JiaXin; ZHAO NanMing; ZHANG XiuFang; GONG YanDao

    2009-01-01

    The amyloid precursor protein (APP) is recognized as the source of Aβ, which plays an important role in Alzheimer's disease. However, the biological function of APP is obscure. Previous studies showed that mitochondria could be a target of APP. In this work, APP knockout mouse embryo fibroblast (MEF) cells were used to test if APP plays any role in maintaining the mitochondrial function. As the result, APP knockout MEF cells (APP-/- cells) showed the abnormal mitochondrial function, including slower cell proliferation, lower mitochondrial membrane potential, lower intracellular ROS, higher mitochon-drial membrane fluidity and lower cytochrome c oxidase activity than their wild-type counterparts. However, no change was found in the amount of mitochondria in MEF APP-/- cells.

  13. Movement of fibroblasts in the periodontal ligament of the mouse incisor is related to eruption

    Movement of fibroblasts in the periodontal ligament of the lower incisor of the mouse was studied by pulse-labeling with tritiated thymidine and proline. 3H-Thymidine was administered to mark the nuclei of the cells in the proliferative compartment near the basal end of the tooth; 3H-proline gave rise to a narrow band of radioactivity in the dentin, which served as a reference line for measurement of eruption. One or three weeks after injection in each animal, the lower right incisor was prevented from further eruption by being pinned to its alveolar process. The animals were killed 0, 1, or 2 weeks later, and their mandibles processed for LM-radioautography. It was found that in the left incisors, which were not inhibited in their eruption, labeled cells in the tooth-half of the periodontal ligament moved incisally at a rate similar to the eruption rate. In the pinned incisors, no further incisal migration could be established. It is concluded that fibroblast migration in the tooth-half of the ligament is strictly coupled to the eruptive process

  14. Genomic organization of the mouse fibroblast growth factor receptor 3 (Fgfr3) gene

    Perez-Castro, A.V.; Wilson, J.; Altherr, M.R. [Los Alamos National Lab., NM (United States)

    1995-11-20

    The fibroblast growth factor receptor 3 (Fgfr3) protein is a tyrosine kinase receptor involved in the signal transduction of various fibroblast growth factors. Recent studies suggest its important role in normal development. In humans, mutation in Fgfr3 is responsible for growth disorders such as achondroplasia, hypoachondroplasia, and thanatophoric dysplasia. Here, we report the complete genomic organization of the mouse Fgfr3 gene. The murine gene spans approximately 15 kb and consists of 19 exons and 18 introns. One major and one minor transcription initiation site were identified. Position +1 is located 614 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exons 2 and 19, respectively. Five Sp1 sites, two AP2 sites, one Zeste site, and one Krox 24 site were observed in the 5{prime}-flanking region. The Fgfr3 promoter appears to be contained within a CpG island and, as is common in genes having multiple Sp1-binding sites, lacks a TATA box. 35 refs., 3 figs., 1 tab.

  15. Effect of Simavastatin on IL-6 and Adiponectin Secretion and mRNA Expression in 3T3-L1 Adipocytes

    YIN Xiaoming; TU Ling; YANG Huiqing

    2007-01-01

    In order to investigate the effects of simvastatin on secretion and mRNA expression of interleukin-6 (IL-6) and adiponectin in 3T3-L1 adipocytes, mouse 3T3-L1 adipocytes were stimulated with lipopolysaccharide (LPS). Production and mRNA expression of IL-6 and adiponectin in 3T3-L1 adipocytes were measured using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. The results showed that simvastatin could significantly suppress LPS-induced IL-6 production and mRNA expression in adipocytes (P<0.05), but increase the LPS-induced adiponectin secretion and mRNA expression in a dose-dependent manner (P<0.05). It was suggested that simvastatin could exert beneficial effects on prevention of obesity-induced metabolic changes in adipocytes.

  16. Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene

    Reznickova, Alena, E-mail: alena.reznickova@vscht.cz [Department of Solid State Engineering, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic); Novotna, Zdenka, E-mail: zdenka1.novotna@vscht.cz [Department of Solid State Engineering, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic); Kolska, Zdenka [Faculty of Science, J.E. Purkyně University, 400 96 Usti nad Labem (Czech Republic); Kasalkova, Nikola Slepickova [Department of Solid State Engineering, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic); Rimpelova, Silvie [Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic); Svorcik, Vaclav [Department of Solid State Engineering, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic)

    2015-07-01

    Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells. - Highlights: • Plasma activation of LDPE, HDPE and UHMWPE • Study of surface properties by several techniques: ARXPS, AFM, zeta-potential, and goniometry • Investigation of adhesion and spreading of vascular smooth muscle cells (VSMCs) and mouse fibroblasts (L929)

  17. Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene

    Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells. - Highlights: • Plasma activation of LDPE, HDPE and UHMWPE • Study of surface properties by several techniques: ARXPS, AFM, zeta-potential, and goniometry • Investigation of adhesion and spreading of vascular smooth muscle cells (VSMCs) and mouse fibroblasts (L929)

  18. Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into "osteocyte-like" cells.

    Alvares, Keith; Ren, Yinshi; Feng, Jian Q; Veis, Arthur

    2016-01-01

    P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, syncytium formation, and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP, a protein common to all vertebrate teeth, and crucial for their mineralization. Here, we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture, the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18-fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN), and β-catenin decreased by 70%, 64%, and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an "osteocyte-like" phenotype, an important process in bone biology. The mechanisms involved are presently under study. PMID:26581835

  19. The effects of Ganoderma lucidum herba pharmacopuncture on 3T3-L1 preadipocyte differentiation

    Chea-woo Lee

    2008-09-01

    Full Text Available Objective : The purpose of this study is to investigate the effects of Ganoderma lucidum herba pharmacopuncture (GHP on the adipogenesis in 3T3-L1 preadipocytes. Methods : 3T3- L1 preadipocytes were differentiated with adipogenic reagents by incubating for 2 days in the absence or presence of GHP ranging from 1 and 2%. The effect of GHP on cell proliferation of 3T3-L1 preadipocytes was investigated using MTT assay. The effect of GHP on adipogenesis was examined by Oil red O staining and measuring glycerol-3-phosphate dehydrogenase (GPDH and intracellular triglyceride (TG content. Results : Following results were obtained from the preadipocyte proliferation and adipocyte differentiation of 3T3-L1. We observed no effect of GHP on preadipocyte proliferation. GHP inhibited adipogenesis, the activity of GPDH and accumulation of intracellular TG content. Conclusions : These results suggest that GHP inhibit differentiation of preadipocyte.

  20. FRS2α is Essential for the Fibroblast Growth Factor to Regulate the mTOR Pathway and Autophagy in Mouse Embryonic Fibroblasts

    Xiang Lin, Yongyou Zhang, Leyuan Liu, Wallace L. McKeehan, Yuemao Shen, Siyang Song, Fen Wang

    2011-01-01

    Full Text Available Although the fibroblast growth factor (FGF signaling axis plays important roles in cell survival, proliferation, and differentiation, the molecular mechanism underlying how the FGF elicits these diverse regulatory signals is not well understood. By using the Frs2α null mouse embryonic fibroblast (MEF in conjunction with inhibitors to multiple signaling pathways, here we report that the FGF signaling axis activates mTOR via the FGF receptor substrate 2α (FRS2α-mediated PI3K/Akt pathway, and suppresses autophagy activity in MEFs. In addition, the PI3K/Akt pathway regulated mTOR is crucial for the FGF signaling axis to suppress autophagy in MEFs. Since autophagy has been proposed to play important roles in cell survival, proliferation, and differentiation, the findings suggest a novel mechanism for the FGF signaling axis to transmit regulatory signals to downstream effectors.

  1. 56Co-labelled radioactive Fe3O4 nanoparticles for in vitro uptake studies on Balb/3T3 and Caco-2 cell lines

    Magnetite nanoparticles (Fe3O4 NPs) are manufactured nanomaterials increasingly used in healthcare for different medical applications ranging from diagnosis to therapy. This study deals with the irradiation of Fe3O4 NPs with a proton beam in order to produce 56Co as radiolabel and also with the possible use of nuclear techniques for the quantification of Fe3O4 NPs in biological systems. Particular attention has been focused on the size distribution (in the range of 100 nm) and the surface charge of the NPs characterizing them before and after the irradiation process in order to verify if these essential properties would be preserved during irradiation. Moreover, X-ray diffraction studies have been performed on radioactive and non-radioactive NPs, to assess if major changes in NPs structure might occur due to thermal and/or radiation effects. The radiation emitted from the radiolabels has been used to quantify the cellular uptake of the NPs in in vitro studies. As for the biological applications two cell lines have been selected: immortalized mouse fibroblast cell line (Balb/3T3) and human epithelial colorectal adenocarcinoma cell line (Caco-2). The cell uptake has been quantified by radioactivity measurements of the 56Co radioisotope performed with high resolution γ-ray spectrometry equipment. This study has showed that, under well-established irradiation conditions, Fe3O4 NPs do not undergo significant structural modifications and thus the obtained results are in line with the uptake studies carried out with the same non-radioactive nanomaterials (NMs). Therefore, the radiolabelling method can be fruitfully applied to uptake studies because of the low-level exposure where higher sensitivity is required.

  2. Platelet-derived growth factor stimulation of [3H]-glucosamine incorporation in density-arrested BALB/c-3T3 cells

    G0/G1 traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G0/G1. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. The authors have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10 fold more [3H]-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca3 (PO4)2 and was not due to cell-cycle traverse. Stimulation of [3H]-GlcN incorporation by PDGF was time dependent, and increased incorporation of [3H]-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation

  3. Intranuclear Localization of EGFP-mouse PPARγ1 in Bovine Fibroblast Cells

    Sorayya Ghasemi

    2010-01-01

    Full Text Available Objective: The aim of this study was to clone PPARγ1 cDNA in an appropriate mammalianexpression vector, with a chimeric cDNA form, encompassing PPARγ with enhanced greenfluorescent protein (EGFP cDNA. This recombinant plasmid will be used for further analysesto investigate the molecular mechanism of PPARγ1 for neural differentiation process.Moreover, the nuclear localization of the PPARγ1 protein linked to EGFP marker was chasedby using transient transfection of a constructed plasmid into bovine fibroblast cells.Materials and Methods: Total RNA was extracted from the fatty tissue of an adult mouse.Using specific pair primers, PPARγ1 cDNA was synthesized and amplified to producethe entire length of ORF. RT-PCR products containing PPARγ1 cDNA were treated byenzymatic digestion and inserted into the pEGFP-C1 downstream from EGFP cDNA. Theconstructed vector was used for transformation into bacterial competent cells. Positivecolonies which showed inserted PPARγ1 cDNA were selected for plasmid preparationsand additional analysis was performed to ensure that PPARγ1 cDNA was inserted properly.Finally, to confirm the intracellular localization of EGFP-PPARγ1, bovine fibroblastcells were transfected with the recombinant plasmid.Results: Our results from enzymatic digestion and sequencing confirmed, as expected, thatPPARγ1 cDNA was amplified and cloned correctly. This cDNA gene encompassed 1428 bp.The related product was entered into the nucleus of bovine fibroblasts after transfection ofits cDNA.

  4. Effects of Ghrelin on the Proliferation and Differentiation of 3T3-L1 Preadipocytes

    Jing LIU; Hanhua LIN; Peixuan CHENG; Xiufen HU; Huiling LU

    2009-01-01

    The effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and the possible mechanisms were investigated in this study.3T3-L1 preadipocytes were cultured in vitro and treated with different concentrations of ghrelin.Proliferation of 3T3-L1 preadipocytes was evaluated by MTT method and mRNA levels of c-myc and thymidine kinase were detected by RT-PCR.Morphological changes of 3T3-L1 preadipocytes were observed and cell differentiation was measured by oil red O staining.The mRNA levels of peroxisome proliferator-activated receptor γ (PPARγ) and CAAT/enhancer binding protein (C/EBPα) in the cells at different differentiation stages were detected by RT-PCR.The results showed that ghrelin at concentrations of 10-7 to 10-15 mol/L could significantly promote preadipocyte proliferation (P<0.05),with the most pronounced effect observed at 1011mol/L (P<0.01).Treatment of 3T3-L1 preadipocytes with ghrelin significantly in-creased the mRNA levels of c-myc and thymidine kinase (P<0.01).Morphological findings demonstrated that the great amount of lipid droplets appeared in the 3T3-L1 preadipocytes treated with ghrelin.Ghrelin could morphologically induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes.Ghrelin significantly increased the mRNA levels of PPART and C/EBPα during the differentiation,when compared with control group (P<0.05).The mRNA levels of PPARγ and C/EBPα were obviously up-regulated with the differentiation of preadipocytes after the treatment of ghrelin.There were significant difference in the mRNA levels of PPARγ and C/EBPα on day 2 and day 8 of the differentiation of 3T3-L1 preadipocytes (P<0.01).In conclusion,ghrelin could promote the proliferation and differentiation of 3T3-L1 preadipocytes by increasing the mRNA levels of PPARγ and C/EBPα and therefore enhance the sensitivity of adipocytes against insulin.

  5. Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

    spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis

  6. Effects of phytoestrogens and environmental estrogens on osteoblastic differentiation in MC3T3-E1 cells

    Phytoestrogens and environmental estrogens, which have in part some structural similarity to 17β-estradiol, are reported to act as agonists/antagonists of estrogen in animals and humans. Estrogen is known to play an important role in maintaining bone mass, since the concentration of serum estrogen decreases after menopause and the estrogen deficiency results in bone loss. In this study, we report the effects of phytoestrogens (genistein, daidzein, and coumestrol) and environmental estrogens (bisphenol A (BPA), p-n-nonylphenol (NP) and bis(2-ethylhexyl)phthalate (DEHP)) on osteoblast differentiation using MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line. Coumestrol (10-10 to 10-6 M) slightly enhanced cell proliferation, while neither the other phytoestrogens (daidzein, genistein) nor environmental estrogens increased cell proliferation. Alkaline phosphatase (ALP) activity and cellular calcium (Ca) and phosphorus (P) contents were increased by phytoestrogens and BPA; however, neither NP nor DEHP affected those osteoblastic indicators. The effects of estrogenic potency, using the cell proliferation of MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line, indicate that coumestrol has the highest estrogenic potency among those phytoestrogens and environmental estrogens. The estrogenic potency of NP and DEHP were lower than the others. In conclusion, phytoestrogens, such as coumestrol, genistein and daidzein, and BPA increased ALP activity and enhanced bone mineralization in MC3T3-E1 cells, suggesting that not only phytoestrogen but also BPA, an environmental estrogen, is implicated in bone metabolism

  7. Effects of UVC-irradiation on cultured mouse embryonic cells

    Effects of UVC-irradiation on the cultured differentiating mouse embryonic cells were investigated. Embryonic mesenchymal cells, isolated from fore-and hind-limbs or mid brain of Day 11 mouse embryos, and 3T3 cells, a reference mouse fibroblast cell line, were irradiated with UVC at a dose range of 0∼30 J/m2. Dose-dependent inhibition was found for both cellular proliferation and differentiation, dose-dependent induction of DNA cyclobutane pyrimidine dimers and (6-4) photoproducts were found in the embryonic cells. Mesenchymal chondrogenesis was more sensitive to the UVC than proliferation, and the UVC-induced DNA damage and their repair kinetics in the cultured embryonic cells were similar to those in mouse 3T3 cells. No effects of treatments by the fluorescent light pre or post UVC-irradiation were found on the repair kinetics of DNA damage in all of the cells

  8. An electron microscopic radioautographic study of collagen secretion in periodontal ligament fibroblasts of the mouse: I. Normal fibroblasts

    Analysis of electron microscopic radioautographs revealed a maximum labeling with 3H-proline of rough endoplasmic reticulum (RER) at 3 minutes, Golgi saccules 1 and 2 at 10 minutes, Golgi saccules type 3 at 20 minutes, and presecretory and secretory granules at 30 minutes. Labeling of the extra-cellular collagen matrix occurred at 30 minutes and increased with time. These observations suggest that pro-a-chains of collagen in periodontal ligament fibroblasts are synthesized in the RER and transported to the Golgi apparatus within 10 minutes. These chains then undergo parallel alignment in Golgi saccules type 2 and form segment-long-spacing-like crystallites in Golgi saccules type 3 between 10 and 20 minutes. The peak labeling of presecretory granules and mature secretory granules in small amounts at 30 minutes and the rapid increase in labeling of extracellular collagen matrix which begins at 30 minutes, indicates that the formation of secretory granules requires approximately 30 minutes and that a rapid system of secretory granule translocation exists in periodontal ligament fibroblasts. This evidence further supports the previously published morphologic evidence for a microtubule-dependent system of collagen secretion in periodontal ligament fibroblasts

  9. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

  10. Microarray data on altered transcriptional program of Phgdh-deficient mouse embryonic fibroblasts caused by ʟ-serine depletion.

    Hamano, Momoko; Sayano, Tomoko; Kusada, Wataru; Kato, Hisanori; Furuya, Shigeki

    2016-06-01

    Inherent ʟ-Ser deficiency culminates in intrauterine growth retardation, severe malformation of multiple organs particularly the central nervous system, and perinatal or early postnatal death in human and mouse. To uncover the molecular mechanisms underlying the growth-arrested phenotypes of l-Ser deficiency, we compared gene expression profiles of mouse embryonic fibroblasts deficient in 3-phosphoglycerate dehydrogenase (Phgdh), the first enzyme of de novo ʟ-Ser synthetic pathway, between ʟ-Ser-depleted and -supplemented conditions. The datasets (CEL and CHP files) from this study are publicly available on the Gene Expression Omnibus repository (accession number GEO: GSE55687). PMID:27222860

  11. Effects of Apatite Cement Containing Atelocollagen on Attachment to and Proliferation and Differentiation of MC3T3-E1 Osteoblastic Cells

    Masaaki Takechi; Yoshiaki Ninomiya; Kouji Ohta; Misato Tada; Kazuki Sasaki; Mohammad Zeshaan Rahman; Akira Ohta; Kanji Tsuru; Kunio Ishikawa

    2016-01-01

    To improve the osteoconductivity of apatite cement (AC) for reconstruction of bone defects after oral maxillofacial surgery, we previously fabricated AC containing atelocollagen (AC(ate)). In the present study, we examined the initial attachment, proliferation and differentiation of mouse osteoblastic cells (MC3T3-E1 cells) on the surface of conventional AC (c-AC), AC(ate) and a plastic cell dish. The number of osteoblastic cells showing initial attachment to AC(ate) was greater than those at...

  12. SILAC based protein profiling data of MKK3 knockout mouse embryonic fibroblasts.

    Srivastava, Anup; Shinn, Amanda S; Lam, TuKiet T; Lee, Patty J; Mannam, Praveen

    2016-06-01

    This data article reports changes in the phospho and total proteome of MKK3 knock out (MKK3(-) (/) (-)) mouse embryonic fibroblasts (MEFs). The dataset generated highlights the changes at protein level which can be helpful for understanding targets of the MAP kinase signaling pathway. Data was collected after TiO2-based phosphopeptide enrichment of whole cell lysate at baseline condition with bottom-up SILAC-based LC MS/MS quantitative mass spectrometry. We report all the proteins and peptides identified and quantified in MKK3(-/-) and WT MEFs. The altered pathways in MKK3(-/-) MEFs were analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7) and Ingenuity Pathway Analysis (IPA) and are presented as a table and graph, respectively. The data reported here is related to the published work [1]. All the associated mass spectrometry data has been deposited in the Yale Protein Expression Database (YPED) with the web-link to the data: http://yped.med.yale.edu/repository/ViewSeriesMenu.do;jsessionid=6A5CB07543D8B529FAE8C3FCFE29471D?series_id=5044&series_name=MMK3+Deletion+in+MEFs. PMID:26977448

  13. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    Kukat, Alexandra [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Edgar, Daniel [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Bratic, Ivana [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Maiti, Priyanka [Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany); Trifunovic, Aleksandra, E-mail: aleksandra.trifunovic@ki.se [Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institute, S-17171 Stockholm (Sweden); Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), Cologne University Clinic, D-50674 Cologne (Germany)

    2011-06-10

    Highlights: {yields} Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. {yields} This process is independent of endogenous ROS production. {yields} Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O{sub 2}) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  14. Increasing mouse embryonic fibroblast cells adhesion on superhydrophilic vertically aligned carbon nanotube films

    Lobo, A.O., E-mail: loboao@yahoo.com [Laboratory of Biomedical Nanotechnology (NanoBio), Instituto de Pesquisa e Desenvolvimento (IP and D), Universidade do Vale do Paraiba UniVap, Avenida Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, SP (Brazil) and Laboratory of Biomedical Vibrational Spectroscopy (LEVB), Instituto de Pesquisa e Desenvolvimento (IP and D), Universidade do Vale do Paraiba UniVap, Avenida Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, SP (Brazil); Marciano, F.R. [Laboratory of Biomedical Nanotechnology (NanoBio), Instituto de Pesquisa e Desenvolvimento (IP and D), Universidade do Vale do Paraiba UniVap, Avenida Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, SP (Brazil); Laboratory of Biomedical Vibrational Spectroscopy LEVB, Instituto de Pesquisa e Desenvolvimento (IP and D), Universidade do Vale do Paraiba (UniVap), Avenida Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, SP (Brazil); Ramos, S.C. [Laboratorio Associado de Sensores e Materiais (LAS), Instituto Nacional de Pesquisas Espaciais (INPE), Avenida dos Astronautas 1758, Sao Jose dos Campos, 12.245-970, SP (Brazil); Machado, M.M. [Centro Multidisciplinar para Investigacao Biologica na Area da Ciencia em Animais de Laboratorio (CEMIB), Universidade Estadual de Campinas (UNICAMP), Rua 05 de Junho s/no, Cidade Universitaria ' Zeferino Vaz' , 13083-877, Campinas (Brazil); Corat, E.J. [Laboratorio Associado de Sensores e Materiais (LAS), Instituto Nacional de Pesquisas Espaciais (INPE), Avenida dos Astronautas 1758, Sao Jose dos Campos, 12.245-970, SP (Brazil); Corat, M.A.F. [Centro Multidisciplinar para Investigacao Biologica na Area da Ciencia em Animais de Laboratorio (CEMIB), Universidade Estadual de Campinas (UNICAMP), Rua 05 de Junho s/no, Cidade Universitaria ' Zeferino Vaz' , 13083-877, Campinas (Brazil)

    2011-10-10

    We have analyzed the adhesion of mouse embryonic fibroblasts (MEFs) genetically modified by green fluorescence protein (GFP) gene cultured on vertically-aligned carbon nanotubes (VACNTs) after 6 days. The VACNTs films grown on Ti were obtained by microwave plasma chemical vapor deposition process using Fe catalyst and submitted to an oxygen plasma treatment, for 2 min, at 400 V and 80 mTorr, to convert them to superhydrophilic. Cellular adhesion and morphology were analyzed by scanning electron, fluorescence microscopy, and thermodynamics analysis. Characterizations of superhydrophilic VACNTs films were evaluated by contact angle and X-Ray Photoelectron Spectroscopy. Differences of crowd adhered cells, as well as their spreading on superhydrophilic VACNTs scaffolds, were evaluated using focal adhesion analysis. This study was the first to demonstrate, in real time, that the wettability of VACNTs scaffolds might have enhanced and differential adherence patterns to the MEF-GFP on VACNTs substrates. Highlights: {yields} A simple oxygen plasma treatment was used to obtain superhydrophilic CNT films. {yields} Superhydrophilic CNTs films were successfully produced by incorporation of carboxylic groups. {yields} Cellular adhesion on superhydrophilic VACNT films was analyzed in real time. {yields} Wettability of CNT films directly affects the cellular migration, proliferation and adhesion.

  15. Cerium oxide nanoparticles stimulate proliferation of primary mouse embryonic fibroblasts in vitro.

    Popov, Anton L; Popova, Nelly R; Selezneva, Irina I; Akkizov, Azamat Y; Ivanov, Vladimir K

    2016-11-01

    The increasing application of cell therapy technologies in the treatment of various diseases requires the development of new effective methods for culturing primary cells. The major limitation for the efficient use of autologous cell material is the low rate of cell proliferation. Successful cell therapy requires sufficient amounts of cell material over a short period of time with the preservation of their differentiation and proliferative potential. In this regard, the development of novel, highly efficient stimulators of proliferative activity in stem cells is a truly urgent task. In this paper we have demonstrated that citrate-stabilized cerium oxide nanoparticles (nanoceria) enhance the proliferative activity of primary mouse embryonic fibroblasts in vitro. Cerium oxide nanoparticles stimulate cell proliferation in a wide range of concentrations (10(-3)М-10(-9)M) through reduction of intracellular levels of reactive oxygen species (ROS) during the lag phase of cell growth and by modulating the expression level of the major antioxidant enzymes. We found the optimal concentration of nanoceria, which provides the greatest acceleration of cell proliferation in vitro, while maintaining the levels of intracellular ROS and mRNA of antioxidant enzymes in the physiological range. Our results confirm that nanocrystalline ceria can be considered as a basis for effective and inexpensive supplements in cell culturing. PMID:27524035

  16. Pol β associated complex and base excision repair factors in mouse fibroblasts.

    Prasad, Rajendra; Williams, Jason G; Hou, Esther W; Wilson, Samuel H

    2012-12-01

    During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an 'affinity-capture' procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3'-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3'-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3'-blocked intermediate. PMID:23042675

  17. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    Highlights: → Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. → This process is independent of endogenous ROS production. → Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O2) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  18. Increasing mouse embryonic fibroblast cells adhesion on superhydrophilic vertically aligned carbon nanotube films

    We have analyzed the adhesion of mouse embryonic fibroblasts (MEFs) genetically modified by green fluorescence protein (GFP) gene cultured on vertically-aligned carbon nanotubes (VACNTs) after 6 days. The VACNTs films grown on Ti were obtained by microwave plasma chemical vapor deposition process using Fe catalyst and submitted to an oxygen plasma treatment, for 2 min, at 400 V and 80 mTorr, to convert them to superhydrophilic. Cellular adhesion and morphology were analyzed by scanning electron, fluorescence microscopy, and thermodynamics analysis. Characterizations of superhydrophilic VACNTs films were evaluated by contact angle and X-Ray Photoelectron Spectroscopy. Differences of crowd adhered cells, as well as their spreading on superhydrophilic VACNTs scaffolds, were evaluated using focal adhesion analysis. This study was the first to demonstrate, in real time, that the wettability of VACNTs scaffolds might have enhanced and differential adherence patterns to the MEF-GFP on VACNTs substrates. Highlights: → A simple oxygen plasma treatment was used to obtain superhydrophilic CNT films. → Superhydrophilic CNTs films were successfully produced by incorporation of carboxylic groups. → Cellular adhesion on superhydrophilic VACNT films was analyzed in real time. → Wettability of CNT films directly affects the cellular migration, proliferation and adhesion.

  19. A combination of small molecules directly reprograms mouse fibroblasts into neural stem cells.

    Zheng, Jie; Choi, Kyung-Ah; Kang, Phil Jun; Hyeon, Solji; Kwon, Suhyun; Moon, Jai-Hee; Hwang, Insik; Kim, Yang In; Kim, Yoon Sik; Yoon, Byung Sun; Park, Gyuman; Lee, JangBo; Hong, SungHoi; You, Seungkwon

    2016-07-15

    The generation of induced neural stem cells (iNSCs) from somatic cells using defined factors provides new avenues for basic research and cell therapies for various neurological diseases, such as Parkinson's disease, Huntington's disease, and spinal cord injuries. However, the transcription factors used for direct reprogramming have the potential to cause unexpected genetic modifications, which limits their potential application in cell therapies. Here, we show that a combination of four chemical compounds resulted in cells directly acquiring a NSC identity; we termed these cells chemically-induced NSCs (ciNSCs). ciNSCs expressed NSC markers (Pax6, PLZF, Nestin, Sox2, and Sox1) and resembled NSCs in terms of their morphology, self-renewal, gene expression profile, and electrophysiological function when differentiated into the neuronal lineage. Moreover, ciNSCs could differentiate into several types of mature neurons (dopaminergic, GABAergic, and cholinergic) as well as astrocytes and oligodendrocytes in vitro. Taken together, our results suggest that stably expandable and functional ciNSCs can be directly reprogrammed from mouse fibroblasts using a combination of small molecules without any genetic manipulation, and will provide a new source of cells for cellular replacement therapy of neurodegenerative diseases. PMID:27207831

  20. IL-17A synergistically enhances TNFα-induced IL-6 and CCL20 production in 3T3-L1 adipocytes.

    Shinjo, Takanori; Iwashita, Misaki; Yamashita, Akiko; Sano, Tomomi; Tsuruta, Mitsudai; Matsunaga, Hiroaki; Sanui, Terukazu; Asano, Tomoichiro; Nishimura, Fusanori

    2016-08-19

    Interleukin-17A (IL-17A) is known to induce inflammatory responses and to be involved in the pathogenesis of not only autoimmune diseases, but also several metabolic and infectious diseases. In this study, IL-17A is shown to induce IL-6 expression in 3T3-L1 mature adipocytes. Interestingly, we found that IL-17A synergistically amplified TNFα-induced secretion of IL-6 and upregulation of IL-17RA expression in 3T3-L1 adipocytes. Its synergistic effects on IL-6 production were inhibited by pre-treatment with inhibitors of IκBα and JNK. Furthermore, IL-17A cooperatively enhanced LPS-mediated IL-6 production in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. In addition, IL-17A also enhanced CCL20 production in 3T3-L1 adipocytes stimulated with TNFα or co-cultured with LPS-stimulated RAW macrophages. In high-fat diet-fed mouse epididymal adipose tissues, IL-17RA and RORγt mRNA levels were significantly increased and the serum level of CCL20 was also upregulated. Taken together, these data show that, in adipose tissues, IL-17A contributes to exacerbating insulin resistance-enhancing IL-6 production and promotes the infiltration of Th17 cells in cooperation with TNFα; these findings represent a novel hypothesis for the association between IL-17A-producing cells and type 2 diabetes. PMID:27311858

  1. Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

    Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

    2014-03-01

    Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

  2. Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

    Soon-Jeong JEONG; Su-Gwan KIM; Jiyun YOO; Mi-Young HAN; Joo-Cheol PARK; Heung-Joong KIM; Seong Soo KANG; Baik-Dong CHOI; Moon-Jin JEONG

    2006-01-01

    Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells.Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins.The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ interacted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway,was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.

  3. Isoproterenol Increases Uncoupling, Glycolysis, and Markers of Beiging in Mature 3T3-L1 Adipocytes.

    Colette N Miller

    Full Text Available Beta-adrenergic activation stimulates uncoupling protein 1 (UCP1, enhancing metabolic rate. In vitro, most work has studied brown adipocytes, however, few have investigated more established adipocyte lines such as the murine 3T3-L1 line. To assess the effect of beta-adrenergic activation, mature 3T3-L1s were treated for 6 or 48 hours with or without isoproterenol (10 and 100 μM following standard differentiation supplemented with thyroid hormone (T3; 1 nM. The highest dose of isoproterenol increased lipid content following 48 hours of treatment. This concentration enhanced UCP1 mRNA and protein expression. The increase in UCP1 following 48 hours of isoproterenol increased oxygen consumption rate. Further, coupling efficiency of the electron transport chain was disturbed and an enhancement of glycolytic rate was measured alongside this, indicating an attempt to meet the energy demands of the cell. Lastly, markers of beige adipocytes (protein content of CD137 and gene transcript of CITED1 were also found to be upregulated at 48 hours of isoproterenol treatment. This data indicates that mature 3T3-L1 adipocytes are responsive to isoproterenol and induce UCP1 expression and activity. Further, this finding provides a model for further pharmaceutical and nutraceutical investigation of UCP1 in 3T3-L1s.

  4. Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

    The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 106 cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture

  5. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

    Lee K

    2015-03-01

    Full Text Available Kunwoo Lee,1,2 Pengzhi Yu,3 Nithya Lingampalli,1 Hyun Jin Kim,1 Richard Tang,1 Niren Murthy1,2 1Department of Bioengineering, University of California, Berkeley, CA, USA; 2UC Berkeley and UCSF Joint Graduate Program in Bioengineering, Berkeley/San Francisco, CA, USA; 3Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA Abstract: The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from a-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. Keywords: direct cardiac

  6. AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase β knockout mouse embryonic fibroblasts

    Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3'-OH and 5'-deoxyribose phosphate (5'-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5'-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER. (author)

  7. Biosynthesis of a mannolipid containing a metabolite of retinoic acid by 3T12 mouse fibroblasts

    Retinol and retinoic acid (RA) increase the adhesive properties of spontaneously transformed mouse fibroblasts and the incorporation of [2-3H]mannose into cellular glycoconjugates. Therefore we searched for a mannolipid of retinoic acid similar to mannosylretinylphosphate (MRP) in these cells. Labeled RA was incorporated into a compound similar to standard MRP. This metabolite contained the same 3H:14C ratio as the precursor [11, 12-3H, 15-14C]retinoic acid, demonstrating that no decarboxylation had occurred. A doubly labeled mannolipid was obtained from cells incubated with [2-3H]mannose and [15-14C]retinoic acid. This mannolipid was readily cleaved by mild acid, yielding [3H]mannosephosphate and a compound that migrated as standard anhydroretinol at Rf 0.93. Standard all trans-MRP yields all-trans-anhydroretinol under these conditions. A HPLC system was developed to further characterize the mannolipids obtained from retinol and retinoic acid in 3T12 cells. [15-3H]Retinol and [15-14C]retinoic acid were incorporated into mannolipids that cochromatographed with standard MRP. The mixture of the [15-3H]retinol and [15-14C]retinoic acid derived mannolipids was subjected to mild acid hydrolysis, after purification by HPLC yielding all-trans-[3H]anhydroretinol and a [14C]labeled product which was eluted from HPLC as a slightly more polar compound than all-trans-anhydroretinol. The retinoic acid-derived mannolipid (MXP) represented approximately 4% of the total radioactivity in the methanolic extract of 3T12 cells incubated in labeled retinoic acid. However, if the cells were incubated for an additional 20 hours in the absence of the radioactive precursor, MXP represented 40% of the total extracted radioactivity

  8. Construction of a eukaryotic expression plasmid pcDNA3.1-HuR-FLAG and its transient expression in NIH3T3 cells

    Tao LI

    2011-04-01

    Full Text Available Objective To construct a eukaryotic expression vector for HuR and analyze its expression and biological function in NIH3T3 cells.Methods The total RNA was extracted from NIH3T3 cells and reverse transcribed to cDNAs.The coding region sequence of mouse HuR was then amplified by PCR and subcloned into the pcDNA3.1-FLAG plasmid.The recombinant plasmid pcDNA3.1-HuR-FLAG was verified by PCR and restriction endonuclease analysis,confirmed by DNA sequence analysis,and then transiently transfected into NIH3T3 cells with Lipofectamine LTX.The expression of HuR protein was determined by Western blotting,and the mRNA level of HuR and DUSP1 were analyzed by using real-time PCR.Result The recombinant plasmid pcDNA3.1-HuR-FLAG was correctly constructed.Twenty-four hours after transfection of the recombinant plasmid into NIH3T3 cells,the fusion protein was found to have highly expressed in the cells as revealed by Western blotting.Real-time PCR results detected that the over-expression of HuR could up-regulate the expression of DUSP1.Conclusion The eukaryotic expression vector for HuR-FLAG fusion protein has been successfully constructed and transiently expressed in NIH3T3 cells.It can be used in further analysis of the posttranscriptional regulation of DUSP1 by HuR in cancer cells.

  9. PPARγ partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes

    Milton, Flora Aparecida [Faculdade de Ciências da Saúde, Laboratório de Farmacologia Molecular, Universidade de Brasília (Brazil); Genomic Medicine, Houston Methodist Research Institute, Houston, TX (United States); Cvoro, Aleksandra [Genomic Medicine, Houston Methodist Research Institute, Houston, TX (United States); Amato, Angelica A. [Faculdade de Ciências da Saúde, Laboratório de Farmacologia Molecular, Universidade de Brasília (Brazil); Sieglaff, Douglas H.; Filgueira, Carly S.; Arumanayagam, Anithachristy Sigamani [Genomic Medicine, Houston Methodist Research Institute, Houston, TX (United States); Caro Alves de Lima, Maria do; Rocha Pitta, Ivan [Laboratório de Planejamento e Síntese de Fármacos – LPSF, Universidade Federal de Pernambuco (Brazil); Assis Rocha Neves, Francisco de [Faculdade de Ciências da Saúde, Laboratório de Farmacologia Molecular, Universidade de Brasília (Brazil); Webb, Paul, E-mail: pwebb@HoustonMethodist.org [Genomic Medicine, Houston Methodist Research Institute, Houston, TX (United States)

    2015-08-28

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1 adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPARγ partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. - Highlights: • GQ-16 is an insulin sensitizing PPARγ ligand with reduced harmful side effects. • GQ-16 displays a continuum of weak partial agonist activities at PPARγ-induced genes. • GQ-16 exerts strong repressive effects at a subset of genes. • These inhibitor actions should be evaluated in models of adipose tissue inflammation.

  10. PPARγ partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1 adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPARγ partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. - Highlights: • GQ-16 is an insulin sensitizing PPARγ ligand with reduced harmful side effects. • GQ-16 displays a continuum of weak partial agonist activities at PPARγ-induced genes. • GQ-16 exerts strong repressive effects at a subset of genes. • These inhibitor actions should be evaluated in models of adipose tissue inflammation

  11. Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells

    A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangement. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas

  12. Cytotoxic effects of the synthetic oestrogens and androgens on Balb/c 3T3 and HepG2 cells

    Minta Maria

    2014-12-01

    Full Text Available The aim of the study was to test and compare the cytotoxic potential of two synthetic oestrogens: diethylstilboestrol (DES and ethinyloestradiol (EE2 and two androgens: testosterone propionate (TP and trenbolone (TREN on two cell lines. The fibroblast cell line Balb/c 3T3 and the hepatoma cell line HepG2 were selected. To get more insight into the mode of toxic action, four methods were used, which evaluated different biochemical endpoints: mitochondrial activity (3-(4,5-dimethylthiazol-2-yl- 2,5-diphenyltetrazolium bromide reduction assay, lysosomal activity (neutral red uptake assay, total protein content, and lactate dehydrogenase release. Cytotoxicity was assessed after 24, 48, and 72 h exposure to eight concentrations ranging from 0.78 to 100 μg/mL. Concentration- and time- dependent effects were observed. Depending on the line and assay used, half maximal effective concentration after 72 h (EC50-72h values ranged as follows: DES 1-13.7 μg/mL (Balb/c 3T3 and 3.7-5.2 μg/mL (HepG2; EE2 2.1-14.3 μg/mL (Balb/c 3T3 and 1.8-7.8 μg/mL (HepG2; TP-14.9-17.5 μg/mL (Balb/c 3T3, and 63.9- 100 μg/mL (HepG2; and TREN 11.3-31.4 μg/mL (Balb/c 3T3 and 12.5-59.4 μg/mL (HepG2. The results revealed that oestrogens were more toxic than androgens and the most affected endpoint was mitochondrial activity. In contrast to oestrogens, for which EC50-72h values were similar in both lines and by all assays used, Balb/c 3T3 cells were more sensitive than HepG2 cells to TP.

  13. Wounding a fibroblast monolayer results in the rapid induction of the c-fos proto-oncogene.

    Verrier, B; Müller, D; Bravo, R.; Müller, R.

    1986-01-01

    The c-fos gene has previously been shown to be transiently induced within minutes after the stimulation of mouse fibroblasts with growth factors. Induction of c-fos was observed specifically with competence factors (e.g., platelet-derived growth factor), not with progression factors (e.g., platelet-poor plasma), suggesting a role for c-fos in conferring competence on fibroblasts. To test this hypothesis we have analyzed c-fos expression in NIH 3T3 cells that were made competent in a different...

  14. Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

    Ishii, Ikumi; Ikeguchi, Yoshihiko; Mano, Hiroshi; Wada, Masahiro; Pegg, Anthony E.; Shirahata, Akira

    2011-01-01

    Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on a...

  15. Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.

    Deutsch, P J; Wan, C F; Rosen, O M; Rubin, C S

    1983-01-01

    Cell surface and cryptic insulin receptors were solubilized from the particulate fraction of murine 3T3-L1 adipocytes with buffer containing 1% Triton X-100. Solubilized receptors were affinity crosslinked with 125I-labeled insulin and disuccinimidyl suberate and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography after specific immunoprecipitation. Two insulin-binding polypeptides were identified: the more abundant protein had a Mr of 130,000, corre...

  16. Rosiglitazone Balances Insulin-Induced Exo- And Endocytosis In Single 3t3-L1 Adipocytes

    Velebit, Jelena; Chowdhury, Helena H.; Kreft, Marko; Zorec, Robert

    2011-01-01

    Abstract Rosiglitazone (Rosi) improves insulin sensitivity and increases the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This involves the fusion of membrane-bound compartments with the plasma membrane, thus increasing the plasma membrane area. However, recent work has shown that in Rosi-pretreated 3T3-L1 adipocytes membrane area did not increase following insulin application, suggesting that the rates of exo- and endocytosis are balanced. Here we ex...

  17. Stevioside from Stevia rebaudiana Bertoni Increases Insulin Sensitivity in 3T3-L1 Adipocytes

    Nabilatul Hani Mohd-Radzman; Wan Iryani Wan Ismail; Siti Safura Jaapar; Zainah Adam; Aishah Adam

    2013-01-01

    Stevioside from Stevia rebaudiana has been reported to exert antihyperglycemic effects in both rat and human subjects. There have been few studies on these effects in vitro. In this paper, radioactive glucose uptake assay was implemented in order to assess improvements in insulin sensitivity in 3T3-L1 cells by elevation of glucose uptake following treatment with stevioside. Oil Red-O staining and MTT assay were utilized to confirm adipocyte differentiation and cell viability, respectively. Fi...

  18. Vasopressin induces selective desensitization of its mitogenic response in Swiss 3T3 cells.

    Collins, M.K.; Rozengurt, E

    1983-01-01

    Prior incubation of quiescent cultures of Swiss 3T3 cells with vasopressin leads to loss of mitogenic stimulation on its subsequent addition in the presence of a synergistic growth factor. This desensitization is selective for vasopressin, requires prolonged incubation (half-maximal desensitization after 12 hr of treatment) for its induction, and is reversed after a 48-hr incubation in the absence of vasopressin. It is elicited by concentrations of vasopressin, and several analogues, similar ...

  19. The intracellular mechanism of alpha-fetoprotein promoting the proliferation of NIH 3T3 cells

    2002-01-01

    AIM The existence and properties of alpha-fetoprotein (AFP) receptor on the surface of NIH 3T3 cells and the effects of AFP on cellular signal transduction pathway were investigated. METHODS The effect of AFP on the proliferation of NIH 3T3 cells was measured by incorporation of 3H-TdR. Receptor-binding assay of 125I-AFP was performed to detect the properties of AFP receptor in NIH 3T3 cells. The influences of AFP on the [cAMP]i and the activities of protein kinase A (PKA) were determined. Western blot was used to detect the change of K-ras P21 protein expression. RESULTS The proliferation of NIH 3T3 cells treated with 0-80 mg/L of AFP was significantly enhanced. The Scatchard analysis indicated that there were two classes of binding sites with KD of 2.722×10-9M (Bmax=12810 sites per cell) and 8.931× 10-SM (Bmax=l19700 sites per cell) respectively. In the presence of AFP (20 mg/L), the content of cAMP and activities of PKA were significantly elevated . The level of K-ras P21 protein was upregulated by AFP at the concentration of 20 mg/L. The monoclonal antibody against AFP could reverse the effects of AFP on the cAMP content, PKA activity and the expression of K-ras p21 gene. CONCLUSION The effect of AFP on the cell proliferation was achieved by binding its receptor to trigger the signal transduction pathway of cAMP-PKA and alter the expression of K- ras p21 gene.

  20. Xylitol does not directly affect adiponectin productionand adipogenesis in 3T3-L1 cells

    Pilaiwan Siripurkpong; Sompoch Prajan; Sudawadee Kongkhum

    2014-01-01

    Xylitol is widely used as a low-calorie sweetener in various kinds of food products, including diabetic foods. Adiponectin, secreted by adipocytes, plays a key role in carbohydrate and lipid metabolism. Low levels of plasma adiponectin are associated with cardiovascular disease and type II diabetes. The aims of this study were to determine effects of xylitol on the adipogenesis of pre-adipocytes, adiponectin synthesis and secretion. To assess adipogenesis, pre-adipocyte 3T3-L1 cel...

  1. An electron microscopic radioautographic study of collagen secretion in periodontal ligament fibroblasts of the mouse: II. Colchicine-treated fibroblasts

    Colchicine administered intravenously depolymerized microtubules and disrupted the normal organization of the Golgi apparatus in periodontal ligament fibroblasts. Radioautography with 3H-proline indicated that collagen secretion was completely inhibited during a period of approximately 4 hours following the onset of the colchicine effect. During this period of secretory inhibition, labeled collagen precursors were present within a variety of dense bodies, primarily located in a juxtanuclear location replacing the normal Golgi complex. The time course of 3H-proline labeling from 2 to 8 hours suggested that small, newly formed dense bodies fused to form larger dense bodies and pleomorphic structures (zebra bodies), within which collagen precursors appeared to undergo partial polymerization. Autophagosomes, many labeled with 3H-proline, also increased in number after colchicine administration. A gradual decline in 3H-proline label occurred from 4 to 24 hours, presumably due to exocytosis of dense bodies or by the digestion of labeled collagen precursors within autophagosomes. These results support the concept that an intact microtubular network is essential for the organized transport of collagen precursors, from the rough endoplasmic reticulum to the Golgi apparatus, and the eventual transport and exocytosis of collagen secretory granules

  2. Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway

    Chian-Jiun Liou

    2015-01-01

    Full Text Available Ginkgolide C, isolated from Ginkgo biloba leaves, is a flavone reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the antiadipogenic effect of ginkgolide C in 3T3-L1 adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to adipogenesis and lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated adipocytes. It also decreased adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK, resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK inhibitor (compound C, ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing lipolysis and inhibiting adipogenesis in adipocytes through the activated AMPK pathway.

  3. Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation

    Prostaglandins (PGs) have been shown to play various roles in adipogenesis. In this study, we investigated on which PGE receptor subtypes are involved in the inhibition of 3T3-L1 preadipocyte differentiation. The triglyceride content of cells, used as an index of differentiation, was decreased when PGE2, the FP-agonist fluprostenol or dibutyryl cAMP, was exogenously added to differentiation cocktails. 3T3-L1 preadipocyte cells express mRNAs for the prostanoid EP4, FP, and IP receptors. PGE2 and the EP4 agonist AE1-329 increased cAMP levels in preadipocytes in a dose-dependent manner. AE1-329 suppressed the expression induction of differentiation marker genes such as resistin and peroxisome proliferator-activated receptor-γ. The inhibitory effect of PGE2 but not that of fluprostenol was reversed by the addition of the EP4 antagonist AE3-208. AE3-208 mimicked the differentiation-promoting effects of indomethacin. These results suggest that the EP4 receptor mediates the suppressive action of PGE2 in 3T3-L1 adipocyte differentiation

  4. Stevioside from Stevia rebaudiana Bertoni Increases Insulin Sensitivity in 3T3-L1 Adipocytes

    Nabilatul Hani Mohd-Radzman

    2013-01-01

    Full Text Available Stevioside from Stevia rebaudiana has been reported to exert antihyperglycemic effects in both rat and human subjects. There have been few studies on these effects in vitro. In this paper, radioactive glucose uptake assay was implemented in order to assess improvements in insulin sensitivity in 3T3-L1 cells by elevation of glucose uptake following treatment with stevioside. Oil Red-O staining and MTT assay were utilized to confirm adipocyte differentiation and cell viability, respectively. Findings from this research showed a significant increase in absorbance values in mature adipocytes following Oil Red-O staining, confirming the differentiation process. Stevioside was noncytotoxic to 3T3-L1 cells as cell viability was reduced by a maximum of 17%, making it impossible to determine its IC50. Stevioside increased glucose uptake activities by 2.1 times (p<0.001 in normal conditions and up to 4.4 times (p<0.001 in insulin-resistant states. At times, this increase was higher than that seen in positive control group treated with rosiglitazone maleate, an antidiabetic agent. Expressions of pY20 and p-IRS1 which were measured via Western blot were improved by stevioside treatment. In conclusion, stevioside has direct effects on 3T3-L1 insulin sensitivity via increase in glucose uptake and enhanced expression of proteins involved in insulin-signalling pathway.

  5. Radiation transformation of BALB/3T3 cells activates dominant transforming gene

    Radiation is known to induce DNA strand breaks, and cause cellular transformation. The biochemical events responsible for these effects are not well-established. The authors investigated the possible roles of gene rearrangement and gene amplification in the process of radiation transformation. BALB/3T3 cells were exposed to 100 to 400 cGy of gamma radiation, and transformed cell lines were established. These were characterized by colony-formation in soft agar and induction of in nude mice. DNA was isolated and transfected onto pre-neoplastic NIH3T3 cells. The transformation frequency increased from a control of 0.01 foci/microgram DNA (normal BALB/3T3) to 0.26 foci/microgram of radiation transformed DNA. To investigate whether a single dominant transforming gene was involved, transfection was repeated following digestion of DNA with multiple restriction endonucleases. No gross rearrangement in the DNAs of transformed cell lines was observed by the Southern-blot hybridization technique for six representative cellular oncogenes. In addition, the expression of these oncogenes was not altered as revealed by cytodot blot analysis

  6. Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayoshi [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

    2013-02-01

    Highlights: ► Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ► Adipose lipin-1 expression is reduced in obesity. ► ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ► Activation of PPAR-γ recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-α and interleukin-1β reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

  7. Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

    Ukita, Mayumi; Yamaguchi, Taihiko; Ohata, Noboru; Tamura, Masato

    2016-06-01

    Sclerostin, a secreted protein encoded by the Sost gene, is produced by osteocytes and is inhibited by osteoblast differentiation and bone formation. Recently, a functional association between bone and fat tissue has been suggested, and a correlation between circulating sclerostin levels and lipid metabolism has been reported in humans. However, the effects of sclerostin on adipogenesis remain unexplored. In the present study, we examined the role of sclerostin in regulating adipocyte differentiation using 3T3-L1 preadipocytes. In these cells, sclerostin enhanced adipocyte-specific gene expression and the accumulation of lipid deposits. Sclerostin also upregulated CCAAT/enhancer binding protein β expression but not cell proliferation and caspase-3/7 activities. Sclerostin also attenuated canonical Wnt3a-inhibited adipocyte differentiation. Recently, the transcriptional modulator TAZ has been involved in the canonical Wnt signaling pathway. Sclerostin reduced TAZ-responsive transcriptional activity and TAZ-responsive gene expression. Transfection of 3T3-L1 cells with TAZ siRNA increased the lipid deposits and adipogenic gene expression. These results show that sclerostin upregulates adipocyte differentiation in 3T3-L1 cells, suggesting a possible role for the osteocyte-derived sclerostin as a regulator of fat metabolism and as a reciprocal regulator of bone and adipose tissues metabolism. J. Cell. Biochem. 117: 1419-1428, 2016. © 2015 Wiley Periodicals, Inc. PMID:26553151

  8. Extract of Chaga mushroom (Inonotus obliquus) stimulates 3T3-L1 adipocyte differentiation.

    Joo, Jeong In; Kim, Dong Hyun; Yun, Jong Won

    2010-11-01

    Chaga mushroom (Inonotus obliquus) has long been used as a folk medicine due to its numerous biological functions such as antibacterial, antiallergic, antiinflammatory and antioxidative activities. In the present study, it was found that the I. obliquus hot water extract (IOWE) activated adipogenesis of 3T3-L1 preadipocytes. Even in the absence of adipogenic stimuli by insulin, the IOWE strongly induced adipogenesis of 3T3-L1 preadipocytes. The major constituent of IOWE was glucose-rich polysaccharides with a molecular mass of 149  kDa. IOWE enhanced the differentiation of 3T3-L1 preadipocytes, increasing TG (triacylglycerol) accumulation that is critical for acquisition of the adipocyte phenotype, in a dose-dependent manner. IOWE stimulated gene expression of C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome proliferator-activated receptors γ) during adipocyte differentiation, and induced the expression of PPARγ target genes such as aP2 (adipocyte protein 2), LPL (lipoprotein lipase) and CD36 (fatty acid translocase). Immunoblot analysis revealed that IOWE increased the expression of adipogenic makers such as PPARγ and GLUT4 (glucose transporter 4). The luciferase reporter assay demonstrated that IOWE did not exhibit PPARγ ligand activity. Although these results require further investigation, the ability of natural mushroom product to increase PPARγ transcriptional activities may be expected to be therapeutic targets for dyslipidemia and type 2 diabetes. PMID:21031614

  9. Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes

    Highlights: ► Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ► Adipose lipin-1 expression is reduced in obesity. ► ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ► Activation of PPAR-γ recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-α and interleukin-1β reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion

  10. PLA2 - a major regulator of volume-sensitive taurine release in NIH3T3 fibroblasts

    Lambert, I. H.

    2006-01-01

    -lipoxygenase (5-LO) system is prevented by the 5-LO inhibitor ETH 615-139 and is reduced under hypertonic conditions. Exposure to the amphiphilic bee venom peptide melittin, which has no effect on the kinetic properties of PLA2 but promotes substrate replenishment, induces release of arachidonic acid and...

  11. High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

    Christensen, Søren Tvorup; Voss, Jesper W.; Teilmann, Stefan C.;

    2005-01-01

    Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na+-dependent taurine transporter...... TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium....

  12. Quantification of effects of irradiation of low power laser about fibroblast 3T3 in culture: Growth parameters

    This work focuses on the practical determination protocol of parameters two basic cell culture growth (latency time and period of exponential duplication or doubling time), for the monitoring of samples subjected to LLLT. (Author)

  13. Macerated-Pineapple Core Crude Extract-derived Bromelain Has Low Cytotoxic Effect in NIH-3T3 Fibroblast

    Dewi Liliany Margaretta; Angliana Chouw; Yanni Dirgantara; Melanie Sadono Djamil; Ferry Sandra

    2015-01-01

    BACKGROUND: Bromelain is a sulfhydryl proteolytic enzyme that can hydrolyze protein, protease or peptide. Bromelain can be found in pineapple stem, fruit and core. Bromelain is composed of 212 amino acid residues with cysteine-25 forming a polypeptide chain that can hydrolyze peptide bonds by H2O. In medicine, bromelain has been developed as antibiotic, cancer drug, anti-inflammatory agent and immunomodulator. In dentistry, bromelain has potential to reduce plaque formation on the teeth and t...

  14. The aporphine alkaloid boldine induces adiponectin expression and regulation in 3T3-L1 cells.

    Yu, Bangning; Cook, Carla; Santanam, Nalini

    2009-10-01

    Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  15. Hydrogen sulfide promotes adipogenesis in 3T3L1 cells.

    Chin-Yi Tsai

    Full Text Available The effect of hydrogen sulfide (H2S on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H2S was increased after 3T3L1 differentiation. The expression of the H2S-synthesising enzymes, cystathionine γ-lyase (CSE, cystathionine β-synthase (CBS and 3-mercaptopyruvate sulfurtransferase (3-MST, was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2, a key regulator of this process, was increased by GYY4137 (a slow-releasing H2S donor compound and sodium hydrosulfide (NaHS, a classical H2S donor but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor, DL-propargylglycine (PAG, CSE inhibitor as well as by CSE small interference RNA (siCSE and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H2S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H2S modulate adipogenesis and adipocyte maturation.

  16. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    Highlights: •Nebivolol may act as a partial agonist of β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors

  17. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Yang, Ying, E-mail: yangying_sh@yahoo.com [Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Shen, Weili, E-mail: weili_shen@hotmail.com [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)

    2013-08-16

    Highlights: •Nebivolol may act as a partial agonist of β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

  18. A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation

    2006-01-01

    Background Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor.Methods Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide.Results Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation.Conclusion RBP could effectively rescue the promoted differentiation of resistin overxepressed 3T3-L1 preadipocyte.

  19. Cell Volume Regulation and Signaling in 3T3-L1 Pre-adipocytes and Adipocytes

    Eduardsen, Kathrine; Larsen, Susanne; Novak, Ivana;

    2011-01-01

    osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required...... had no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity...

  20. Mitotic defects lead to pervasive aneuploidy and accompany loss of RB1 activity in mouse LmnaDhe dermal fibroblasts.

    C Herbert Pratt

    Full Text Available BACKGROUND: Lamin A (LMNA is a component of the nuclear lamina and is mutated in several human diseases, including Emery-Dreifuss muscular dystrophy (EDMD; OMIM ID# 181350 and the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS; OMIM ID# 176670. Cells from progeria patients exhibit cell cycle defects in both interphase and mitosis. Mouse models with loss of LMNA function have reduced Retinoblastoma protein (RB1 activity, leading to aberrant cell cycle control in interphase, but how mitosis is affected by LMNA is not well understood. RESULTS: We examined the cell cycle and structural phenotypes of cells from mice with the Lmna allele, Disheveled hair and ears (Lmna(Dhe. We found that dermal fibroblasts from heterozygous Lmna(Dhe (Lmna(Dhe/+ mice exhibit many phenotypes of human laminopathy cells. These include severe perturbations to the nuclear shape and lamina, increased DNA damage, and slow growth rates due to mitotic delay. Interestingly, Lmna(Dhe/+ fibroblasts also had reduced levels of hypophosphorylated RB1 and the non-SMC condensin II-subunit D3 (NCAP-D3, a mitosis specific centromere condensin subunit that depends on RB1 activity. Mitotic check point control by mitotic arrest deficient-like 1 (MAD2L1 also was perturbed in Lmna(Dhe/+ cells. Lmna(Dhe/+ fibroblasts were consistently aneuploid and had higher levels of micronuclei and anaphase bridges than normal fibroblasts, consistent with chromosome segregation defects. CONCLUSIONS: These data indicate that RB1 may be a key regulator of cellular phenotype in laminopathy-related cells, and suggest that the effects of LMNA on RB1 include both interphase and mitotic cell cycle control.

  1. Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims

    Priscila Cardoso Cristovam

    2008-10-01

    Full Text Available OBJETIVO: Avaliar a importância da presença de células 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo obtido de rimas córneo-esclerais. MÉTODOS: Rimas de diferentes doadores tiveram seus estroma posterior e endotélio removidos (n=6. Cada rima foi dividida em três segmentos iguais, que foram colocados em cultura em três diferentes condições: um segmento foi colocado na placa de cultura com o lado epitelial para cima (Grupo A. Os dois segmentos restantes foram tripsinizados e a suspensão de células obtida foi cultivada com (Grupo B ou sem (Grupo C células 3T3 irradiadas. As células foram mantidas em meio de cultura "supplemental hormonal epithelial médium" (SHEM, a migração epitelial e a formação de clones nos grupos A, B e C foram avaliadas pela microscopia de contraste de fase e por coloração pela rodamina B. Os resultados foram comparados estatisticamente. RESULTADOS: O crescimento de células epiteliais foi observado em 4/6 rimas (Grupo A. Todas as suspensões de células epiteliais que foram cultivadas com células 3T3 (Grupo B formaram clones. Nenhuma adesão ou formação de clones verdadeiros (holo ou meroclones foi observada na cultura de células que foi cultivada sem 3T3 (Grupo C (p=0,009. CONCLUSÕES: Suspensão de células epiteliais límbicas obtidas de rimas córneo-esclerais no modelo utilizado precisa ser cultivada com células 3T3 para formar clones e estabelecer colônias epiteliais com perspectivas para uso terapêutico na reconstrução da superfície ocular.PURPOSE: To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR. METHODS: Corneo-scleral rims from different donors (n=6 had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the

  2. Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

    Xing ZHONG; Ling-ling XIU; Guo-hong WEI; Yuan-yuan LIU; Lei SU; Xiao-pei CAO; Yan-bing LI; Hai-peng XIAO

    2011-01-01

    Aim: To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.Methods: MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.Results: Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezaflbrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARa inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 pmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast prolifera-tion.Conclusion: Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.

  3. Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells.

    Owen, R D; Ostrowski, M C

    1987-01-01

    Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-r...

  4. NMR studies of pH regulation in NIH 3T3 cells

    In order to understand the correlation of intracellular pH (pH/sub in/) and mitogenic stimulation, they have undertaken NMR studies of the regulation of the intracellular pH in perfused cultures of 3T3 cells anchored on microcarrier beads. Because these cells have very low levels of intracellular inorganic phosphate under well energized conditions, an added pH indicator is required in this system. The 31P NMR indicators, deoxyglucose 6P, and methyl phosphonate, have, for different reasons, also proven unsatisfactory. Of a series of phosphono-amino acids studied as possible alternative indicators, the best thus far have proven to be 2-amino-5-phosphonovaleric acid and 2-amino-6 phosphono-hexanoic acid. Their properties include: (a) physiological pKa, 7.1 and 7.5, respectively; (b) sensitivity greater than 1 ppm/1 pH unit; (c) resonant frequency far from the phosphate region; (d) low toxicity. In confluent cultures of 3T3 cells grown on 200 μm diameter polystyrene beads the pH/sub in/ remains approximately constant at 7.05 as the external pH is varied between 7.0 and 7.8. No pH shift is observed in this system upon addition of 10% FBS to cells previously incubated for 24 hours in 1% FBS. This finding is at variance with previous reports of an intracellular alkalinization following stimulation by serum or other mitogens

  5. Behavior of MC3T3-E1 Osteoblast Cultured on Chitosan Modified with Polyvinylpyrrolidone

    XI Jing; GAO Yuan; KONG Lijun; GONG Yandao; ZHAO Nanming; ZHANG Xiufang

    2005-01-01

    The physical and chemical properties of four kinds of modified chitosan materials made by blending chitosan with polyvinylpyrrolidone (PVP) were investigated. All four of these modified chitosan materials were hydrophilic with water contact angles ranging from 59° to 69°. Fourier transform-infrared spectra of the modified materials showed a new band at 1288 cm-1, implying formation of a surface physical interpenetrating network structure. Enzyme linked immunosorbent assay results indicated that much less fibronectin was adsorbed on the modified materials than on only chitosan. The viability of MC3T3-E1 osteoblasts cultured on the materials was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide assay. The results show that adding PVP10000 into the chitosan promotes adhesion of MC3T3-E1 osteoblasts on the modified materials, but has no effect on cell growth and proliferation; while adding PVP40000 reduces cell adhesion, growth, and proliferation. The results suggest that the increased hydrophilicity of the material surface does not always improve its biocompatibility, which will influence the selection and design of biomaterials.

  6. Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells.

    Ishii, Ikumi; Ikeguchi, Yoshihiko; Mano, Hiroshi; Wada, Masahiro; Pegg, Anthony E; Shirahata, Akira

    2012-02-01

    Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N(1)-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis. PMID:21809076

  7. High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells

    Gastrin-releasing peptide (GRP) labeled with 125I at tyrosine-15 (125I-GRP) binds to intact quiescent Swiss 3T3 cells in a specific and saturable manner. Scatchard analysis indicates the presence of a single class of high-affinity binding sites of Kd = 0.5 X 10(-9) M and a value for the number of sites per cell of about 100,000. 125I-GRP binding was not inhibited by other mitogens for these cells, and cell lines that are mitogenically unresponsive to GRP do not exhibit specific GRP binding. Structure-activity relationships show a close parallel between the ability of a range of GRP-related peptides to both inhibit GRP binding and to stimulate mitogenesis. Further, GRP binding is selectively blocked in a competitive fashion by a novel bombesin antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P. In addition, this compound selectively inhibits GRP and bombesin-induced mitogenesis. These results demonstrate that the mitogenic response of Swiss 3T3 cells to peptides of the bombesin family is mediated by a class of receptors distinct from those of other mitogens for these cells

  8. Xylitol does not directly affect adiponectin productionand adipogenesis in 3T3-L1 cells

    Pilaiwan Siripurkpong

    2014-08-01

    Full Text Available Xylitol is widely used as a low-calorie sweetener in various kinds of food products, including diabetic foods. Adiponectin, secreted by adipocytes, plays a key role in carbohydrate and lipid metabolism. Low levels of plasma adiponectin are associated with cardiovascular disease and type II diabetes. The aims of this study were to determine effects of xylitol on the adipogenesis of pre-adipocytes, adiponectin synthesis and secretion. To assess adipogenesis, pre-adipocyte 3T3-L1 cells were treated with xylitol during cell differentiation and fat droplets in the mature adipocytes were stained with oil red O. Adiponectin levels were determined by Western blot in both culture media and mature adipocytes treated with xylitol. There were no significant differences in the levels of adipogenesis, adiponectin synthesis and secretion in the xylitol-treated 3T3-L1 cells compared with the untreated control cells. This suggests that xylitol does not have a direct effect on adipogenesis or on adiponectin synthesis and secretion.

  9. Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes.

    Cui, Ju; Pang, Jing; Lin, Ya-Jun; Jiang, Ping; Gong, Huan; Wang, Zai; Li, Jian; Cai, Jian-Ping; Huang, Jian-Dong; Zhang, Tie-Mei

    2016-08-01

    Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. PMID:27264953

  10. Genes Responsive to Low-Intensity Pulsed Ultrasound in MC3T3-E1 Preosteoblast Cells

    Yoshiaki Tabuchi

    2013-11-01

    Full Text Available Although low-intensity pulsed ultrasound (LIPUS has been shown to enhance bone fracture healing, the underlying mechanism of LIPUS remains to be fully elucidated. Here, to better understand the molecular mechanism underlying cellular responses to LIPUS, we investigated gene expression profiles in mouse MC3T3-E1 preosteoblast cells exposed to LIPUS using high-density oligonucleotide microarrays and computational gene expression analysis tools. Although treatment of the cells with a single 20-min LIPUS (1.5 MHz, 30 mW/cm2 did not affect the cell growth or alkaline phosphatase activity, the treatment significantly increased the mRNA level of Bglap. Microarray analysis demonstrated that 38 genes were upregulated and 37 genes were downregulated by 1.5-fold or more in the cells at 24-h post-treatment. Ingenuity pathway analysis demonstrated that the gene network U (up contained many upregulated genes that were mainly associated with bone morphology in the category of biological functions of skeletal and muscular system development and function. Moreover, the biological function of the gene network D (down, which contained downregulated genes, was associated with gene expression, the cell cycle and connective tissue development and function. These results should help to further clarify the molecular basis of the mechanisms of the LIPUS response in osteoblast cells.

  11. Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

    Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-α stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-α-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-α and BMP signaling pathways.

  12. Platelet-derived growth factor mediates interleukin-13-induced collagen I production in mouse airway fibroblasts

    Jiamei Lu; Yanting Zhu; Wei Feng; Yilin Pan; Shaojun Li; Dong Han; Lu Liu; Xinming Xie; Guizuo Wang; Manxiang Li

    2014-09-01

    Interleukin-13 (IL-13) is associated with the production of collagen in airway remodelling of asthma. Yet, the molecular mechanisms underlying IL-13 induction of collagen remain unclear; the aim of this study is to address this issue. IL-13 dose- and time-dependently-induced collagen I production in primary cultured airway fibroblasts; this was accompanied with the STAT6 phosphorylation, and pre-treatment of cells with JAK inhibitor suppressed IL-13-induced collagen I production. Further study indicated that IL-13 stimulated JAK/STAT6-dependent PDGF production and subsequent ERK1/2 MAPK activation in airway fibroblasts, and the presence of either PDGF receptor blocker or MEK inhibitor partially suppressed IL-13-induced collagen I production. Taken together, our study suggests that activation of JAK/STAT6 signal pathway and subsequent PDGF generation and resultant ERK1/2 MAPK activation mediated IL-13-induced collagen I production in airway fibroblasts.

  13. Mechanism underlying defective interferon gamma-induced IDO expression in non-obese diabetic mouse fibroblasts.

    Azadeh Hosseini-Tabatabaei

    Full Text Available Indoleamine 2,3-dioxygenase (IDO can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD mice can be attributed to the impaired interferon-gamma (IFN-γ- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age female or male, and diabetic female or male (12 and 24 wks of age respectively NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I and type I collagen (COL-I. The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1 phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway.

  14. Generation of iPSCs from mouse fibroblasts with a single gene,Oct4,and small molecules

    Yanqin Li; Xu Zhang; Yetao Wu; Honggang Li; Kang Liu; Chen Wu; Zhihua Song; Yang Zhao; Yan Shi; Hongkui Deng; Qiang Zhang; Xiaolei Yin; Weifeng Yang; Yuanyuan Du; Pingping Hou; Jian Ge; Chun Liu; Weiqi Zhang

    2011-01-01

    The introduction of four transcription factors Oct4,Klf4,Sox2 and c-Myc by viral transduction can induce reprogramming of somatic cells into induced pluripotent stem cells(iPSCs),but the use of iPSCs is hindered by the use of viral delivery systems.Chemical-induced reprogramming offers a novel approach to generating iPSCs without any viral vector-based genetic modification.Previous reports showed that several small molecules could replace some of the reprogramming factors although at least two transcription factors,Oct4 and Klf4,are still required to generate iPSCs from mouse embryonic fibroblasts.Here,we identify a specific chemical combination,which is sufficient to permit reprogramming from mouse embryonic and adult fibroblasts in the presence of a single transcription factor,Oct4,within 20 days,replacing Sox2,Klf4 and c-Myc.The iPSCs generated using this treatment resembled mouse embryonic stem cells in terms of global gene expression profile,epigenetic status and pluripotency both in vitro and in vivo.We also found that 8 days of Oct4 induction was sufficient to enable Oct4-induced reprogramming in the presence of the small molecules,which suggests that reprogramming was initiated within the first 8 days and was independent of continuous exogenous Oct4 expression.These discoveries will aid in the future generation of iPSCs without genetic modification,as well as elucidating the molecular mechanisms that underlie the reprogramming process.

  15. Capsaicin induces "brite" phenotype in differentiating 3T3-L1 preadipocytes.

    Ritesh K Baboota

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

  16. Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells

    Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 μg/mL and 100 μg/mL SNP, respectively, although with minor decrease in confluence. IC50 values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 μg/mL and 449 μg/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC50 SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with ∼ 1/2 IC50 concentration of SNP (i.e. 30 μg/mL and 225 μg/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH (∼ 1.2 fold) and depletion of lipid peroxidation (∼ 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD (∼ 1.4 fold) and GSH ( 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 μg/mL in primary fibroblasts, 12.5 μg/mL in primary liver cells) than the necrotic concentration (100 μg/mL in primary fibroblasts, 500 μg/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC50 SNP (resulting in apoptosis) and 2x IC50) cells (resulting in necrosis). These results clearly

  17. The interaction of /sup 125/I-insulin with cultured 3T3-L1 adipocytes: quantitative analysis by the hypothetical grain method

    Fan, J.Y.; Carpentier, J.L.; Van Obberghen, E.; Blackett, N.M.; Grunfeld, C.; Gorden, P.; Orci, L.

    1983-07-01

    The murine 3T3-L1 fibroblast under appropriate incubation conditions differentiates into an adipocyte phenotype. This 3T3-L1 adipocyte exhibits many of the morphologic, biochemical, and insulin-responsive features of the normal rodent adipocyte. Using quantitative electron microscopic (EM) autoradiography we find that, when /sup 125/I-insulin is incubated with 3T3-L1 adipocytes, the ligand at early times of incubation localizes to the plasma membrane of the cell preferentially to microvilli and coated pits. When the incubation is continued at 37 degrees C, /sup 125/I-insulin is internalized by the cells and preferential binding to the villous surface is lost. With the internalization of the ligand, two intracellular structures become labeled, as determined by the method of hypothetical grain analysis. These include large clear, presumably endocytotic, vesicles and multivesicular bodies. Over the first hour of incubation the labeling of these structures increases in parallel, but in the second hour they diverge: the labeling of multivesicular bodies and other lysosomal forms continuing to increase and the labeling of large clear vesicles decreasing. At 3 hours limited but significant labeling occurs in small Golgi-related vesicles that have the typical distribution of GERL. The distinct morphologic features of this cell make it ideal for a quantitative morphologic analysis and allow for an unambiguous view of the sequence of events involved in receptor-mediated endocytosis of a polypeptide hormone. These events are likely to be representative of the processing of insulin by the mature rodent adipocyte.

  18. Flavanone exhibits PPARγ ligand activity and enhances differentiation of 3T3-L1 adipocytes

    Flavanones are class of polyphenolic compounds, some of which are found in foods and provide health benefits. In this study, we show that flavanone significantly enhances differentiation of 3T3-L1 preadipocytes. During adipogenesis, flavanone enhanced expression of genes and accumulation of proteins that are involved in adipocyte function. Some reports have indicated that flavanone inhibits proliferation of mammalian cells, and down-regulates expression of growth-related proteins. Such proteins include phosphorylated ERK1/2, cyclins, and Cdks that are important for an early event in adipogenesis, mitotic clonal expansion (MCE). We demonstrated that flavanone did not inhibit MCE or expression of MCE-related proteins, except for a modest inhibition of cyclin D1 expression. Using luciferase reporter assays, we found that flavanone acted as a peroxisome proliferator-activated receptor γ (PPARγ) ligand in a dose-dependent manner. Together, our results suggest that flavanone enhances adipogenesis, at least in part, through its PPARγ ligand activity.

  19. Anthraquinones from Morinda officinalis roots enhance adipocyte differentiation in 3T3-L1 cells.

    Liu, Qing; Kim, Seon Beom; Ahn, Jong Hoon; Hwang, Bang Yeon; Kim, Sung Yeon; Lee, Mi Kyeong

    2012-01-01

    To search for anti-diabetic and insulin-sensitising natural products, the effect on adipocyte differentiation was investigated by assessing fat accumulation in 3T3-L1 preadipocytes using Oil Red O staining. Fractionation and separation of n-hexane and CHCl₃ fractions of Morinda officinalis (Rubiaceae) using several chromatographic methods led to the isolation of three anthraquinones, 1,2-dimethoxyanthraquinone (1), alizarin-2-methyl ether (2) and rubiadin-1-methyl ether (3). Among them, alizarin-2-methyl ether (2) showed the strongest enhancing activity, followed by rubiadin-1-methyl ether (3) and 1,2-dimethoxyanthraquinone (1). At a concentration of 100 µM, alizarin-2-methyl ether (2) enhanced adipocyte differentiation by up to 131% (compared to insulin-treated cells). Thus, these compounds could be beneficial in the treatment of diabetes. PMID:22008000

  20. Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells.

    Inoue, Chisato; Kozaki, Tomomi; Morita, Yukiko; Shirouchi, Bungo; Fukami, Katsuya; Shimizu, Kuniyoshi; Sato, Masao; Katakura, Yoshinori

    2015-08-01

    Adipocyte size is closely related to the occurrence of diabetes, metabolic syndrome, and insulin resistance. Thus, researchers are searching for active substances that function to reduce adipocyte size. In the present study, we focused on sugar cane vinegar, Kibizu, and evaluated the function of Kibizu to reduce adipocyte size by using an in vitro model system, because people in Amami Oshima famous for longevity regularly consume Kibizu. Results showed that Kibizu treatment significantly reduced the size and number of lipid droplets in 3T3-L1 cells, relative to treatment with Kurozu, another traditional vinegar. Results of an extraction experiment suggest that the active components in Kibizu are lipophilic and hydrophobic. In addition, an in vivo experiment on rats treated with Kibizu showed that the active components were contained in large vein blood. Results of an additional in vivo experiment suggest that metabolites generated by Kibizu-treated rats are primarily contained or modified specifically in the large vein blood. PMID:25672941

  1. High extracellular calcium attenuates adipogenesis in 3T3-L1 preadipocytes

    We studied the effect of extracellular Ca2+ concentration ([Ca2+]e) on adipocyte differentiation. Preadipocytes exposed to continuous [Ca2+]e higher than 2.5 mmol/l accumulated little or no cytoplasmic lipid compared to controls in 1.8 mmol/l [Ca2+]e. Differentiation was monitored by Oil Red O staining of cytoplasmic lipid and triglyceride assay of accumulated lipid, by RT-PCR analysis of adipogenic markers, and by the activity of glycerol-3-phosphate dehydrogenase (GPDH). Elevated [Ca2+]e inhibited expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and steroid regulatory binding element protein. High [Ca2+]e significantly inhibited differentiation marker expression including adipocyte fatty acid binding protein, and GPDH. The decrease in Pref-1 expression that accompanied differentiation also was prevented by high [Ca2+]e. Treatment of 3T3-L1 cells with high [Ca2+]e did not significantly affect cell number or viability and did not trigger apoptosis. Levels of intracellular Ca+2 remained unchanged in various [Ca2+]e. Treatment of 3T3-L1 with pertussis toxin (PTX) partially restored lipid accumulation and increased differentiation markers in cells treated with 5 mmol/l [Ca2+]e. 'Classical' parathyroid cell Ca2+ sensing receptors (CaSR) were not detected either by RT-PCR or by Western blotting. These results suggest that continuos exposure to high [Ca2+]e inhibits preadipocyte differentiation and that this may involve a G-protein-coupled mechanism mediated by a novel Ca2+ sensor or receptor

  2. Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells

    Yeong-Min Yoo

    2016-04-01

    Full Text Available Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3, suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2, indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR (Ser2448, phosphorylation of extracellular signal-regulated kinase (p-ERK, and phosphorylation of serine-threonine protein kinase (p-Akt (Ser473 were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD, and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis.

  3. mTOR complex 2 phosphorylates IMP1 cotranslationally to promote IGF2 production and the proliferation of mouse embryonic fibroblasts

    Dai, Ning; Christiansen, Jan; Nielsen, Finn;

    2013-01-01

    Lack of IGF2 in mice results in diminished embryonic growth due to diminished cell proliferation. Here we show that mouse embryonic fibroblasts lacking the RNA-binding protein IMP1 (IGF2 mRNA-binding protein 1) have defective splicing and translation of IGF2 mRNAs, markedly reduced IGF2 polypeptide...

  4. Effects of Chowiseungcheng-tang Extracts on the Preadipocytes Proliferation in 3T3-L1 cell line, Lipolysis of Adipocytes in rat, and Localized Fat Accumulation by extraction methods

    Jae-eun, Lee

    2008-03-01

    Full Text Available Objectives : The purpose of this study is to investigate the effects of Chowiseungcheng-tang extracts on the preadipocytes proliferation in 3T3-L1 cell line, lipolysis of adipocytes in rat’s epididymal adipocytes and localized fat accumulation of porcine by extraction methods(alcohol and water. Methods : Diminish preadipocytes proliferation and promote lipolysis of adipocytes do primary role to reduce obesity. So, we used 3T3-L1 mouse embryo fibroblasts(preadipocytes and rat epididymal adipocytes from Sprague-Dawley rats to investigate the effects of Chowiseungcheng-tang extracts on the preadipocytes proliferation, lipolysis of adipocytes. They were treated with 0.01, 0.1, 1.0㎎/㎖ Chowiseungcheng-tang alcohol and water extracts. And for the purpose of investigating the effects of Chowiseungcheng-tang alcohol and water extracts on the localized fat accumulation, we injected 0.1, 1.0, 10.0㎎/㎖ Chowiseungcheng-tang extracts to porcine fat tissues and observed histological changes of them. Results : Following results were obtained from the preadipocytes proliferation and lipolysis of adipocytes and histological investigation of fat tissues. 1. Chowiseungcheng-tang extracts suppressed preadipocytes proliferation on the high dosage(especially 1.0㎎/㎖, and especially alcohol extracts had better effects. 2. The alcohol extracts of Chowiseungcheng-tang decreased the activity of glycerol-3-phosphate dehydrogenase(GPDH on the concentrations of 0.1, 1.0㎎/㎖. Alcohol extracts had better effects than water extracts. 3. Chowiseungcheng-tang extracts increased lipolysis of adipocytes on the concentrations of 0.1, 1.0㎎/㎖, and especially on the concentration of 1.0㎎/㎖ alcohol extract of Chowiseungcheng-tang had better effect. 4. The water extract of Chowiseungcheng-tang had significant activity to the destruction of porcine fat cell membranes only on the concentration of 10.0㎎/㎖, but alcohol extracts of Chowiseungcheng-tang had it on all

  5. Mitochondrial DNA from colorectal cancer cells induces the mutation of NIH3T3 cells%大肠癌线粒体DNA诱导NIH3T3细胞D-环突变

    姬宏莉; 姬宏娟; 宋卫兵; 马永全; 杜芳; 王辉; 肖冰

    2011-01-01

    Objective To investigate the mtDNA mutation in NIH3T3 cell, which was transfected with mutated mtDNA from colorectal cancer cell line. Methods Recombinant eukaryotic vector, expressing plasmid of the mutated mtDNA, was transfected into NIH3T3 cell using Lipofection 2000 TM and screened by G418. The mutated mtDNA of transfected NIH3T3 cell was detected by PCR. Results Mutation and polymorphism was observed in NIH3T3 cell transfected with mutated mtDNA. Conclusion MtDNA mutations in colorectal cancer cell affects NIH3T3 cell mtDNA loci, though further study is required.%目的 观察突变的大肠癌细胞线粒体DNA(mtDNA)转染 NIH3T3 细胞后转染细胞mtDNA D-环突变特性.方法 通过脂质体法(Lipofection 2000TM) 将大肠癌细胞突变的 mtDNA 真核表达载体转染 NIH3T3细胞,利用 G418 抗性筛选克隆细胞;用PCR法检测转染细胞线粒体突变情况.结果 突变mtDNA 导致转染细胞 mtDNA 的突变和多态性变化.结论 突变的大肠癌 mtDNA可致NIH3T3细胞mtDNA位点变化,但具体的机制和过程有待于进一步研究.

  6. Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M; Abdallah, Basem M; Kassem, Moustapha

    2012-01-01

    from E13.5 embryos after removing heads and viscera, followed by plastic adherence. Compared to BMSC, MEF exhibited telomerase activity and improved cell proliferation as assessed by q-PCR based TRAP assay and cell number quantification, respectively. FACS analysis revealed that MEF exhibited surface....../tricalcium phosphate, in immune deficient mice. In conclusion, MEF contain a population of stem cells that behave in ex vivo and in vivo assays, similar but not identical, to BMSC. Due to their enhanced cell growth, they may represent a good alternative for BMSC in studying molecular mechanisms of stem cell commitment......Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been...

  7. Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts

    Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing

  8. A mouse model for achondroplasia produced by targeting fibroblast growth factor receptor 3

    Wang, Yingcai; Spatz, Michal K.; Kannan, Karuppiah; Hayk, Hovhannisyan; Avivi, Aaron; Gorivodsky, Marat; Pines, Mark; Yayon, Avner; Lonai, Peter; Givol, David

    1999-01-01

    Achondroplasia, the most common form of dwarfism in man, is a dominant genetic disorder caused by a point mutation (G380R) in the transmembrane region of fibroblast growth factor receptor 3 (FGFR3). We used gene targeting to introduce the human achondroplasia mutation into the murine FGFR3 gene. Heterozygotes for this point mutation that carried the neo cassette were normal whereas neo+ homozygotes had a phenotype similar to FGFR3-deficient mice, exhibiting bone overgrowth. This was because o...

  9. Hypoxia induces NO-dependent release of heparan sulfate in fibroblasts from the Alzheimer mouse Tg2576 by activation of nitrite reduction.

    Cheng, Fang; Bourseau-Guilmain, Erika; Belting, Mattias; Fransson, Lars-Åke; Mani, Katrin

    2016-06-01

    There is a functional relationship between the heparan sulfate proteoglycan glypican-1 and the amyloid precursor protein (APP) of Alzheimer disease. In wild-type mouse embryonic fibroblasts, expression and processing of the APP is required for endosome-to-nucleus translocation of anhydromannose-containing heparan sulfate released from S-nitrosylated glypican-1 by ascorbate-induced, nitrosothiol-catalyzed deaminative cleavage. In fibroblasts from the transgenic Alzheimer mouse Tg2576, there is increased processing of the APP to amyloid-β peptides. Simultaneously, there is spontaneous formation of anhydromannose-containing heparan sulfate by an unknown mechanism. We have explored the effect of hypoxia on anhydromannose-containing heparan sulfate formation in wild-type and Tg2576 fibroblasts by deconvolution immunofluorescence microscopy and flow cytometry using an anhydromannose-specific monoclonal antibody and by (35)SO4-labeling experiments. Hypoxia prevented ascorbate-induced heparan sulfate release in wild-type fibroblasts, but induced an increased formation of anhydromannose-positive and (35)S-labeled heparan sulfate in Tg2576 fibroblasts. This appeared to be independent of glypican-1 S-nitrosylation as demonstrated by using a monoclonal antibody specific for S-nitrosylated glypican-1. In hypoxic wild-type fibroblasts, addition of nitrite to the medium restored anhydromannose-containing heparan sulfate formation. The increased release of anhydromannose-containing heparan sulfate in hypoxic Tg2576 fibroblasts did not require addition of nitrite. However, it was suppressed by inhibition of the nitrite reductase activity of xanthine oxidoreductase/aldehyde oxidase or by inhibition of p38 mitogen-activated protein kinase or by chelation of iron. We propose that normoxic Tg2576 fibroblasts maintain a high level of anhydromannose-containing heparan sulfate production by a stress-activated generation of nitric oxide from endogenous nitrite. This activation is enhanced

  10. Role of oxygen tension and genetic background during the epigenetic conversion of mouse fibroblasts into insulin secreting cells

    Alessandro Zenobi

    2015-07-01

    Full Text Available Epigenetic cell conversion overcomes the stability of a mature cell phenotype transforming a somatic cell in an unlimited source of autologous cells of a different type. It is based on the exposure to a demethylating agent followed by an induction protocol. In our work we exposed mouse dermal fibroblasts to the demethylating agent 5-azacytidine. Cell differentiation was directed toward the endocrine pancreatic lineage with a sequential combination of Activin A, Retinoic Acid, B27 supplement, ITS and bFGF. The overall duration of the process was 10 days. Aim of this work was to evaluate the role of oxygen during differentiation of dermal fibroblasts derived from two different mouse strains, NOD and C57 BL/6J. During differentiation, both cell lines were cultured either in the standard in vitro culture 20% oxygen concentration or in the lower and more physiological 5% of oxygen. Our results show that C57 BL/6J cells are able to differentiate into insulin secreting cells in both oxygen tensions with a higher amount of insulin release in low oxygen conditions. On the other hand, cells of NOD mice, which are physiologically predisposed to the onset of diabetes, differentiate in 20% of oxygen but not in low oxygen and they died after three days of culture. However, if these cells are moved to 5% of oxygen after their differentiation in high oxygen they remain viable for up to four days. Furthermore, their capacity to release insulin remains unchanged for 24 hours. Results suggest that genetic background has a profound effect on the role of oxygen during the in vitro differentiation process, possibly reflecting the different susceptibility to the disease of the strains used in the experiment.Supported by EFSD and Carraresi Foundation