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Sample records for cell line over-expression

  1. Over-expression of LPTS-L in hepatocellular carcinoma cell line SMMC-7721 induces crisis

    Institute of Scientific and Technical Information of China (English)

    Cheng Liao; Mu-Jun Zhao; Jing Zhao; Di Jia; Hai Song; Zai-Ping Li

    2002-01-01

    AIM: To evaluate the function of the longer transcripts LPTS-Lin hepatocellular carcinoma cell line SMMC-7721.METHODS: SMMC-7721 cells were transfected with LPTSL expression construct and stably transfected cells were selected by G418. Multiple single clones formed and were checked for their phenotype. In the study of the effect on telomerase activity of LPTS-Lin vitro, GST-LPTS-L fusion protein was expressed in E.coli and purified by glutathioneagarose column. Telomeric repeat amplification protocol (TRAP) assays were performed to study the influence of telomerase activity in SMMC-7721 cells.RESULTS: Over-expression of LPTS-L induced SMMC-7721 cells into crisis. LPTS-L could inhibit the telomerase activity in SMMC-7721 cellsin vitro.CONCLUSION: LPTS-L is a potent telomeraseinhibitor. Over-expression of LPTS-L can induce hepatoma cells into crisis due to the reduction of telomerase activity.

  2. MECHANISMS OF MRP OVER-EXPRESSION IN 4 HUMAN LUNG-CANCER CELL-LINES AND ANALYSIS OF THE MRP AMPLICON

    NARCIS (Netherlands)

    EIJDEMS, EWHM; DEHAAS, M; COCOMARTIN, JM; OTTENHEIM, CPE; ZAMAN, GJR; DAUWERSE, HG; BREUNING, MH; TWENTYMAN, PR; BORST, P; BAAS, F

    1995-01-01

    Some multidrug resistant cell lines over-express the gene encoding the multidrug-resistance-associated protein (MRP). In all cell lines reported thus far, over-expression is associated with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human lung-cancer c

  3. Characterization of a human cell line stably over-expressing the candidate oncogene, dual specificity phosphatase 12.

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    Erica L Cain

    Full Text Available BACKGROUND: Analysis of chromosomal rearrangements within primary tumors has been influential in the identification of novel oncogenes. Identification of the "driver" gene(s within cancer-derived amplicons is, however, hampered by the fact that most amplicons contain many gene products. Amplification of 1q21-1q23 is strongly associated with liposarcomas and microarray-based comparative genomic hybridization narrowed down the likely candidate oncogenes to two: the activating transcription factor 6 (atf6 and the dual specificity phosphatase 12 (dusp12. While atf6 is an established transcriptional regulator of the unfolded protein response, the potential role of dusp12 in cancer remains uncharacterized. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the oncogenic potential of dusp12, we established stable cell lines that ectopically over-express dusp12 in isolation and determined whether this cell line acquired properties frequently associated with transformed cells. Here, we demonstrate that cells over-expressing dusp12 display increased cell motility and resistance to apoptosis. Additionally, over-expression of dusp12 promoted increased expression of the c-met proto-oncogene and the collagen and laminin receptor intergrin alpha 1 (itga1 which is implicated in metastasis. SIGNIFICANCE: Collectively, these results suggest that dusp12 is oncologically relevant and exposes a potential association between dusp12 and established oncogenes that could be therapeutically targeted.

  4. Acetoacetate reduces growth and ATP concentration in cancer cell lines which over-express uncoupling protein 2

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    Quadros Edward V

    2009-05-01

    Full Text Available Abstract Background Recent evidence suggests that several human cancers are capable of uncoupling of mitochondrial ATP generation in the presence of intact tricarboxylic acid (TCA enzymes. The goal of the current study was to test the hypothesis that ketone bodies can inhibit cell growth in aggressive cancers and that expression of uncoupling protein 2 is a contributing factor. The proposed mechanism involves inhibition of glycolytic ATP production via a Randle-like cycle while increased uncoupling renders cancers unable to produce compensatory ATP from respiration. Methods Seven aggressive human cancer cell lines, and three control fibroblast lines were grown in vitro in either 10 mM glucose medium (GM, or in glucose plus 10 mM acetoacetate [G+AcA]. The cells were assayed for cell growth, ATP production and expression of UCP2. Results There was a high correlation of cell growth with ATP concentration (r = 0.948 in a continuum across all cell lines. Controls demonstrated normal cell growth and ATP with the lowest density of mitochondrial UCP2 staining while all cancer lines demonstrated proportionally inhibited growth and ATP, and over-expression of UCP2 (p Conclusion Seven human cancer cell lines grown in glucose plus acetoacetate medium showed tightly coupled reduction of growth and ATP concentration. The findings were not observed in control fibroblasts. The observed over-expression of UCP2 in cancer lines, but not in controls, provides a plausible molecular mechanism by which acetoacetate spares normal cells but suppresses growth in cancer lines. The results bear on the hypothesized potential for ketogenic diets as therapeutic strategies.

  5. Effects of Smac gene over-expression on the radiotherapeutic sensitivities of cervical cancer cell line HeLa

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    ZHENG Li-duan; XIONG Zhou-fang; ZHU Jian-wen; WANG Ze-hua

    2005-01-01

    Background The second mitochondria-derived activator of caspases (Smac) is a novel proapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation on tumor cells. The purpose of this study was to investigate the effects of extrinsic Smac gene transfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells. Methods After the Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtained by persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Western blot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatment with X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazolium bromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry. Results Smac mRNA and protein levels in HeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higher than those of HeLa (P0.05). However, after irradiation with 8 Gy X-ray, growth activities of HeLa/Smac were reduced by 22.42% (P<0.01). When compared with those of HeLa, partial HeLa/Smac cells presented characteristic morphological changes of apoptosis under electronic microscope, with higher apoptosis rates (16.4% vs. 6.2%, P<0.01); the caspase-3 expression levels in HeLa/Smac cells were improved significantly (P<0.01), while its activities were increased by 3.42 times (P<0.01).Conclusions Stable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance the expression and activities of cellular caspase-3 and ameliorate apoptosis-inducing effects of irradiation on cancer cells, which was a novel strategy to improve radiotherapeutic effects on cervical cancer.

  6. Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line.

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    Uchida, D; Kawamata, H; Omotehara, F; Miwa, Y; Hino, S; Begum, N M; Yoshida, H; Sato, M

    2000-06-01

    We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.

  7. Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway

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    Mennerich Detlev

    2007-01-01

    Full Text Available Abstract Background Stromelysin-3 (ST-3 is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized. Methods The present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed. Results Using both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts. Conclusion These data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour

  8. Establishment of a robust hepatitis C virus replicon cell line over-expressing P-glycoprotein that facilitates analysis of P-gp drug transporter effects on inhibitor antiviral activity.

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    Hernandez, Dennis; Falk, Paul; Yu, Fei; Zhai, Guangzhi; Quan, Yong; Faria, Teresa; Cao, Kai; Scola, Paul; McPhee, Fiona

    2013-01-01

    P-glycoprotein (P-gp) is an active efflux pump affecting the pharmacokinetic (PK) profiles of drugs that are P-gp substrates. The Caco-2 bi-directional assay is widely used to identify drug-P-gp interactions in vitro. For molecules exhibiting non-classical drug properties however, ambiguous results limit its use in lead optimization. The goal of this study was to develop a robust cell-based assay system to directly measure the role of P-gp-driven efflux in reducing the potency of hepatitis C virus (HCV) replication inhibitors. Vinblastine (Vin) was employed to select for a Vin-resistant HCV replicon (313-11) from the parental cell line (377-2). The 313-11 cell line was >50-fold resistant to Vin and over-expressed P-gp, as determined by Western immunoblots. Increased expression of P-gp was mediated by up-regulation of the MDR1 transcript. The reduced potency of different classes of HCV replication inhibitors in the 313-11 P-gp cell line was restored in the presence of known P-gp inhibitors. Addition of the P-gp inhibitor, tariquidar, increased the uptake of a radiolabeled HCV replication inhibitor by 14-fold in the 313-11 replicon cell line. Finally, a positive correlation was demonstrated between potency in the 313-11 replicon and the bi-directional Caco-2 efflux ratio for a panel of HCV protease inhibitors. In conclusion, a robust P-gp HCV replicon cell-based assay has been developed to measure the effect of the P-gp efflux pump on the potency of different classes of HCV replication inhibitors. This system establishes a direct correlation between antiviral activity and the effect of P-gp efflux in a single cell line.

  9. Effects of resistin-like molecule β over-expression on gastric cancer cells in vitro

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    Li-Duan Zheng; Chun-Lei Yang; Teng Qi; Meng Qi; Ling Tong; Qiang-Song Tong

    2012-01-01

    AIM:To investigate the effects of resistin-like molecule β (RELMβ) over-expression on the invasion,metastasis and angiogenesis of gastric cancer cells.METHODS:Human RELMβ encoding expression vector was constructed and transfected into the RELMβ lowly-expressed gastric cancer cell lines SGC-7901 and MKN-45.Gene expression was measured by Western blotting,reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR.Cell proliferation was measured by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry,colony formation and 5-ethynyl-20-deoxyuridine incorporation assays.The in vitro migration,invasion and metastasis of cancer cells were measured by cell adhesion assay,scratch assay and matrigel invasion assay.The angiogenic capabilities of cancer cells were measured by tube formation of endothelial cells.RESULTS:Transfection of RELMβ vector into SGC-7901 and MKN-45 cells resulted in over-expression of RELMβ,which did not influence the cellular proliferation.However,over-expression of RELMβ suppressed the in vitro adhesion,invasion and metastasis of cancer cells,accompanied by decreased expression of matrix metalloproteinase-2 (MMP-2) and MMP-9.Moreover,transfection of RELMβ attenuated the expression of vascular endothelial growth factor and in vitro angiogenic capabilities of cancer cells.CONCLUSION:Over-expression of RELMβ abolishes the invasion,metastasis and angiogenesis of gastric cancer cells in vitro,suggesting its potentials as a novel therapeutic target for gastric cancer.

  10. Establishment of stable over-expressing human SIRT1 HEK293 cell line%稳定过表达人SIRT1基因的HEK293细胞系的建立

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    林蓉; 张凯帆; 王维蓉; 林琴琴; 杨莉娜; 任峰; 张建丰

    2013-01-01

    Objective To construct a plasmid expressing human SIRT1 and to establish HEK293 cell line with stable over-expressing human SIRT1. Methods Full length of human SIRT1 gene cDNA was ligated into an expressing vector pcDNA3.1 ( + ). After confirmed by restriction analysis and sequencing, the recombinant plasmid was transfected into HEK293 cells mediated with liposome. G418-resistant clones of HEK293 cells were then detected by real-time PCR and Western blot for the expression level of SIRT1. Results The accuracy of constructed and selected plasmids was confirmed by restriction enzymatic analyses and DNA sequencing. As compared with untransfected HEK293 cells, the levels of SIRT1 mRNA and SIRT1 protein of transfected HEK293 cells were significantly increased (P<0.01). Conclusions The pcDNA3.1 ( + )-SIRTl plasmid is successfully constructed. HEK293 cell line with stable over-expressing SIRT1 is established, which provides a useful tool for further study on the effect of SIRT1 on cardiovascular diseases.%目的:构建人沉默信息调节因子2同源蛋白1(SIRT1)基因的真核表达载体,建立稳定过表达人SIRT1的HEK293细胞系.方法:将含有SIRT1的克隆载体pCRⅡ-TOPO-SIRT1双酶切后,连接至真核表达载体pcDNA3.1(+)中,连接产物经酶切鉴定后进行测序.构建的重组真核表达质粒pcDNA3.1(+)-SIRT1转染HEK293细胞.G418进行筛选.Real-time PCR和Western Blot分别从mRNA和蛋白水平检测SIRTI的表达.结果:酶切分析和测序结果证实,SIRT1基因成功插入真核表达质粒pcDNA3.1(+)中.Real-time PCR和Western Blot结果显示稳定转染pc:DNA3.1(+)-SIRT1的HEK293细胞SIRT1的表达水平明显高于未转染细胞(P<0.01).结论:成功构建了真核表达载体pcDNA3.1(+)-SIRT1,并建立了稳定过表达SIRT1基因的HEK293细胞系.为进-步研究SIRT1基因在心血管疾病中的作用及其机制奠定了基础.

  11. ABCC2基因过表达细胞株的建立及鉴定%Construction and identification of the recombinant cell line over-expressing ABCC2 gene

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    魏丹芸; 张洪; 彭锐; 张英

    2016-01-01

    Objective To construct the lentiviral vecter carrying human ABCC2 gene,then to screen out the stable cell line over -ex-pressing ABCC2 after package and transfection to HEK293 cells,thus to provide cell model to determine substrate drugs of multidrug re-sistance protein 2(MRP2)in vitro.Methods The primers were designed according to the cDNA sequence of ABCC2 gene from Gen-Bank.The gene were amplified by PCR and connected to lentiviral vector PEZ -LV105.The recombined lentiviral vector was pack-aged and transfected to HEK293 cells.After puromycin screening,HEK293 cells over -expressing human ABCC2 gene were selected and cloned.Finally,gene sequencing,real -time quantitative PCR(RTQ -PCR)and Western blot assays were performed for identifica-tion.Results RTQ -PCR showed that the ABCC2 mRNA in HEK293 cells transfected with exogenous ABCC2 gene was about 320 times higher than that of normal HEK293 cells and blank carrier HEK293 cells.MRP2 protein level in HEK293 cells with ABCC2 gene transfection was nearly 150 times higher than that of normal HEK293 cells and blank carrier HEK293 cells according to the Western blot.Conclusions Stable recombinant cell line over -expressing ABCC2 was successfully generated.The cell line could be useful in the confirmation of substrate drugs of multidrug resistance protein 2(MRP2)and the transport mechanism in vitro.%目的:构建携带 ABCC2基因的慢病毒载体,转染 HEK293细胞,筛选出稳定过表达 ABCC2基因的细胞株并进行鉴定,为体外实验确定与多药耐药相关蛋白2(MRP2)外排转运相关的底物药物及其转运机制提供细胞模型。方法根据 GenBank提供的 ABCC2基因 cDNA 序列设计引物,PCR 扩增该基因并将其连接至慢病毒载体 PZE -LV105,包装病毒并感染 HEK293细胞。用嘌呤霉素进行筛选得到过表达 ABCC2基因的稳转细胞株,通过基因测序、实时荧光定量 PCR(RTQ -PCR)和蛋白免疫印迹(Western blot)对稳转细

  12. IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells.

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    Long, Jun; Zhang, Xulong; Wen, Mingjie; Kong, Qingli; Lv, Zhe; An, Yunqing; Wei, Xiao-Qing

    2013-01-01

    Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

  13. GLUT 5 is not over-expressed in breast cancer cells and patient breast cancer tissues.

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    Gayatri Gowrishankar

    Full Text Available F18 2-Fluoro 2-deoxyglucose (FDG has been the gold standard in positron emission tomography (PET oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.

  14. Anosmin-1 over-expression regulates oligodendrocyte precursor cell proliferation, migration and myelin sheath thickness.

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    Murcia-Belmonte, Verónica; Esteban, Pedro F; Martínez-Hernández, José; Gruart, Agnès; Luján, Rafael; Delgado-García, José María; de Castro, Fernando

    2016-04-01

    During development of the central nervous system, anosmin-1 (A1) works as a chemotropic cue contributing to axonal outgrowth and collateralization, as well as modulating the migration of different cell types, fibroblast growth factor receptor 1 (FGFR1) being the main receptor involved in all these events. To further understand the role of A1 during development, we have analysed the over-expression of human A1 in a transgenic mouse line. Compared with control mice during development and in early adulthood, A1 over-expressing transgenic mice showed an enhanced oligodendrocyte precursor cell (OPC) proliferation and a higher number of OPCs in the subventricular zone and in the corpus callosum (CC). The migratory capacity of OPCs from the transgenic mice is increased in vitro due to a higher basal activation of ERK1/2 mediated through FGFR1 and they also produced more myelin basic protein (MBP). In vivo, the over-expression of A1 resulted in an elevated number of mature oligodendrocytes with higher levels of MBP mRNA and protein, as well as increased levels of activation of the ERK1/2 proteins, while electron microscopy revealed thicker myelin sheaths around the axons of the CC in adulthood. Also in the mature CC, the nodes of Ranvier were significantly longer and the conduction velocity of the nerve impulse in vivo was significantly increased in the CC of A1 over-expressing transgenic mice. Altogether, these data confirmed the involvement of A1 in oligodendrogliogenesis and its relevance for myelination.

  15. The effect of a DNA repair gene on cellular invasiveness: XRCC3 over-expression in breast cancer cells.

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    Veronica L Martinez-Marignac

    Full Text Available Over-expression of DNA repair genes has been associated with resistance to radiation and DNA-damage induced by chemotherapeutic agents such as cisplatin. More recently, based on the analysis of genome expression profiling, it was proposed that over-expression of DNA repair genes enhances the invasive behaviour of tumour cells. In this study we present experimental evidence utilizing functional assays to test this hypothesis. We assessed the effect of the DNA repair proteins known as X-ray complementing protein 3 (XRCC3 and RAD51, to the invasive behavior of the MCF-7 luminal epithelial-like and BT20 basal-like triple negative human breast cancer cell lines. We report that stable or transient over-expression of XRCC3 but not RAD51 increased invasiveness in both cell lines in vitro. Moreover, XRCC3 over-expressing MCF-7 cells also showed a higher tumorigenesis in vivo and this phenotype was associated with increased activity of the metalloproteinase MMP-9 and the expression of known modulators of cell-cell adhesion and metastasis such as CD44, ID-1, DDR1 and TFF1. Our results suggest that in addition to its' role in facilitating repair of DNA damage, XRCC3 affects invasiveness of breast cancer cell lines and the expression of genes associated with cell adhesion and invasion.

  16. Over-expression of tetraspanin 8 in malignant glioma regulates tumor cell progression

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    Pan, Si-Jian [Department of Neurosurgery, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China); Wu, Yue-Bing [Department of Internal Medicine Oncology, Hubei Cancer Hospital, Wuhan, Hubei 430079 (China); Cai, Shang [Department of Radiotherapy and Oncology, the Second Affiliated Hospital of Soochow University, Suzhou 21500 (China); Pan, Yi-Xin; Liu, Wei [Department of Stereotactic and Functional Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Bian, Liu-Guan [Department of Neurosurgery, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China); Sun, Bomin [Department of Stereotactic and Functional Neurosurgery, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Sun, Qing-Fang, E-mail: sunqingfang11@163.com [Department of Neurosurgery, Rui Jin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China)

    2015-03-13

    Tumor cell invasion and proliferation remain the overwhelming causes of death for malignant glioma patients. To establish effective therapeutic methods, new targets implied in these processes have to be identified. Tetraspanin 8 (Tspn8) forms complexes with a large variety of trans-membrane and/or cytosolic proteins to regulate several important cellular functions. In the current study, we found that Tspn8 was over-expressed in multiple clinical malignant glioma tissues, and its expression level correlated with the grade of tumors. Tspn8 expression in malignant glioma cells (U251MG and U87MG lines) is important for cell proliferation and migration. siRNA-mediated knockdown of Tspn8 markedly reduced in vitro proliferation and migration of U251MG and U87MG cells. Meanwhile, Tspn8 silencing also increased the sensitivity of temozolomide (TMZ), and significantly increased U251MG or U87MG cell death and apoptosis by TMZ were achieved with Tspn8 knockdown. We observed that Tspn8 formed a complex with activated focal adhesion kinase (FAK) in both human malignant glioma tissues and in above glioma cells. This complexation appeared required for FAK activation, since Tspn8 knockdown inhibited FAK activation in U251MG and U87MG cells. These results provide evidence that Tspn8 contributes to the pathogenesis of glioblastoma probably by promoting proliferation, migration and TMZ-resistance of glioma cells. Therefore, targeting Tspn8 may provide a potential therapeutic intervention for malignant glioma. - Highlights: • Tspn8 is over-expressed in multiple clinical malignant glioma tissues. • Tspn8 expression is correlated with the grade of malignant gliomas. • Tspn8 knockdown suppresses U251MG/U87MG proliferation and in vitro migration. • Tspn8 knockdown significantly increases TMZ sensitivity in U251MG/U87MG cells. • Tspn8 forms a complex with FAK, required for FAK activation.

  17. Over-expression of hypoxia-inducible factor 1 alpha increases angiogenesis of LNCaP cells

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    Yili Han; Dalin He; Yong Luo; Hepeng Cheng; Guangfeng Zhu

    2007-01-01

    Objective:To evaluate the effect of HIF-1 α over-expression on angiogenesis in human prostate cancer cells. Methods:LNCaP cells(a human prostate cancer cell line) were transfected with the recombinant plasmid pcDNA3.1(-)-HIF-1α with Lipofectamine 2000 system. The positive clones were selected by G418 being further confirmed by Western blot and immunofluorescence. The expression levels of VEGF, iNOS and Ang- Ⅱ were determined. Results:The expression of HIF-1α in the LNCaP/HIF1α cells was significantly increased in transfected cells, which induced the up-regulation of VEGF, iNOS, whereas Ang- Ⅱ expression remained un- changed. Conclusion :Over-expression of HIF-1α can induce angiogenesis proteins and may improve the angiogenesis potency of prostate cancer.

  18. Marked over expression of uncoupling protein-2 in beta cells exerts minor effects on mitochondrial metabolism

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    Hals, Ingrid K., E-mail: ingrid.hals@ntnu.no [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim (Norway); Ogata, Hirotaka; Pettersen, Elin [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim (Norway); Ma, Zuheng; Bjoerklund, Anneli [Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm (Sweden); Skorpen, Frank [Department of Laboratory Medicine, NTNU, Trondheim (Norway); Egeberg, Kjartan Wollo [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim (Norway); Grill, Valdemar [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim (Norway); Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm (Sweden)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The impact of UCP-2 over expression on mitochondrial function is controversial. Black-Right-Pointing-Pointer We tested mitochondrial functions at defined levels of overexpression. Black-Right-Pointing-Pointer We find minor increases of fatty acid oxidation and uncoupling. Black-Right-Pointing-Pointer Effects were seen only at high level (fourfold) of over expression. Black-Right-Pointing-Pointer Hence it is doubtful whether these effects are of importance in diabetes. -- Abstract: Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 {mu}g/ml of doxycycline (dox) induced UCP-2 fourfold (424 {+-} 113%, mean {+-} SEM) and 0.1 {mu}g/ml twofold (178 {+-} 29%, n = 3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 {+-} 11%) as well as D-[U-{sup 14}C]-glucose oxidation (+5 {+-} 9% at 11 mM glucose). Oxidation of [1-{sup 14}C]-oleate was increased from 4088 to 5797 fmol/{mu}g prot/2 h at 3.3 mM glucose, p < 0.03. Oxidation of L-[{sup 14}C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p < 0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p < 0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the

  19. Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells

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    Panula, Sarita; Reda, Ahmed; Stukenborg, Jan-Bernd; Ramathal, Cyril; Sukhwani, Meena; Albalushi, Halima; Edsgärd, Daniel; Nakamura, Michiko; Söder, Olle; Orwig, Kyle E.; Yamanaka, Shinya; Reijo Pera, Renee A.; Hovatta, Outi

    2016-01-01

    The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo. PMID:27768780

  20. Human neural stem cells over-expressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model.

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    Hong J Lee

    Full Text Available BACKGROUND: Intracerebral hemorrhage (ICH is a lethal stroke type. As mortality approaches 50%, and current medical therapy against ICH shows only limited effectiveness, an alternative approach is required, such as stem cell-based cell therapy. Previously we have shown that intravenously transplanted human neural stem cells (NSCs selectively migrate to the brain and induce behavioral recovery in rat ICH model, and that combined administration of NSCs and vascular endothelial growth factor (VEGF results in improved structural and functional outcome from cerebral ischemia. METHODS AND FINDINGS: We postulated that human NSCs overexpressing VEGF transplanted into cerebral cortex overlying ICH lesion could provide improved survival of grafted NSCs, increased angiogenesis and behavioral recovery in mouse ICH model. ICH was induced in adult mice by unilateral injection of bacterial collagenase into striatum. HB1.F3.VEGF human NSC line produced an amount of VEGF four times higher than parental F3 cell line in vitro, and induced behavioral improvement and 2-3 fold increase in cell survival at two weeks and eight weeks post-transplantation. CONCLUSIONS: Brain transplantation of F3 human NSCs over-expressing VEGF near ICH lesion sites provided differentiation and survival of grafted human NSCs and renewed angiogenesis of host brain and functional recovery of ICH animals. These results suggest a possible application of the human neural stem cell line, which is genetically modified to over-express VEGF, as a therapeutic agent for ICH-stroke.

  1. Over-expressed and truncated midkines promote proliferation of BGC823 cells in vitro and tumor growth in vivo

    Institute of Scientific and Technical Information of China (English)

    Qing-Ling Wang; Hui Wang; Shu-Li Zhao; Ya-Hong Huang; Ya-Yi Hou

    2008-01-01

    AIM: To determine whether midkine (MK) and its truncated form (tMK) contribute to gastric tumorigenesis using in vitro and in vivo models.METHODS: Human MK and tMK plasmids were constructed and expressed in BGC823 (a gastric adenocarcinoma cell line) to investigate the effect of over-expressed MK or tMK on cell growth and turmorigenesis in nude mice.RESULTS: The growth of MK-transfected or tMK-transfected cells was significantly increased compared with that of the control cells, and tMK-transfected cells grew more rapidly than MK-transfected cells. The number of colony formation of the cells transfected with MK or tMK gene was larger than the control cells. In nude mice injected with MK-transfected or tMK-transfected cells, visible tumor was observed earlier and the tumor tissues were larger in size and weight than in control animals that were injected with cells without the transfection of either genes.CONCLUSION: Over-expressed MK or tMK can promote human gastric cancer cell growth in vitro and in vivo, and tMK has greater effect than MK. tMK may be a more promising gene therapeutic target compared with MK for treatment of malignant tumors.

  2. Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein.

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    Entsar Saheb

    2015-06-01

    Full Text Available Acanthamoeba castellanii forms a resistant cyst that protects the parasite against the host's immune response. Acanthamoeba Type-I metacaspase (Acmcp is a caspase-like protein that has been found to be expressed during the encystations. Dictyostelium discoideum is an organism closely related to Acanthamoeba useful for studying the molecular function of this protozoan caspase-like protein.The full length of Acmcp and a mutated version of the same gene, which lacks the proline rich N-terminal region (Acmcp-dpr, were cloned into the pDneo2a-GFP vector separately. The pDneo2a-GFP-Acmcp and pDneo2a-GFPAcmcp-dpr were electro-transfected into wild type D. discoideum cells to create cell lines that over-expressed Acmcp or Acmcp-dpr.Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells. Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena. Quantitative cell death analysis provided additional support for these findings.Acmcp is involved in the processes of endocytosis and phagocytosis. In addition, the proline rich region in Acmcp is important for cellular development in Dictyostelium. Given its important role in the development process, metacaspase protein is proposed as a candidate drug target against infections caused by A. castellanii.

  3. Over-Expression of Catalase in Myeloid Cells Confers Acute Protection Following Myocardial Infarction

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    E. Bernadette Cabigas

    2014-05-01

    Full Text Available Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger of hydrogen peroxide (H2O2, has been met with mixed results. When over-expressed in cardiomyocytes from birth, catalase improves function following injury. When expressed in the same cells in an inducible manner, catalase showed a time-dependent response with no acute benefit, but a chronic benefit due to altered remodeling. In myeloid cells, catalase over-expression reduced angiogenesis during hindlimb ischemia and prevented monocyte migration. In the present study, due to the large inflammatory response following infarction, we examined myeloid-specific catalase over-expression on post-infarct healing. We found a significant increase in catalase levels following infarction that led to a decrease in H2O2 levels, leading to improved acute function. This increase in function could be attributed to reduced infarct size and improved angiogenesis. Despite these initial improvements, there was no improvement in chronic function, likely due to increased fibrosis. These data combined with what has been previously shown underscore the need for temporal, cell-specific catalase delivery as a potential therapeutic option.

  4. Effect of SMYD3 over-expression on DNMT3B levels and proliferation ability in human cholangiocarcinoma cell line FRH0201%上调SMYD3对人胆管癌FRH0201细胞中DNMT3B表达及细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    程帝; 李志花; 陈汝福; 郭宁; 廖巧芳; 郑礼平; 周泉波; 周嘉嘉

    2012-01-01

    目的:研究人胆管癌细胞株FRH0201中SET和MYND结构域含有蛋白3(SET and MYND domain-containing protein 3,SMYD3)过度表达对DNA甲基化转移酶3B(DNA methyltransferase 3B,DNMT3B)表达及细胞增殖能力的影响.方法:瞬时转染SMYD3真核表达质粒后,RT-PCR检测细胞中DNMT3B mRNA水平的变化;Western blotting检测细胞中DNMT3B蛋白水平的变化;CCK-8检测细胞增殖能力的改变;流式细胞术检测细胞周期的改变.结果:以未处理组为对照,胆管癌细胞FRH0201在转染pEGFP-C3-SMYD3质粒后,DNMT3B蛋白及mRNA表达均显著上升(P<0.01);细胞的增殖能力显著提高、细胞增殖速度加快(P<0.05);进入G2/M期的细胞明显增多(P<0.05).结论:过度表达SMYD3,可引起细胞中DNMT3B的表达上调并增强细胞增殖能力.%AIM:To explore ihe effect of SET and MYND domain - containing protein 3 ( SMYD3 ) over - expression on the expression of DNA methyltransferase 3B (DNMT3B) and the proliferation ability in human cholangiocarci-noma cell line FRH0201. METHODS; Transient transfection of SMYD3 eukaryotic expression plasmid pEGFP - C3 - SMYD3 into human cholangiocarcinoma cell line FRH0201 was performed. The expression of DNMT3B at mRNA and protein levels was detected by RT - PCR and Western blotting,respectively. Cell proliferation was examined by CCK -8 method and cell cycle situation was checked by flow cytometry. RESULTS; After transfected with SMYD3 eukaryotic expression plasmid pEGFP - C3 - SMYD3 , the over - expression of SMYD3 in FRH0201 cells was observed. Compared with the un-transfected cells, the expression of DNMT3B was significantly increased (P <0. 01) , the proliferation rate was obviously accelerated (P<0. 05) and the number of the cells in G2/M phase was significantly increased (P<0. 05) in FRH0201 cells transiently transfected with pEGFP - C3 - SMYD3 plasmid. CONCLUSION; The transient transfection of pEGFP -C3 - SMYD3 plasmid induces over - expression of DNMT3B and

  5. Over-expression of human endosulfatase-1 exacerbates cadmium-induced injury to transformed human lung cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Huiying [Department of Molecular Biomedical Sciences, Center for Comparative Molecular Translational Research, College of Veterinary Medicine, NC State University, Raleigh, NC 27607 (United States); Department of Environmental and Molecular Toxicology, College of Agriculture and Life Sciences, NC State University, Raleigh, NC 27695 (United States); Newman, Donna R. [Department of Molecular Biomedical Sciences, Center for Comparative Molecular Translational Research, College of Veterinary Medicine, NC State University, Raleigh, NC 27607 (United States); Bonner, James C. [Department of Environmental and Molecular Toxicology, College of Agriculture and Life Sciences, NC State University, Raleigh, NC 27695 (United States); Sannes, Philip L., E-mail: philip_sannes@ncsu.edu [Department of Molecular Biomedical Sciences, Center for Comparative Molecular Translational Research, College of Veterinary Medicine, NC State University, Raleigh, NC 27607 (United States)

    2012-11-15

    Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals. -- Highlights: ► Primary human lung alveolar type 2 (hAT2) cells and H292 and A549 cells were used. ► Cadmium induced apoptosis in hAT2 cells but not in H292 or A549 cells. ► HSulf-1exacerbates apoptosis induced by cadmium in H292 and A549 but not hAT2 cells.

  6. Over-expression of putative transcriptional coactivator KELP interferes with Tomato mosaic virus cell-to-cell movement.

    Science.gov (United States)

    Sasaki, Nobumitsu; Ogata, Takuya; Deguchi, Masakazu; Nagai, Shoko; Tamai, Atsushi; Meshi, Tetsuo; Kawakami, Shigeki; Watanabe, Yuichiro; Matsushita, Yasuhiko; Nyunoya, Hiroshi

    2009-03-01

    Tomato mosaic virus (ToMV) encodes a movement protein (MP) that is necessary for virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the virus. In co-bombardment experiments, the over-expression of KELP inhibited virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for virus cell-to-cell movement, but not on virus amplification in individual cells.

  7. Analysis of membrane proteome and secretome in cells over-expressing ADAM17 using quantitative proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, R.; Simabuco, F.M. [Laboratorio Nacional de Biociencias - LNBIO, Campinas, SP (Brazil); Yokoo, S.; Paes Leme, A.F. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil); Sherman, N. [University of Virginia, Charlottesville, VA (United States)

    2012-07-01

    Full text: A disintegrin and metalloproteinase (ADAM) protease is involved in proteolytic ectodomain shedding of several membrane-associated proteins and modulation of key cell signaling pathways in the tumor microenvironment. In this study, we examined the effect of over-expressing the full length human ADAM17 in membrane and secreted proteins. To this end, we constructed a stable Flp-In T-RExHEK293 cells expressing ADAM17 by tetracycline induction. These cells were grown in Dulbeccos modified Eagles medium containing light lysine, arginine or heavy, L-Arg-13C615N4 and L-Lys -13C615N2 (SILAC: stable isotope labeling with amino acid in cell culture) media and they were treated with an ADAM17 activator, phorbolester (PMA). Controls such as Flp-In T-RExHEK293 cell without PMA treatment and without ADAM17 cloned were cultivated in light medium. The ADAM17 overexpression was induced with tetracycline 500 ng/ml for 24 hours. Cells in a heavy condition were treated with PMA 50 ng/ml for 1 hour and vehicle DMSO was used as control in a light cell condition. The extracellular media were collected, concentrated and used to evaluate the secretome and a cell surface biotinylation-based approach was used to capture cell surface-associated proteins. The biotinylated proteins were eluted with dithiothreitol, alkylated with iodoacetamide and then digested with trypsin. The resulting peptides were subjected to LC-MS/MS analysis on an ETD enabled Orbitrap Velos instrument. The results showed different proteins up or down regulated in membrane and secretome analysis which might represent potential molecules involved in signaling or ADAM17 regulation events. (author)

  8. Over-expression of miR-106b promotes cell migration and metastasis in hepatocellular carcinoma by activating epithelial-mesenchymal transition process.

    Directory of Open Access Journals (Sweden)

    Wing Lung Yau

    Full Text Available Hepatocellular carcinoma (HCC is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. In vitro functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis in vivo. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005, and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018. Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. In vivo functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration in vitro and metastasis in vivo.

  9. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo.

    Science.gov (United States)

    McDole, B; Isgor, C; Pare, C; Guthrie, K

    2015-09-24

    Olfactory bulb granule cells (GCs) are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on GC spines. These contacts are established in the distal apical dendritic compartment, while GC basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong GC neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb GC spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF in transgenic mice produces a marked increase in GC spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on GCs, suggesting a role for this factor in modulating GC functional connectivity within adult olfactory circuitry.

  10. The DNA methylation inhibitor induces telomere dysfunction and apoptosis of leukemia cells that is attenuated by telomerase over-expression.

    Science.gov (United States)

    Zhang, Xiaolu; Li, Bingnan; de Jonge, Nick; Björkholm, Magnus; Xu, Dawei

    2015-03-10

    DNA methyltransferase inhibitors (DNMTIs) such as 5-azacytidine (5-AZA) have been used for treatment of acute myeloid leukemia (AML) and other malignancies. Although inhibiting global/gene-specific DNA methylation is widely accepted as a key mechanism behind DNMTI anti-tumor activity, other mechanisms are likely involved in DNMTI's action. Because telomerase reverse transcriptase (TERT) plays key roles in cancer through telomere elongation and telomere lengthening-independent activities, and TERT has been shown to confer chemo- or radio-resistance to cancer cells, we determine whether DNMTIs affect telomere function and whether TERT/telomerase interferes with their anti-cancer efficacy. We showed that 5-AZA induced DNA damage and telomere dysfunction in AML cell lines by demonstrating the presence of 53-BP1 foci and the co-localization of 53-BP1 foci with telomere signals, respectively. Telomere dysfunction was coupled with diminished TERT expression, shorter telomere and apoptosis in 5-AZA-treated cells. However, 5-AZA treatment did not lead to changes in the methylation status of subtelomere regions. Down-regulation of TERT expression similarly occurred in primary leukemic cells derived from AML patients exposed to 5-AZA. TERT over-expression significantly attenuated 5-AZA-mediated DNA damage, telomere dysfunction and apoptosis of AML cells. Collectively, 5-AZA mediates the down-regulation of TERT expression, and induces telomere dysfunction, which consequently exerts an anti-tumor activity.

  11. Cell type-specific over-expression of chromosome 21 genes in fibroblasts and fetal hearts with trisomy 21

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    Zigman Warren B

    2006-03-01

    Full Text Available Abstract Background Down syndrome (DS is caused by trisomy 21 (+21, but the aberrations in gene expression resulting from this chromosomal aneuploidy are not yet completely understood. Methods We used oligonucleotide microarrays to survey mRNA expression in early- and late-passage control and +21 fibroblasts and mid-gestation fetal hearts. We supplemented this analysis with northern blotting, western blotting, real-time RT-PCR, and immunohistochemistry. Results We found chromosome 21 genes consistently over-represented among the genes over-expressed in the +21 samples. However, these sets of over-expressed genes differed across the three cell/tissue types. The chromosome 21 gene MX1 was strongly over-expressed (mean 16-fold in senescent +21 fibroblasts, a result verified by northern and western blotting. MX1 is an interferon target gene, and its mRNA was induced by interferons present in +21 fibroblast conditioned medium, suggesting an autocrine loop for its over-expression. By immunohistochemistry the p78MX1 protein was induced in lesional tissue of alopecia areata, an autoimmune disorder associated with DS. We found strong over-expression of the purine biosynthesis gene GART (mean 3-fold in fetal hearts with +21 and verified this result by northern blotting and real-time RT-PCR. Conclusion Different subsets of chromosome 21 genes are over-expressed in different cell types with +21, and for some genes this over-expression is non-linear (>1.5X. Hyperactive interferon signaling is a candidate pathway for cell senescence and autoimmune disorders in DS, and abnormal purine metabolism should be investigated for a potential role in cardiac defects.

  12. Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants.

    Science.gov (United States)

    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2003-04-01

    Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.

  13. Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Guseva, Daria [Kazan State Medical University, Kazan, Republic of Tatarstan (Russian Federation); Hannover Medical School, Hannover (Germany); Rizvanov, Albert A.; Salafutdinov, Ilnur I.; Kudryashova, Nezhdana V. [Kazan Federal University, Kazan, Republic of Tatarstan (Russian Federation); Palotás, András, E-mail: palotas@asklepios-med.eu [Kazan Federal University, Kazan, Republic of Tatarstan (Russian Federation); Asklepios-Med (Private Medical Practice and Research Center), Szeged (Hungary); Islamov, Rustem R., E-mail: islamru@yahoo.com [Kazan State Medical University, Kazan, Republic of Tatarstan (Russian Federation)

    2014-09-05

    Highlights: • Gene and cell-based therapies comprise innovative aspects of regenerative medicine. • Genetically modified hUCB-MCs enhanced differentiation of cells in a mouse model of ALS. • Stem cells successfully transformed into micro-glial and endothelial lines in spinal cords. • Over-expressing oct4 and sox2 also induced production of neural marker PGP9.5. • Formation of new nerve cells, secreting trophic factors and neo-vascularisation could improve symptoms in ALS. - Abstract: Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.

  14. Selective over-expression of endothelin-1 in endothelial cells exacerbates inner retinal edema and neuronal death in ischemic retina.

    Directory of Open Access Journals (Sweden)

    Simon S F Cheung

    Full Text Available The level of endothelin-1 (ET-1, a potent vasoconstrictor, was associated with retinopathy under ischemia. The effects of endothelial endothelin-1 (ET-1 over-expression in a transgenic mouse model using Tie-1 promoter (TET-1 mice on pathophysiological changes of retinal ischemia were investigated by intraluminal insertion of a microfilament up to middle cerebral artery (MCA to transiently block the ophthalmic artery. Two-hour occlusion and twenty-two-hour reperfusion were performed in homozygous (Hm TET-1 mice and their non-transgenic (NTg littermates. Presence of pyknotic nuclei in ganglion cell layer (GCL was investigated in paraffin sections of ipsilateral (ischemic and contralateral (non-ischemic retinae, followed by measurement of the thickness of inner retinal layer. Moreover, immunocytochemistry of glial fibrillary acidic protein (GFAP, glutamine synthetase (GS and aquaporin-4 (AQP4 peptides on retinal sections were performed to study glial cell reactivity, glutamate metabolism and water accumulation, respectively after retinal ischemia. Similar morphology was observed in the contralateral retinae of NTg and Hm TET-1 mice, whereas ipsilateral retina of NTg mice showed slight structural and cellular changes compared with the corresponding contralateral retina. Ipsilateral retinae of Hm TET-1 mice showed more significant changes when compared with ipsilateral retina of NTg mice, including more prominent cell death in GCL characterized by the presence of pyknotic nuclei, elevated GS immunoreactivity in Müller cell bodies and processes, increased AQP-4 immunoreactivity in Müller cell processes, and increased inner retinal thickness. Thus, over-expression of endothelial ET-1 in TET-1 mice may contribute to increased glutamate-induced neurotoxicity on neuronal cells and water accumulation in inner retina leading to edema.

  15. TOX3 (TNRC9) Over Expression in Bladder Cancer Cells Decreases Cellular Proliferation and Triggers an Interferon-Like Response

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Mansilla Castaño, Francisco; Dyrskjøt, Lars

    2013-01-01

    urothelium. Microarray expression profiling of human bladder cancer cells over expressing TOX3 followed by Pathway analysis showed that TOX3 Overexpression mainly affected the Interferon Signaling Pathway. TOX3 up regulation induced the expression of several genes with a gamma interferon activation site (GAS......), e.g. STAT1. In vitro functional studies showed that TOX3 was able to bind to the GAS-sequence located at the STAT1 promoter. siRNA mediated knockdown of TOX3 in RT4 bladder cancer cells decreased STAT1 expression suggesting a direct impact of TOX3 on STAT1. Immunoprecipitation of TOX3 over......Background: Human TOX3 (TOX high mobility group box family member 3) regulates Ca2+ dependent transcription in neurons and has been associated with breast cancer susceptibility. Aim of the study was to investigate the expression of TOX3 in bladder cancer tissue samples and to identify genes...

  16. Over-expressed Genes Detected by Suppression Subtractive Hybridization in Carcinoma Derived From Transformed 16HBE Cells Induced by BPDE

    Institute of Scientific and Technical Information of China (English)

    SHE-JUAN AN; JIA-KUN CHEN; LI-LI LIU; YAN-FENG ZHAO; XUE-MIN CHEN

    2005-01-01

    Objective To screen the over differentially expressed genes in carcinoma induced by BPDE-transformed 16HBE cells (16HBE-C cells). Methods The suppression subtractive hybridization (SSH) method was performed to profile differentially expressed genes between 16HBE-C cells and 16HBE cells. The cDNA fragments of differentially expressed genes were inserted into TA cloning vector and transformed competent E. coli strain. Positive clones were randomly picked up and identified by the colony PCR method. Dot blot was used to test the same source with the tester. The differentially expressed cDNA fragments were sequenced and compared with known genes and EST database in Genbank. Results Eight known genes were over-expressed in 16HBE-C cells including eukaryotic translation elongation factor 1 alpha 1, HIF-1 responsive RTP801, ribosomal protein L10 (RPL10), ribosomal protein S29 (RPS29), mitochondrion related genes, and laminin receptor 1. Three differentially expressed cDNA fragments could not be matched to the known genes but to the EST database. Conclusion The SSH method can detect differentially expressed genes between 16HBE-C and 16HBE cells. BPDE-induced carcinogenesis may be related to alteration of at least eight known genes and three unknown genes. These expression data provide a clue to further cloning novel genes and studying functions in BPDE-induced carcinoma.

  17. Pleiotropic effect of sigE over-expression on cell morphology, photosynthesis and hydrogen production in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Osanai, Takashi; Kuwahara, Ayuko; Iijima, Hiroko; Toyooka, Kiminori; Sato, Mayuko; Tanaka, Kan; Ikeuchi, Masahiko; Saito, Kazuki; Hirai, Masami Yokota

    2013-11-01

    Over-expression of sigE, a gene encoding an RNA polymerase sigma factor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, is known to activate sugar catabolism and bioplastic production. In this study, we investigated the effects of sigE over-expression on cell morphology, photosynthesis and hydrogen production in this cyanobacterium. Transmission electron and scanning probe microscopic analyses revealed that sigE over-expression increased the cell size, possibly as a result of aberrant cell division. Over-expression of sigE reduced respiration and photosynthesis activities via changes in gene expression and chlorophyll fluorescence. Hydrogen production under micro-oxic conditions is enhanced in sigE over-expressing cells. Despite these pleiotropic phenotypes, the sigE over-expressing strain showed normal cell viability under both nitrogen-replete and nitrogen-depleted conditions. These results provide insights into the inter-relationship among metabolism, cell morphology, photosynthesis and hydrogen production in this unicellular cyanobacterium.

  18. Distinct cell clusters touching islet cells induce islet cell replication in association with over-expression of Regenerating Gene (REG protein in fulminant type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Kaoru Aida

    Full Text Available BACKGROUND: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs, extracellular matrix (ECM, and possible cell clusters, are unclear. PROCEDURES: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.

  19. Over-expression of PUMA correlates with the apoptosis of spinal cord cells in rat neuropathic intermittent claudication model.

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    Bin Ma

    Full Text Available BACKGROUND: Neuropathic intermittent claudication (NIC is a typical clinical symptom of lumbar spinal stenosis and the apoptosis of neurons caused by cauda equina compression (CEC has been proposed as an important reason. Whereas, the factors and the mechanism involved in the process of apoptosis induced by CEC remain unclear. METHODOLOGY AND RESULTS: In our modified rat model of NIC, a trapezoid-shaped silicon rubber was inserted into the epidural space under the L5 and L6 vertebral plate. Obvious apoptosis was observed in spinal cord cells after compression by TUNEL assay. Simultaneously, qRT-PCR and immunohistochemistry showed that the expression levels of PUMA (p53 up-regulated modulator of apoptosis and p53 were upregulated significantly in spinal cord under compression, while the expression of p53 inhibitor MDM2 and SirT2 decreased in the same region. Furthermore, CEC also resulted in the upregulation of Bcl-2 pro-apoptotic genes expression and caspase-3 activation. With the protection of Methylprednisolone, the upregulation of PUMA and p53 expression as well as the decrease of MDM2 and SirT2 in spinal cord were partially rescued in western bolt analysis. CONCLUSIONS: These results suggest that over-expression of PUMA correlates with CEC caused apoptosis of spinal cord cells, which is characterized by the increase of p53, Bax and Bad expression. PUMA upregulation might be crucial to induce apoptosis of spinal cord cells through p53-dependent pathway in CEC.

  20. No dramatic age-related loss of hair cells and spiral ganglion neurons in Bcl-2 over-expression mice or Bax null mice

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    Ohlemiller Kevin K

    2010-07-01

    Full Text Available Abstract Age-related decline of neuronal function is associated with age-related structural changes. In the central nervous system, age-related decline of cognitive performance is thought to be caused by synaptic loss instead of neuronal loss. However, in the cochlea, age-related loss of hair cells and spiral ganglion neurons (SGNs is consistently observed in a variety of species, including humans. Since age-related loss of these cells is a major contributing factor to presbycusis, it is important to study possible molecular mechanisms underlying this age-related cell death. Previous studies suggested that apoptotic pathways were involved in age-related loss of hair cells and SGNs. In the present study, we examined the role of Bcl-2 gene in age-related hearing loss. In one transgenic mouse line over-expressing human Bcl-2, there were no significant differences between transgenic mice and wild type littermate controls in their hearing thresholds during aging. Histological analysis of the hair cells and SGNs showed no significant conservation of these cells in transgenic animals compared to the wild type controls during aging. These data suggest that Bcl-2 overexpression has no significant effect on age-related loss of hair cells and SGNs. We also found no delay of age-related hearing loss in mice lacking Bax gene. These findings suggest that age-related hearing loss is not through an apoptotic pathway involving key members of Bcl-2 family.

  1. Largescale Transcriptomics Analysis Suggests Over-Expression of BGH3, MMP9 and PDIA3 in Oral Squamous Cell Carcinoma.

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    Yuan He

    Full Text Available Oral squamous cell carcinoma (OSCC has been reported as the most prevalent cancer of the head and neck region, while early diagnosis remains challenging. Here we took a comprehensive bioinformatics study on microarray data of 326 OSCC clinical samples with control of 165 normal tissues. The cell interaction pathways of ECM-receptor interaction and focal adhesion were found to be significantly regulated in OSCC samples. Further analysis of the topological properties and expression consistency identified that three hub genes in the gene interaction network, MMP9, PDIA3 and BGH3, were consistently up-expressed in OSCC samples. When being validated on additional microarray datasets of 41 OSCC samples, the validation rate of over-expressed BGH3, MMP9, and PDIA3 reached 90%, 90% and 84% respectively. At last, immuno-histochemical assays were done to test the protein expression of the three genes on newly collected clinical samples of 35 OSCC, 20 samples of pre-OSCC stage, and 12 normal oral mucosa specimens. Their protein expression levels were also found to progressively increase from normal mucosa to pre-OSCC stage and further to OSCC (ANOVA p = 0.000, suggesting their key roles in OSCC pathogenesis. Based on above solid validation, we propose BGH3, MMP9 and PDIA3 might be further explored as potential biomarkers to aid OSCC diagnosis.

  2. Largescale Transcriptomics Analysis Suggests Over-Expression of BGH3, MMP9 and PDIA3 in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    He, Yuan; Shao, Fangyang; Pi, Weidong; Shi, Cong; Chen, Yujia; Gong, Diping; Wang, Bingjie; Cao, Zhiwei; Tang, Kailin

    2016-01-01

    Oral squamous cell carcinoma (OSCC) has been reported as the most prevalent cancer of the head and neck region, while early diagnosis remains challenging. Here we took a comprehensive bioinformatics study on microarray data of 326 OSCC clinical samples with control of 165 normal tissues. The cell interaction pathways of ECM-receptor interaction and focal adhesion were found to be significantly regulated in OSCC samples. Further analysis of the topological properties and expression consistency identified that three hub genes in the gene interaction network, MMP9, PDIA3 and BGH3, were consistently up-expressed in OSCC samples. When being validated on additional microarray datasets of 41 OSCC samples, the validation rate of over-expressed BGH3, MMP9, and PDIA3 reached 90%, 90% and 84% respectively. At last, immuno-histochemical assays were done to test the protein expression of the three genes on newly collected clinical samples of 35 OSCC, 20 samples of pre-OSCC stage, and 12 normal oral mucosa specimens. Their protein expression levels were also found to progressively increase from normal mucosa to pre-OSCC stage and further to OSCC (ANOVA p = 0.000), suggesting their key roles in OSCC pathogenesis. Based on above solid validation, we propose BGH3, MMP9 and PDIA3 might be further explored as potential biomarkers to aid OSCC diagnosis.

  3. Effects of adipose-derived mesenchymal stem cells over-expressing glial cell line-derived neurotrophic factor on electrically injured sciatic nerve of rats%过表达胶质细胞源性神经营养因子的脂肪源性间充质干细胞对大鼠电损伤坐骨神经的作用

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    杨晨; 胡大海; 郑朝; 白晓智; 王耀军; 汤朝武

    2015-01-01

    Objective To observe the effects of adipose-derived mesenchymal stem cells (ADSCs) with continous over-expression of glial cell line-derived neurotrophic factor (GDNF) on the motor function recovery and nerve regeneration of sciatic nerve of rats after electrical injury.Methods Five SD rats were collected to prepare ADSCs with over-expression of GDNF.One hundred and fifty SD rats were divided into normal control group (N),GDNF-ADSCs group (GA),ADSCs group (A),GDNF group (G),and physiological saline group (P) according to the random number table,with 30 rats in each group.Rats in group N were routinely fed without treatment,and rats in the other 4 groups were inflicted with electrical injury on sciatic nerve of thigh of the right hind leg.Rats in groups GA,A,G,and P were respectively injected with 100 μL suspension of ADSCs with over-expression of GDNF (1 × 107 cells per mL),100 μL ADSCs suspension (1 × 107 cells per mL),100 μL GDNF solution (100 mg/L),and 100 μL physiological saline to the surface of the injured nerves immediately after injury.Six rats of each group were collected for measuring hind limb stride from post injury week (PIW) 1 to 8,and morphology of the sciatic nerves was observed in PIW 8.In PIW 4,the protein expression of GDNF of sciatic nerves of the rest rats in each group was determined with Western blotting.Data were processed with one-way analysis of variance,analysis of variance of repeated measurement,and SNK test.Results Compared with that of group N,the hind limb stride values in groups GA,A,G,and P were significantly lower at each time point (with P values below 0.05).Compared with those of group P,the hind limb stride values in group GA from PIW 3 to 8,in group A in PIW 3,5,and 7,and in group G in PIW 3,5,7,and 8 were significantly longer (with P values below 0.05).The hind limb stride values in group GA from PIW 4 to 8 were respectively (10.83 ± 0.97),(13.25±1.40),(12.86±1.42),(14.06±1.50),and (15.09 ±1.17) cm,which were

  4. Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

    Science.gov (United States)

    Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

    2016-12-30

    This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

  5. Consequences of over-expression of rat Scavenger Receptor, SR-BI, in an adrenal cell model

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    Azhar Salman

    2006-12-01

    Full Text Available Abstract Background The plasma membrane scavenger receptor, SR-BI, mediates the 'selective uptake' process by which cholesteryl esters (CE from exogenously supplied HDL are taken up by target cells. Recent work suggests that dimer and higher order oligomeric forms of the SR-BI protein are important to this process. SR-BI has been shown to be particularly associated with microvilli and microvillar channels found at the cell surface of steroidogenic cells, and a study with the hormone stimulated adrenal gland has shown impressive changes in the size and complexity of the microvillar compartment as the mass of CE uptake (and accompanying steroidogenesis fluctuates. In the present study, we examine a cell line in which we overexpress the SR-BI protein to determine if morphological, biochemical and functional events associated with SR-BI in a controlled cell system are similar to those observed in the intact mammalian adrenal which is responsive to systemic factors. Methods Y1-BS1 mouse adrenocortical cells were transiently transfected using rat SR-BI-pcDNA6-V5-His, rat SR-BI-pcDNA6-cMyc-His or control pcDNA6-V5-His vector construct using a CaPO4 precipitation technique. Twenty four hours after transfection, cells were treated with, or without, Bt2cAMP, and SR-BI expression, CE uptake, and steroidogenesis was measured. SR-BI dimerization and cell surface architectural changes were assessed using immunoelectron microscopic techniques. Results Overexpression of the scavenger receptor protein, SR-BI, in Y1-BS1 cells results in major alterations in cell surface architecture designed to increase uptake of HDL supplied-CEs. Changes include 1 the formation of crater-like erosions of the surface with multiple double membraned channel structures lining the craters, and 2 dimerized formations of SR-BI lining the newly formed craters and associated double membraned channels. Conclusion These data show that overexpression of the scavenger receptor protein, SR

  6. Over-expression of AhR (aryl hydrocarbon receptor induces neural differentiation of Neuro2a cells: neurotoxicology study

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    Ishihara-Sugano Mitsuko

    2006-09-01

    Full Text Available Abstract Background Dioxins and related compounds are suspected of causing neurological disruption in human and experimental animal offspring following perinatal exposure during development and growth. The molecular mechanism(s of the actions in the brain, however, have not been fully investigated. A major participant in the process of the dioxin-toxicity is the dioxin receptor, namely the aryl hydrocarbon receptor (AhR. AhR regulates the transcription of diverse genes through binding to the xenobiotic-responsive element (XRE. Since the AhR has also been detected in various regions of the brain, the AhR may play a key role in the developmental neurotoxicity of dioxins. This study focused on the effect of AhR activation in the developing neuron. Methods The influence of the AhR on the developing neuron was assessed using the Neuro2a-AhR transfectant. The undifferentiated murine neuroblastoma Neuro2a cell line (ATCC was stably transfected with AhR cDNA and the established cell line was named N2a-Rα. The activation of exogenous AhR in N2a-Rα cells was confirmed using RNAi, with si-AhR suppressing the expression of exogenous AhR. The neurological properties of N2a-Rα based on AhR activation were evaluated by immunohistochemical analysis of cytoskeletal molecules and by RT-PCR analysis of mRNA expression of neurotransmitter-production related molecules, such as tyrosine hydroxylase (TH. Results N2a-Rα cells exhibited constant activation of the exogenous AhR. CYP1A1, a typical XRE-regulated gene, mRNA was induced without the application of ligand to the culture medium. N2a-Rα cells exhibited two significant functional features. Morphologically, N2a-Rα cells bore spontaneous neurites exhibiting axon-like properties with the localization of NF-H. In addition, cdc42 expression was increased in comparison to the control cell line. The other is the catecholaminergic neuron-like property. N2a-Rα cells expressed tyrosine hydroxylase (TH mRNA as a

  7. Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

    Science.gov (United States)

    Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

    2016-07-01

    First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835.

  8. Neuroglobin over expressing mice

    DEFF Research Database (Denmark)

    Raida, Zindy; Hundahl, Christian Ansgar; Nyengaard, Jens R

    2013-01-01

    BACKGROUND: Stroke is a major cause of death and severe disability, but effective treatments are limited. Neuroglobin, a neuronal heme-globin, has been advocated as a novel pharmacological target in combating stroke and neurodegenerative disorders based on cytoprotective properties. Using...... thoroughly validated antibodies and oligos, we give a detailed brain anatomical characterization of transgenic mice over expressing Neuroglobin. Moreover, using permanent middle artery occlusion the effect of elevated levels of Neuroglobin on ischemic damage was studied. Lastly, the impact of mouse strain...... genetic background on ischemic damage was investigated. PRINCIPAL FINDINGS: A four to five fold increase in Neuroglobin mRNA and protein expression was seen in the brain of transgenic mice. A β-actin promoter was used to drive Neuroglobin over expression, but immunohistochemistry and in situ hybridization...

  9. The Evaluation of Nerve Growth Factor Over Expression on Neural Lineage Specific Genes in Human Mesenchymal Stem Cells

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    Mortazavi Yousef

    2016-07-01

    Full Text Available Objective Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer’s disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells (MSCs as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor (NGF and assessed their neural lineage genes. Materials and Methods In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein (GFP gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 (HEK-293 packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction (PCR. Results We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor (GDNF, glial fibrillary acidic protein (GFAP and Microtubule-associated protein 2 (MAP2 genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor (BDNF genes reduced. Conclusion Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this

  10. Hypoxia in Tumor Angiogenesis and Metastasis: Evaluation of VEGF and MMP Over-expression and Down-Regulation of HIF-1alpha with RNAi in Hypoxic Tumor Cells

    Science.gov (United States)

    Shah, Shruti

    Background: As tumor mass grows beyond a few millimeters in diameter, the angiogenic "switch" is turned on leading to recruitment of blood vessels from surrounding artery and veins. However, the tumor mass is poorly perfused and there are pockets of hypoxia or lower oxygen concentrations relative to normal tissue. Hypoxia-inducing factor-1a (HIF-1a), a transcription factor, is activated when the oxygen concentration is low. Upon activation of HIF-1a, a number of other genes also turn on that allows the tumor to become more aggressive and resistant to therapy. Purpose: The main objectives of this study were to evaluate the effect of hypoxia-induced HIF-1a followed by over-expression of angiogenic and metastatic markers in tumor cells and down-regulation of HIF-1a using nanoparticle-delivered RNA interference therapy. Methods: Human ovarian (SKOV3) and breast (MDA-MB-231) adenocarcinoma cells were incubated under normoxic and hypoxic conditions. Following hypoxia treatment of the cells, HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), and MMP-9 expression was analyzed qualitatively and quantitatively. For intracellular delivery of HIF-1a gene silencing small interfering RNA (siRNA), type B gelatin nanoparticles were fabricated using the solvent displacement method and the surface was modified with poly(ethylene glycol) (PEG, Mol. wt. 2kDa). Cellular uptake and distribution of the nanoparticles was observed with Cy3-siRNA loaded, FITC-conjugated gelatin nanoparticles. Cytotoxicity of the nanoparticle formulations was evaluated in both the cell lines. siRNA was transfected in the gelatin nanoparticles under hypoxic conditions. Total cellular protein and RNA were extracted for analysis of HIF1a, VEGF, MMP-2 and MMP-9 expression. Results: MDA-MB-231 and SKOV3 cells show increased expression of HIF1a under hypoxic conditions compared to baseline levels at normoxic conditions. ELISA and western blots of VEGF, MMP-2 and MMP-9 appear to

  11. Effects of over-expression of TLR2 in transgenic goats on pathogen clearance and role of up-regulation of lysozyme secretion and infiltration of inflammatory cells

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    Deng Shoulong

    2012-10-01

    Full Text Available Abstract Background Toll-like receptor 2 (TLR2 is important to host recognition of invading gram-positive microbes. In goats, these microbes can cause serious mastitis, anthrax, tetanus, and other problems. Transgenic goats constitutively over-expressing TLR2 in many tissues serve as a suitable model for the study of the role of TLR2 over-expression in bacterial clearance. Results Capra hircus TLR2 over-expression vector (p3S-LoxP-TLR2 was used to generate transgenic goats by egg microinjection. The integration efficiency was 8.57%. Real-time PCR and immunohistochemical results confirmed that the goats over-expressing the TLR2 gene (Tg expressed more TLR2 than wild-type goats (WT. Monocyte-macrophages from the bloodstreams of transgenic goats were stimulated with synthetic bacterial lipoprotein (Pam3CSK4 and by the promotion of interleukin-6 (IL-6 and IL-10 expression in vitro. The oxidative damage was significantly reduced, and lysozyme (LZM secretion was found to be up-regulated. Ear tissue samples from transgenic goats that had been stimulated with Pam3CSK4 via hypodermic injection showed that transgenic individuals can undergo the inflammation response very quickly. Conclusions Over-expression of TLR2 was found to decrease radical damage to host cells through low-level production of NO and MDA and to promote the clearance of invasive bacteria by up-regulating lysozyme secretion and filtration of inflammatory cells to the infected site.

  12. Retraction: "Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells" by Bao et al.

    Science.gov (United States)

    2016-08-01

    The above article, published online on April 18, 2011 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the second author that found Figures 1C and 4C to be inappropriately re-used and re-labeled. REFERENCE Bao B, Wang Z, Ali S, Kong D, Banerjee S, Ahmad A, Li Y, Azmi AS, Miele L, Sarkar FH. 2011. Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells. J Cell Biochem 112:2296-2306; doi: 10.1002/jcb.23150.

  13. Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF

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    Deng, Xuefeng, E-mail: dengxfdoctor@hotmail.com [Department of Thoracic Surgery, Tangdu Hospital, Forth Military Medical University (China); Department of Cardio-thoracic Surgery, Affiliated Hospital of Academy of Military Medical Sciences (China); Ma, Qunfeng [Department of Cardio-thoracic Surgery, Affiliated Hospital of Academy of Military Medical Sciences (China); Zhang, Bo [Department of Pathology, Affiliated Hospital of Academy of Military Medical Sciences (China); Jiang, Hong [College of Life Sciences and Bioengineering, Beijing Jiaotong University (China); Zhang, Zhipei; Wang, Yunjie [Department of Thoracic Surgery, Tangdu Hospital, Forth Military Medical University (China)

    2013-10-15

    Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level of MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression. - Highlights: • MSF expression was upregulated in NSCLC and correlated with TNM stages. • MSF may be a new biomarker for NSCLC progression. • MSF promoted migration and invasion in A549 cells, independent of MMP-2/MMP-9 expression.

  14. Identification and characterization of L985P, a CD20 related family member over-expressed in small cell lung carcinoma.

    Science.gov (United States)

    Bangur, Chaitanya S; Johnson, Jeffrey C; Switzer, Ann; Wang, Yi-Hong; Hill, Beth; Fanger, Gary R; Wang, Tongtong; Retter, Marc W

    2004-12-01

    We recently reported on the use of cDNA subtraction combined with microarray based expression analysis for identifying genes that are differentially over-expressed in small cell lung carcinoma. One of the several hundred genes identified using this approach was termed L985P and its molecular characterization is described in this report. The differential over-expression of L985P mRNA in SCLC, as determined by microarray analysis, was confirmed by real-time RT-PCR and Northern blot analysis. Immunohistochemical analyses show that L985P protein is highly expressed in SCLC with very restricted expression observed in normal lung, which was confined to the apical region of the ciliated bronchiolar epithelium. Flow cytometric and immunohistochemical analysis showed that L985P was localized to the cell surface. Sequence homology comparison indicated that L985P is identical to MS4A8B, a member of the recently described membrane-spanning 4-domain family, subfamily A (MS4A) gene family. The MS4A gene family currently consists of greater than 20 distinct human and mouse proteins that include CD20 and FcepsilonRIbeta. Both CD20 and FcepsilonRIbeta are involved in signaling events that regulate diverse cellular functions including cell growth regulation and differentiation. Collectively, the results presented herein demonstrate that L985P is differentially over-expressed in SCLC and may have potential clinical utility as an immunotherapeutic target for the treatment of SCLC.

  15. Vascular endothelial growth factor-D over-expressing tumor cells induce differential effects on uterine vasculature in a mouse model of endometrial cancer

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    Stacker Steven A

    2010-07-01

    Full Text Available Abstract Background It has been hypothesised that increased VEGF-D expression may be an independent prognostic factor for endometrial cancer progression and lymph node metastasis; however, the mechanism by which VEGF-D may promote disease progression in women with endometrial cancer has not been investigated. Our aim was to describe the distribution of lymphatic vessels in mouse uterus and to examine the effect of VEGF-D over-expression on these vessels in a model of endometrial cancer. We hypothesised that VEGF-D over-expression would stimulate growth of new lymphatic vessels into the endometrium, thereby contributing to cancer progression. Methods We initially described the distribution of lymphatic vessels (Lyve-1, podoplanin, VEGFR-3 and VEGF-D expression in the mouse uterus during the estrous cycle, early pregnancy and in response to estradiol-17beta and progesterone using immunohistochemistry. We also examined the effects of VEGF-D over-expression on uterine vasculature by inoculating uterine horns in NOD SCID mice with control or VEGF-D-expressing 293EBNA tumor cells. Results Lymphatic vessels positive for the lymphatic endothelial cell markers Lyve-1, podoplanin and VEGFR-3 profiles were largely restricted to the connective tissue between the myometrial circular and longitudinal muscle layers; very few lymphatic vessel profiles were observed in the endometrium. VEGF-D immunostaining was present in all uterine compartments (epithelium, stroma, myometrium, although expression was generally low. VEGF-D immunoexpression was slightly but significantly higher in estrus relative to diestrus; and in estradiol-17beta treated mice relative to vehicle or progesterone treated mice. The presence of VEGF-D over-expressing tumor cells did not induce endometrial lymphangiogenesis, although changes were observed in existing vessel profiles. For myometrial lymphatic and endometrial blood vessels, the percentage of profiles containing proliferating

  16. Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IκBα protein

    Institute of Scientific and Technical Information of China (English)

    Jian-Feng Wei; Ke Sun; Shi-Guo Xu; Hai-Yang Xie; Shu-Sen Zheng

    2005-01-01

    AIM: NF-κB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection. In this study, we use pCMV-IκBαM vector to inhibit NF-κB activation and investigate the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells. METHODS: The NF-κB activity was detected with pNF-κB reporter gene and electrophoretic mobility shift assay. Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells. RESULTS: We could find that NF-κB activity is inhibited by over-expression of non-degraded IκBα protein. Expression of adhesion molecules like ICAM-1, VCAM-1, and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV-IκBαM vector. CONCLUSION: Our results indicate that the pCMVIκBαM, which inhibit the activity of NF-κB through over-expression of non-degraded IκBα protein, can be used for gene therapy in diseases involving NF-κB activation abnormally like organ transplantation via inhibiting cell adhesion.

  17. Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Trail,a tumor necrosis factor-related apoptosis-inducing ligand,is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2.Its role,like FasL in activation-induced cell death(AICD),has been demonstrated in immune system.However the mechanism of Trail induced apoptosis remains unclear.In this report,the recombinant Trail protein was expressed and purified.The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro.Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner.Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells.Treatment with PMA(phorbol 12-myristate 13-acetate),a PKC activator,suppressed Trail-induced apoptosis in Jurkat T cells.The inhibition of apoptosis by PMA was abolished by pretreatment with Bis,a PKC inhibitor.Taken together,it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

  18. Targeting Hsp90 with small molecule inhibitors induces the over-expression of the anti-apoptotic molecule, survivin, in human A549, HONE-1 and HT-29 cancer cells

    Directory of Open Access Journals (Sweden)

    Lyu Kevin W

    2010-04-01

    Full Text Available Abstract Background Survivin is a dual functioning protein. It inhibits the apoptosis of cancer cells by inhibiting caspases, and also promotes cancer cell growth by stabilizing microtubules during mitosis. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that up-regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently enhance cancer anti-drug responses. The purpose of this study is to determine whether targeting Hsp90 can alter survivin expression differently in different cancer cell lines and to explore possible mechanisms that cause the alteration in survivin expression. Results Here, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by Western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced proteasomal activity while inhibition of 26S proteasome activity further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines. Conclusions We showed for the first time that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. Instead, targeting Hsp90 with small

  19. Differential effect of over-expressing UGT1A1 and CYP1A1 on xenobiotic assault in MCF-7 cells.

    Science.gov (United States)

    Leung, Hau Y; Wang, Yun; Leung, Lai K

    2007-12-05

    Gene mutation has been considered as a major step of carcinogenesis. Some defective genes may induce spontaneous tumorigenesis, while others are required to interact with the environment to induce cancer. CYP1A1 and UGT1A1 are encoded for the respective phase I and II drug-metabolizing enzymes. Their expressions have been associated with breast cancer incidence in women, and some xenobiotics are substrates of these two enzymes. In the current study, cytochrome P450 (CYP) 1A1 and UDP-glucuronosyltransferase (UGT) 1A1 were over-expressed in the breast cancer MCF-7 cells, and potential interactions between these enzymes and estrogen or polycyclic aromatic hydrocarbon were evaluated. Compared with control cells (MCF-7(VEC)), reduced cell proliferation was seen in cells expressing UGT1A1 (MCF-7(UGT1A1)) under estradiol treatment. 7,12-Dimethylbenz[a]anthracene (DMBA) is an established breast cancer initiator in animal model. Over-expressing UGT1A1 reduced the binding of DMBA to DNA, and increased MCF-7(UGT1A1) intact cells under DMBA treatment was verified by comet assay. On the other hand, intensified DMBA binding and damages were observed in MCF-7(CYP1A1) cells. This study supported that UGT1A1 but not CYP1A1 expression could protect against xenobiotic assault.

  20. The over-expression of FGFR4 could influence the features of gastric cancer cells and inhibit the efficacy of PD173074 and 5-fluorouracil towards gastric cancer.

    Science.gov (United States)

    Li, Jingjing; Ye, Yanwei; Wang, Min; Lu, Lisha; Han, Chao; Zhou, Yubing; Zhang, Jingmin; Yu, Zujiang; Zhang, Xiefu; Zhao, Chunlin; Wen, Jianguo; Kan, Quancheng

    2016-05-01

    The aim was to investigate the function of fibroblast growth factor receptor 4 (FGFR4) in gastric cancer (GC) and explore the treatment value of agent targeted to FGFR4. Function assays in vitro and in vivo were performed to investigate the discrepancy of biological features among the GC cells with different expression of FGFR4. GC cells were treated with the single and combination of PD173074 (PD, an inhibitor of FGFR4) and 5-fluorouracil (5-Fu). The invasion ability were stronger, and the apoptosis rates were lower in MGC803 and BGC823 cells treated with FGFR4-LV5 (over-expression of FGFR4 protein) (P FGFR4-LV5 group was less inhibited compared with control group (P FGFR4-LV5 compared with control group (P FGFR4-LV5 group were much more increased (P FGFR4 enhanced the proliferation ability of GC in vitro and in vivo. The combination of 5-Fu and PD exerted synergetic effect in weakening the proliferation ability and promoting apoptosis in GC cells, while the over-expression of FGFR4 might inhibit the efficacy of two drugs.

  1. Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells.

    Science.gov (United States)

    Yang, Yi-Lin; Serrano, Myrna G; Sheoran, Abhineet S; Manque, Patricio A; Buck, Gregory A; Widmer, Giovanni

    2009-11-01

    The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers the intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously.

  2. Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

    Science.gov (United States)

    Edinger, Nufar; Lebendiker, Mario; Klein, Shoshana; Zigler, Maya; Langut, Yael; Levitzki, Alexander

    2016-01-01

    Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC. PMID:27598772

  3. Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo

    DEFF Research Database (Denmark)

    Perryman, L A; Blair, J M; Kingsley, E A;

    2006-01-01

    This study has investigated the impact of three specific dominant-negative p53 mutants (F134L, M237L, and R273H) on tumorigenesis by LNCaP prostate cancer cells. Mutant p53 proteins were associated with an increased subcutaneous "take rate" in NOD-SCID mice, and increased production of PSA. Tumors...

  4. Oestrogen receptor beta over expression in males with non-small cell lung cancer is associated with better survival

    DEFF Research Database (Denmark)

    Skov, Birgit Guldhammer; Sode, Birgitte M Fischer; Pappot, H.

    2008-01-01

    BACKGROUND: Adenocarcinoma of the lung is more frequent in females than in males and the association with smoking is less pronounced than for the other histological subtypes of lung cancer. Oestrogen induction of cell proliferation has been found in breast adenocarcinomas, and since oestrogen...... receptors (ER) have been demonstrated in lung tumours, a similar role of oestrogens in the development of lung cancer has been suggested. We examined the expression of ERalpha, ERbeta and progesterone in a well defined cohort of patients with NSCLC with more than 15 years of follow up, and related...... of the clinical variables, including survival. None of the 104 patients had tumours positive for progesterone. CONCLUSION: The presence of ERbeta in a tumour seems to be a positive prognostic factor for men with non-small cell lung cancer. The finding confirms another recent study and suggests that the relation...

  5. Adenovirus-Mediated Over-Expression of Nrf2 Within Mesenchymal Stem Cells (MSCs Protected Rats Against Acute Kidney Injury

    Directory of Open Access Journals (Sweden)

    Mohammad Mohammadzadeh-Vardin

    2015-06-01

    Full Text Available Purpose: Recent developments in the field of cell therapy have led to a renewed interest in treatment of acute kidney injury (AKI. However, the early death of transplanted mesenchymal stem cells (MSCs in stressful microenvironment of a recipient tissue is a major problem with this kind of treatment. The objective of this study was to determine whether overexpression of a cytoprotective factor, nuclear factor erythroid-2 related factor 2 (Nrf2, in MSCs could protect rats against AKI. Methods: The Nrf2 was overexpressed in MSCs by recombinant adenoviruses, and the MSCs were implanted to rats suffering from cisplatin-induced AKI. Results: The obtained results showed that transplantation with the engineered MSCs ameliorates cisplatin-induced AKI. Morphologic features of the investigated kidneys showed that transplantation with the MSCs in which Nrf2 had been overexpressed significantly improved the complications of AKI. Conclusion: These findings suggested that the engineered MSCs might be a good candidate to be further evaluated in clinical trials. However, detailed studies must be performed to investigate the possible carcinogenic effect of Nrf2 overexpression.

  6. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Xing Hua Tang

    Full Text Available We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs.Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

  7. Transgenic over-expression of slit2 enhances disruption of blood-brain barrier and increases cell death after traumatic brain injury in mice.

    Science.gov (United States)

    Li, Shuai; Li, Hang; He, Xiao-Fei; Li, Ge; Zhang, Qun; Liang, Feng-Ying; Jia, Huan-Huan; Li, Jiang-Chao; Huang, Ren; Pei, Zhong; Wang, Li-Jing; Zhang, Yu

    2016-09-19

    Traumatic brain injury (TBI) is the leading cause of mortality and disability among male adolescents and young adults; and mild traumatic brain injury is the most common type of traumatic brain injury. The disruption of blood-brain barrier (BBB) plays an important role in brain trauma. Previously, we have found that slit2, a member of slit protein family, increases permeability of BBB. In the present study, we examined the role of slit2 in the pathogenesis of mild TBI in a mouse model of micro TBI. Rhodamine BandPI (PropidiumIodide) staining were used to detect the permeability of BBB and cell death, respectively. The leakage of Rhodamine B and cell death were significantly increased in Slit2-Tg mice than in C57 control mice after micro TBI. The present results suggest that over expression of slit2 plays a detrimental role in the pathophysiology of mild TBI.

  8. OVER-EXPRESSION OF EXTRACELLULAR SIGNAL-REGULATED KINASE IN VASCULAR SMOOTH MUSCLE CELL OF HYPERTENSIVE RATS

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension. Methods Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery. The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively. The control group were sham operated age-matched Wistar rats. Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats. Results Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198.00 ± 33.00 mm Hg at the end of experiment, significantly higher than that in the control rats ( P < 0. 01 ). Blood pressure in SHR4w ( 108.00 ± 11.25 mm Hg) was similar to that in the controls. However, it rose to 122.25 ± 21.75 mm Hg in SHR8w, and even up to 201.75 ± 18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls ( P < 0. 01 ). The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P < 0. 05 ). Hyaline degeneration of the afferent arterioles was found in WHR. In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w,and SHR16w. Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2. The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7. 09% ± 1.75%, 14. 57% ± 4. 58%, 29.44% ± 7. 35%, and 13.63% ±3.85%, respectively) than that of the controls( P < 0. 01 ). The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR

  9. Storage of cell lines.

    Science.gov (United States)

    Parker, Katharine A

    2011-01-01

    The successful storage of cell lines depends upon many factors, including the condition of the cells to be frozen and the experience of the operator. Attempting to freeze down unhealthy, contaminated or poorly labelled cells can have huge implications for a research laboratory. This chapter outlines the importance of good record keeping, vigilant monitoring, aseptic technique, and high-quality reagents in the successful storage and downstream propagation of cell lines.

  10. Lysophosphatidic acid activates peroxisome proliferator activated receptor-γ in CHO cells that over-express glycerol 3-phosphate acyltransferase-1.

    Directory of Open Access Journals (Sweden)

    Cliona M Stapleton

    Full Text Available Lysophosphatidic acid (LPA is an agonist for peroxisome proliferator activated receptor-γ (PPARγ. Although glycerol-3-phosphate acyltransferase-1 (GPAT1 esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.

  11. Abnormal B-cell activation associated with TALL-1 over-expression and SOCS-1 suppression during chronic hepatitis C virus infection.

    Science.gov (United States)

    Moorman, Jonathan; Dong, Zhi P; Ni, Lei; Zhang, Chunlan; Borthwick, Thomas; Yao, Zhi Q

    2009-10-01

    Chronic hepatitis C virus (HCV) infection is associated with cirrhosis, autoimmunity and lymphoproliferative disorders. We have previously reported a differential regulation of T and B lymphocytes by HCV core protein in vitro. In this report, we employed a translational approach to characterize the activation status of peripheral B cells from individuals with chronic HCV infection and to explore potential mechanisms for B-cell dysregulation in the setting of HCV infection. In contrast to the T-cell suppression observed in HCV-infected individuals, B cells exhibit a non-specific polyclonal activation phenotype, characterized by significantly higher levels of (1) the early activation marker, CD69, (2) the costimulatory molecule, CD86, and (3) the CCR5 chemokine receptor, CD195, when compared with B cells from healthy donors in response to phytohaemagglutinin (PHA) stimulation. Importantly, tumour necrosis factor- and Apo-L-related leucocyte-expressed ligand-1 (TALL-1), also known as B-lymphocyte stimulator (BLYS), was found to be up-regulated on the surface of B cells from HCV patients in response to PHA as well as HCV core antigen stimulation. This up-regulation of TALL-1 was associated with vigorous memory B-cell responses to viral antigenic stimulation. Additionally, suppressor of cytokine signalling-1 (SOCS-1), a negative feedback immunoregulator that is inhibited in B lymphocytes by HCV core in vitro, was also inhibited in B cells from HCV patients when compared with healthy donors. These findings suggest that TALL-1 over-expression and SOCS-1 suppression are associated with aberrant B-cell activation, providing a plausible basis for the B-cell clonal expansion underlying the lymphoproliferative disorders and autoimmune phenomena observed during chronic HCV infection.

  12. Over-expression of small ubiquitin-like modifier proteases 1 predicts chemo-sensitivity and poor survival in non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    Mu Juwei; Zuo Yong; Yang Wenjing; Chen Zhaoli; Liu Ziyuan; Tu Jun; Li Yan

    2014-01-01

    Background Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors.Despite the advances in therapy over the years,its mortality remains high.The aim of this study was to evaluate the expression of small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) in NSCLC tissues and its role in the regulation of vascular endothelial growth factor (VEGF) expression.We also investigated the association between the expression level of SENP1 and the clinicopathological features and survival of the patients.Methods A SENP1 small interfering RNA (siRNA) was constructed and transfected into the NSCLC cells.VEGF gene expression was analyzed by real-time polymerase chain reaction (RT-PCR).Immunohistochemistry staining was used to assess the expression of SENP1 in 100 NSCLC patients and its association with the clinicopathological features and survival was analyzed.Results VEGF expression was significantly higher in NSCLC tissues than in normal lung tissues.Inhibition of SENP1 by siRNA was associated with decreased VEGF expression.SENP1 was over-expressed in 55 of the 100 NSCLC samples (55%) and was associated with a moderate and low histological tumor grade (3.6%,38.2%,and 58.2% in high,moderate and low differentiated tumors,respectively,P=0.046),higher T stage (10.9% in T1,and 89.1% in T2 and T3 tumor samples,P <0.001)and TNM stage (10.9% in stage Ⅰ,and 89.1% in stages Ⅱ and Ⅲ tumor samples,P <0.001).The rate of lymph node metastasis was significantly higher in the SENP1 over-expression group (76.4%) than that in the SENP1 low expression group (33.3%,P <0.001).Sixty three patients received postoperative chemotherapy,including 34 with SENP1 over-expression and 29 with SENP1 low expression.Among the 34 patients with SENP1 over-expression,22 (64.7%) patients developed recurrence or metastasis,significantly higher than those in the low expression group 27.6% (8/29) (P=0.005).Multivariate Cox regression analysis showed that lymph

  13. Aberrant over-expression of TRPM7 ion channels in pancreatic cancer: required for cancer cell invasion and implicated in tumor growth and metastasis

    Directory of Open Access Journals (Sweden)

    Nelson S. Yee

    2015-03-01

    Full Text Available Our previous studies in zebrafish development have led to identification of the novel roles of the transient receptor potential melastatin-subfamily member 7 (TRPM7 ion channels in human pancreatic cancer. However, the biological significance of TRPM7 channels in pancreatic neoplasms was mostly unexplored. In this study, we determined the expression levels of TRPM7 in pancreatic tissue microarrays and correlated these measurements in pancreatic adenocarcinoma with the clinicopathological features. We also investigated the role of TRPM7 channels in pancreatic cancer cell invasion using the MatrigelTM-coated transwell assay. In normal pancreas, TRPM7 is expressed at a discernable level in the ductal cells and centroacinar cells and at a relatively high level in the islet endocrine cells. In chronic pancreatitis, pre-malignant tissues, and malignant neoplasms, there is variable expression of TRPM7. In the majority of pancreatic adenocarcinoma specimens examined, TRPM7 is expressed at either moderate-level or high-level. Anti-TRPM7 immunoreactivity in pancreatic adenocarcinoma significantly correlates with the size and stages of tumors. In human pancreatic adenocarcinoma cells in which TRPM7 is highly expressed, short hairpin RNA-mediated suppression of TRPM7 impairs cell invasion. The results demonstrate that TRPM7 channels are over-expressed in a proportion of the pre-malignant lesions and malignant tumors of the pancreas, and they are necessary for invasion by pancreatic cancer cells. We propose that TRPM7 channels play important roles in development and progression of pancreatic neoplasm, and they may be explored as clinical biomarkers and targets for its prevention and treatment.

  14. Hypomethylation and Over-Expression of the Beta Isoform of BLIMP1 is Induced by Epstein-Barr Virus Infection of B Cells; Potential Implications for the Pathogenesis of EBV-Associated Lymphomas

    Directory of Open Access Journals (Sweden)

    Katerina Vrzalikova

    2012-10-01

    Full Text Available B-lymphocyte-induced maturation protein 1 (BLIMP1 exists as two major isoforms, α and β, which arise from alternate promoters. Inactivation of the full length BLIMP1α isoform is thought to contribute to B cell lymphomagenesis by blocking post-germinal centre (GC B cell differentiation. In contrast, the shorter β isoform is functionally impaired and over-expressed in several haematological malignancies, including diffuse large B cell lymphomas (DLBCL. We have studied the influence on BLIMP1β expression of the Epstein-Barr virus (EBV, a human herpesvirus that is implicated in the pathogenesis of several GC-derived lymphomas, including a subset of DLBCL and Hodgkin’s lymphoma (HL. We show that BLIMP1β expression is increased following the EBV infection of normal human tonsillar GC B cells. We also show that this change in expression is accompanied by hypomethylation of the BLIMP1β-specific promoter. Furthermore, we confirmed previous reports that the BLIMP1β promoter is hypomethylated in DLBCL cell lines and show for the first time that BLIMP1β is hypomethylated in the Hodgkin/Reed-Sternberg (HRS cells of HL. Our results provide evidence in support of a role for BLIMP1β in the pathogenesis of EBV-associated B cell lymphomas.

  15. Hepatitis E virus ORF2 protein over-expressed by baculovirus in hepatoma cells, efficiently encapsidates and transmits the viral RNA to naïve cells

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    Emerson Suzanne U

    2011-04-01

    Full Text Available Abstract A recombinant baculovirus(vBacORF2 that expressed the full-length ORF2 capsid protein of a genotype 1 strain of hepatitis E virus(HEV was constructed. Transduction of S10-3 human hepatoma cells with this baculovirus led to large amounts of ORF2 protein production in ~50% of the cells as determined by immune fluorescence microscopy. The majority of the ORF2 protein detected by Western blot was 72 kDa, the size expected for the full-length protein. To determine if the exogenously-supplied ORF2 protein could transencapsidate viral genomes, S10-3 cell cultures that had been transfected the previous day with an HEV replicon of genotype 1 that contained the gene for green fluorescent protein(GFP, in place of that for ORF2 protein, were transduced with the vBacORF2 virus. Cell lysates were prepared 5 days later and tested for the ability to deliver the GFP gene to HepG2/C3A cells, another human hepatoma cell line. FACS analysis indicated that lysates from cell cultures receiving only the GFP replicon were incapable of introducing the replicon into the HepG2/C3A cells whereas ~2% of the HepG2/C3A cells that received lysate from cultures that had received both the replicon and the baculovirus produced GFP. Therefore, the baculovirus-expressed ORF2 protein was able to trans-encapsidate the viral replicon and form a particle that could infect naïve HepG2/C3A cells. This ex vivo RNA packaging system should be useful for studying many aspects of HEV molecular biology.

  16. Over-expression of ATF3 Inhibits the Proliferation of Esophageal Squamous Carcinoma Cells%激活转录因子3的过表达对食管癌细胞生长的抑制作用

    Institute of Scientific and Technical Information of China (English)

    谢仰民; 谢剑君; 周飞; 侯健; 曹君君; 许丽艳; 李恩民

    2009-01-01

    背景与目的:激活转录因子3(activating transcription factor 3,ATF3)在食管癌中表达异常下调,但其功能仍不清楚.本研究拟探讨ATF3在食管癌细胞过表达对癌细胞生长及裸鼠成瘤的影响. 材料与方法:利用分子克隆技术将ATF3基因完整编码区克隆至真核细胞表达载体pcDNA3中,获得重组表达质粒;将该表达质粒转染食管癌EC109细胞并用G418筛选稳定表达的细胞克隆;用Western blot检测ATF3的过表达效果;对ATF3过表达的细胞与相应对照细胞进行细胞克隆形成实验和裸鼠体内成瘤实验,以分析ATF3过表达对食管癌细胞生长的影响. 结果:ATF3的过表达在体外可以降低食管癌细胞的克隆形成能力;在体内可以抑制食管癌细胞在雌鼠中的成瘤能力,但对雄鼠中的成瘤能力没有明显影响.结论:ATF3的过表达可以抑制食管癌细胞的生长,ATF3可能在食管癌的发生发展中发挥重要作用.%BACKGROUND AND AIM: ATF3 was down-regulated in esophageal squamous cell carcinoma (ESCC), but the roles of ATF3 in ESCC cells still remained unclear. The purpose of this study was to explore the effect of ATF3 on the proliferation of ESCC cells. MATERIALS AND METHODS: The recombinant expressing plasmid was constructed by inserting the full coding sequence of ATF3 gene into the eukaryotic expressing vector pcDNA3. Then, the expressing plasmid was stably transfected into EC 109 cells, an ESCC cell line, and the over-expressing ATF3 cell clones were obtained. Colony formation assay and tumor formation assay in nude mice were used to explore the effect of ATF3 over-expression on the proliferation of ESCC cells. RESULTS: With the over-expression of ATF3, the colony formation ability of EC109 cells was decreased and the tumor formation of EC109 cells in female mice was inhibited. CONCLUSION: Over-expression of ATF3 could inhibit the proliferation of ESCC cells and ATF3 may play important roles in the progression of ESCC.

  17. Effects of clusterin over-expression on metastatic progression and therapy in breast cancer

    Directory of Open Access Journals (Sweden)

    Chatterjee Namita

    2010-03-01

    Full Text Available Abstract Background Clusterin is a secreted glycoprotein that is upregulated in a variety of cell lines in response to stress, and enhances cell survival. A second nuclear isoform of clusterin that is associated with cell death has also been identified. The aim of this study was to determine the role(s of the secretory isoform in breast tumor progression and metastasis. Methods To investigate the role of secretory clusterin in the biology of breast cancer tumor growth and resistance to therapy we have engineered an MCF-7 cell line (MCF-7CLU that over-expresses clusterin. We have measured the in vitro effects of clusterin over-expression on cell cycle, cell death, and sensitivity to TNFalpha and tamoxifen. Using an orthotopic model of breast cancer, we have also determined the effects of over-expression of clusterin on tumor growth and metastatic progression. Results In vitro, over-expression of secretory clusterin alters the cell cycle kinetics and decreases the rate of cell death, resulting in the enhancement of cell growth. Over-expression of secretory clusterin also blocks the TNFalpha-mediated induction of p21 and abrogates the cleavage of Bax to t-Bax, rendering the MCF-7CLU cells significantly more resistant to the cytokine than the parental cells. Orthotopic primary tumors derived from MCF-7CLU cells grow significantly more rapidly than tumors derived from parental MCF-7 cells and, unlike the parental cells, metastasize frequently to the lungs. Conclusions These data suggest that secretory clusterin, which is frequently up-regulated in breast cancers by common therapies, including anti-estrogens, may play a significant role in tumor growth, metastatic progression and subsequent drug resistance in surviving cells.

  18. NOK对肺腺癌细胞SPC-A-1增殖的影响%Effect of NOK over expression on proliferation of lung adenocarcinoma cell SPC-A-1

    Institute of Scientific and Technical Information of China (English)

    刘涛; 张志培; 李小飞

    2012-01-01

    目的:建立稳定过表达NOK基因的SPC-A-1-NOK细胞系,观察其增殖变化,探讨NOK基因对SPC-A-l细胞增殖的影响.方法:运用电穿孔仪将pcDNA3.1-NOK质粒转染肺腺癌SPC-A-1细胞,筛选出稳定过表达NOK基因的SPC-A-1-NOK细胞单克隆株.RT-PCR检测转染前后细胞内NOK基因的表达效果.通过流式细胞术检测细胞周期和细胞凋亡,MTT法检测细胞生长并绘制细胞生长曲线.结果:RT-PCR显示SPC-A-1-NOK细胞中NOK基因表达量明显增高.流式细胞术显示SPC-A-1-NOK与SPC-A-1和SPC-A-1-3.1细胞相比,S期细胞增多[(28.00±1.42)% VS(24.93±1.57)%,(23.75±1.20)%,均P<0.05]、细胞增殖指数升高[(56.70±1.43)% VS(46.47士1.32)%,(46.89±1.19)%,均P<0.05]、细胞凋亡比减少[(4.O±0.2)% VS(8.4±0.5)%,(7.9±0.4)%,均P<0.05].MTT结果显示SPC-A-1-NOK细胞较SPC-A-1和SPC-A-1-3.1细胞生长曲线上移(P<0.05).结论:NOK可促进SPC-A-1细胞的增殖,可能在肺癌的发生、发展中发挥重要作用.%Objective: To establish the stable over expression of NOK in lung adenocarcinoma cell line SPC - A -1 and to investigate the effect of NOK over expression on the proliferation of SPC - A - 1. Methods :pcDNA3. 1 -NOK plasmid was transferred into SPC - A -1 cells with electroporation apparatus. The cloned strain SPC - A - 1 -NOK with NOK stable over expression was screened and validated by RT - PCR. The cell cycle and cell apoptosis incidence were detected by flow cytometry and the cell growth curve was determined by MTT. Results;There was stable over expression of NOK in SPC - A - 1 - NOK cells. The number of SPC - A - 1 - NOK cells at S stage was more than that of SPC - A - 1 and SPC -A-l -3.1 cells[ (28.00 ± 1.42)% VS (24.93 ± 1.57)% ,(23. 75 ± 1.20) % , both P < 0.05 ]; The proliferation index (PI) in SPC - A - 1 - NOK cells with the number of cell apoplosis being decreased[ (4.0 ±0.2)% VS (8.4 ±0.5)% ,(7.9 ±0.4)% ,both P<0.05] ,was higher than that in

  19. Ubiquitous Over-Expression of Chromatin Remodeling Factor SRG3 Ameliorates the T Cell-Mediated Exacerbation of EAE by Modulating the Phenotypes of both Dendritic Cells and Macrophages.

    Science.gov (United States)

    Lee, Sung Won; Park, Hyun Jung; Jeon, Sung Ho; Lee, Changjin; Seong, Rho Hyun; Park, Se-Ho; Hong, Seokmann

    2015-01-01

    Although SWI3-related gene (SRG3), a chromatin remodeling factor, is critical for various biological processes including early embryogenesis and thymocyte development, it is unclear whether SRG3 is involved in the differentiation of CD4+ T cells, the key mediator of adaptive immune responses. Because it is known that experimental autoimmune encephalomyelitis (EAE) development is determined by the activation of CD4+ T helper cells, here, we investigated the role of SRG3 in EAE development using SRG3 transgenic mouse models exhibiting two distinct SRG3 expression patterns: SRG3 expression driven by either the CD2 or β-actin promoter. We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3. The specific over-expression of SRG3 using the CD2 promoter facilitated EAE via the induction of Th1 and Th17 cells, whereas the ubiquitous over-expression of SRG3 using the β-actin promoter inhibited EAE by promoting Th2 differentiation and suppressing Th1 and Th17 differentiation. In addition, the ubiquitous over-expression of SRG3 polarized CD4+ T cell differentiation towards the Th2 phenotype by converting dendritic cells (DCs) or macrophages to Th2 types. SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE. In addition, Th2 differentiation in β-actin-SRG3 Tg mice during EAE was associated with an increase in the basophil and mast cell populations and in IL4 production. Furthermore, the increased frequency of Treg cells in the spinal cord of β-actin-SRG3 Tg mice might induce the suppression of and accelerate the recovery from EAE symptoms. Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization

  20. Over-expression of hNGF in adult human olfactory bulb neural stem cells promotes cell growth and oligodendrocytic differentiation

    NARCIS (Netherlands)

    H.E.S. Marei (Hany); A. Althani (Asmaa); N. Afifi (Nahla); A. Abd-Elmaksoud (Ahmed); C. Bernardini (Camilla); F. Michetti (Fabrizio); M. Barba (Marta); M. Pescatori (Mario); G. Maira (Giulio); E. Paldino (Emanuela); L. Manni (Luigi); P. Casalbore (Patrizia); C. Cenciarelli (Carlo)

    2013-01-01

    textabstractThe adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, h

  1. CLO : The cell line ontology

    NARCIS (Netherlands)

    Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

    2014-01-01

    Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

  2. Over-expression of hNGF in adult human olfactory bulb neural stem cells promotes cell growth and oligodendrocytic differentiation.

    Directory of Open Access Journals (Sweden)

    Hany E S Marei

    Full Text Available The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood-brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF and green fluorescent protein (GFP genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia

  3. Over-expression of nm23-H1 in HeLa cells provides cells with higher resistance to oxidative stress possibly due to raising intracellular p53 and GPX1

    Institute of Scientific and Technical Information of China (English)

    Run AN; Yong-lie CHU; Chan TIAN; Xiao-xia DAI; Jing-hong CHEN; Qi SHI; Jun HAN; Xiao-ping DONG

    2008-01-01

    Aim: To determine whether the antitumor factor nm23 is related with antioxi-dation. Methods: Full-length human nm23-Hl was cloned into a mammalian-expressing vector and transiently introduced into HeLa cells. Results: A remark-ably low level of reactive oxygen species (ROS) was detected in the cells over-expressing nm23-Hl. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trypan blue assays found that the cells transfected with a nm23-H1-expressing plasmid had higher viability and stronger resistance to oxidative stress. Immunoprecipitation tests revealed that endogenous nm23-H1 formed a protein complex with p53. Furthermore, the intracellular levels of p53 and p53-regulated gene GPXI were obviously increased in the cells overexpressing nm23-H1. The downregulation of p53 in the cells overexpressing nm23-H1 resulted in a higher cellular ROS level and lower cell viability. Conclusion: The findings suggest that nm23-H1 may act as a cellular protector against oxidative stress, possibly triggering the p53-related antioxidative pathway.

  4. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    Directory of Open Access Journals (Sweden)

    Anita Collavoli

    2011-01-01

    Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

  5. Metabolomic comparison between cells over-expressing isocitrate dehydrogenase 1 and 2 mutants and the effects of an inhibitor on the metabolism.

    Science.gov (United States)

    Wen, He; Cho, Hye Rim; Yun, Taeho; Kim, Hyeonjin; Park, Chul-Kee; Lee, Se-Hoon; Choi, Seung Hong; Park, Sunghyouk

    2015-01-01

    The R132H and R172K mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have neomorphic activity of generating 2-hydroxyglutarate (2-HG) which has been implicated in the oncogenesis. Although similarities in structure and enzyme activity for the two isotypic mutations have been suggested, the difference in their cellular localization and biochemical properties suggests differential effects on the metabolic oncogenesis. Using U87 cells transfected with either wild-type (WT) and mutant (MT) IDH genes, the MT-IDH1 and MT-IDH2 cells were compared with NMR-based metabolomics. When normalized with the respective WT-IDH cells, the general metabolic shifts of MT-IDH1 and IDH2 were almost opposite. Subsequent analysis with LC-MS and metabolic pathway mapping showed that key metabolites in pentose phosphate pathway and tricarboxylic acid cycle are disproportionately altered in the two mutants, suggesting different activities in the key metabolic pathways. Notably, lactate level was lower in MT-IDH2 cells which produced more 2-HG than MT-IDH1 cells, indicating that the Warburg effects can be overridden by the production of 2-HG. We also found that the effect of a mutant enzyme inhibitor is mainly reduction of the 2-HG level rather than general metabolic normalization. Overall, the metabolic alterations in the MT-IDH1 and 2 can be different and seem to be commensurate with the degree of 2-HG production. The R132H and R172K mutations of isocitrate dehydrogenase 1 and 2, respectively, (IDH1 and IDH2) have neomorphic activity of generating 2-hydroxyglutarate (2-HG) which has been implicated in oncogenesis. The mutant cell's metabolic shifts from the respective wild type cells were almost opposite, with lactate level being lower in the IDH2 mutant only, implicating an overridden Warburg effect. The metabolic effect of an IDH1 mutant inhibitor was limited to 2-HG lowering.

  6. ALMS1-deficient fibroblasts over-express extra-cellular matrix components, display cell cycle delay and are resistant to apoptosis.

    Directory of Open Access Journals (Sweden)

    Elisabetta Zulato

    Full Text Available Alström Syndrome (ALMS is a rare genetic disorder (483 living cases, characterized by many clinical manifestations, including blindness, obesity, type 2 diabetes and cardiomyopathy. ALMS is caused by mutations in the ALMS1 gene, encoding for a large protein with implicated roles in ciliary function, cellular quiescence and intracellular transport. Patients with ALMS have extensive fibrosis in nearly all tissues resulting in a progressive organ failure which is often the ultimate cause of death. To focus on the role of ALMS1 mutations in the generation and maintenance of this pathological fibrosis, we performed gene expression analysis, ultrastructural characterization and functional assays in 4 dermal fibroblast cultures from ALMS patients. Using a genome-wide gene expression analysis we found alterations in genes belonging to specific categories (cell cycle, extracellular matrix (ECM and fibrosis, cellular architecture/motility and apoptosis. ALMS fibroblasts display cytoskeleton abnormalities and migration impairment, up-regulate the expression and production of collagens and despite the increase in the cell cycle length are more resistant to apoptosis. Therefore ALMS1-deficient fibroblasts showed a constitutively activated myofibroblast phenotype even if they do not derive from a fibrotic lesion. Our results support a genetic basis for the fibrosis observed in ALMS and show that both an excessive ECM production and a failure to eliminate myofibroblasts are key mechanisms. Furthermore, our findings suggest new roles for ALMS1 in both intra- and extra-cellular events which are essential not only for the normal cellular function but also for cell-cell and ECM-cell interactions.

  7. Over-expression of TRIM37 promotes cell migration and metastasis in hepatocellular carcinoma by activating Wnt/β-catenin signaling

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jianxin; Yu, Chao; Chen, Meiyuan; Tian, She; Sun, Chengyi, E-mail: chenyisun11@163.com

    2015-09-04

    Hepatocellular carcinoma (HCC) is the most common cancer in the world especially in East Asia and Africa. Advanced stage, metastasis and frequent relapse are responsible for the poor prognosis of HCC. However, the precise mechanisms underlying HCC remained unclear. So it is urgent to identify the pathological processes and relevant molecules of HCC. TRIM37 is an E3 ligase and has been observed deregulated expression in various tumors. Recent studies of TRIM37 have implicated that TRIM37 played critical roles in cell proliferation and other processes. In the present study, we demonstrated that TRIM37 expression was notably up-regulated in HCC samples and was associated with advanced stage and tumor volume, which all indicating the poor outcomes. We also found that TRIM37 could serve as an independent prognostic factor of HCC. During the course of in vitro and in vivo work, we showed that TRIM37 promoted HCC cells migration and metastasis by inducing EMT. Furthermore, we revealed that the effect of TRIM37 mediated EMT in HCC cells was achieved by the activation of Wnt/β-catenin signaling. These finding may provide insight into the understanding of TRIM37 as a novel critical factor of HCC and a candidate target for HCC treatment. - Highlights: • Highly expression of TRIM37 is found in HCC samples compared with nontumorous samples. • TRIM37 expression is correlated with advanced HCC stages and could be an independent prognostic factor. • TRIM37 promotes cell proliferation and metastasis. • We report an E3 ligase TRIM37 affects Wnt/β-catenin signaling.

  8. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

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

    1991-12-01

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

  9. Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4{sup +}CD25{sup +} regulatory T cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Yulan; Purohit, Sharad [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA (United States); Chen, Xueqin; Yi, Bing [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); She, Jin-Xiong, E-mail: jshe@georgiahealth.edu [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA (United States)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer This is the first study to provide direct evidence of the role of Stat5b in NOD mice. Black-Right-Pointing-Pointer Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. Black-Right-Pointing-Pointer This protection may be mediated by the up-regulation of CD4{sup +}CD25{sup +} Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4{sup +} T cells and especially CD8{sup +} T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4{sup +} and CD8{sup +} T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-{gamma}, TNF-{alpha} and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4{sup +}CD25{sup +} regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4{sup +}CD25{sup +} regulatory T cells.

  10. Over-expression of Pygo2 Promotes C6 Cells Proliferation of Glioma%Pygo2过表达促进大鼠胶质瘤C6细胞增殖

    Institute of Scientific and Technical Information of China (English)

    陈玉英; 王海东; 王占祥; 谭国伟; 刘希尧; 沈上杭

    2012-01-01

    Objective To up-regulate expression of Pygopus2 (Fygo2) by construction of the recombinanl vectors of over-expression of Py-go2 protein,and to explore the role and mechanism of over-expression of Pygo2 in C6 cells proliferation of glioma. Methods The recombi-nant plasmids were digested with EcoR I and Hind III to execute the restriction endonuclease identification,then the sequence analysis was assayed by DNA sequencing. The recombinant plasmids were transfected into cultured gliohlastoma C6 cells using lipofectamineTM 2000. The exogenous Pygo2 protein level of C6 cells was detected by Western blot analysis. Colony framing assay and MTT assay were used to detect the cell proliferation,and cell cycle analysis was performed by flow cytometry analysis. The effect of Pygo2 over-expression on the level of cy-clinD1 and β-catenin of C6 cells was detected by Western blot analysis,and the expression and subcellular location of cyclinD1 and (3-catenin of C6 cells were further quantified by immunofluorescent staining. Results The recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis,which up-regulated Pygo2 expression of C6 cells efficiently. After Pygo2 expression were up-regulated by transfected C6 cells with the recombinant plasmids,cells proliferation was promoted and colony forming was increased significantly,cell cycle progression from G, to S transition was enhanced notably. Furthermore,the expression level of cyclinD1 was significantly increased without change of subcellular location,and the expression level and subcellular location of β-catenin were not changed obviously. Concluson The recombinant vectors of Pygo2 over-expression were constructed successfully. Over-expression of Pygo2 promotes the growth of glioma cells by an increased expression of cyclinD1 to improve G1/S transition.%目的 通过构建过表达Pygo2的重组体上调Pygo2表达,探讨其在大鼠胶质瘤C6细胞增

  11. Presenilin-1 mutations alter K+ currents in the human neuroblastoma cell line, SH-SY5Y

    DEFF Research Database (Denmark)

    Plant, Leigh D; Boyle, John P; Thomas, Natasha M

    2002-01-01

    Mutations in presenilin 1 (PS1) are the major cause of autosomal dominant Alzheimer's disease. We have measured the voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y using whole-cell patch-clamp. When cells were stably transfected to over-express PS1, no change in K+ current...... membrane distribution when the deltaE9 over-expressing cells were compared to control cells. Intracellular retention of Kv3.1 is consistent with the notion that PS1 can modulate the activity and trafficking of ion channels in central neurones and implicates a compromise in electrical signalling...

  12. Over expression of minichromosome maintenance genes is clinically correlated to cervical carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Mitali Das

    Full Text Available Minichromosome Maintenance (MCM proteins play important roles in cell cycle progression by mediating DNA replication initiation and elongation. Among 10 MCM homologues MCM 2-7 form a hexamer and assemble to the pre-replication complex acting as replication licensing factors. Binding and function of MCM2-7 to pre-replication complex is regulated by MCM10 mediated binding of RECQL4 with MCM2-7. The purpose of this study is to explore the role of MCMs in cervical cancer and their correlation with the clinical parameters of cervical cancer. We have investigated sixty primary cervical cancer tissue samples, eight cervical cancer cell lines and thirty hysterectomised normal cervical tissue. The expression profiling of MCMs was done using semi-quantitative RT-PCR, immunoblotting and immunohistochemistry. MCM2, 4, 5, 6, 7, 10 and RECQL4 are significantly over-expressed in cervical cancer. Among these, MCM4, 6 and 10 show increased frequency of over expression along with advancement of tumor stages. MCM4, 5 and 6 also show differential expression in different types of lesion, while MCM2 and MCM10 are over expressed in cervical cancer irrespective of clinico-pathological parameters. Our data indicates the role of MCM4, MCM5, MCM6, MCM10 and RECQL4 in the progression of cervical cancer.

  13. Effects of K562 cells with over-expression of MHC class Ⅰ chain-related protein A on phagocytosis of dendritic cells%K562细胞过表达MHCⅠ类相关抗原A对树突状细胞吞噬功能的影响

    Institute of Scientific and Technical Information of China (English)

    张跃; 邵小青; 陈贝; 季明春; 龚卫娟

    2012-01-01

    Objective To observe how apoptosed K562 cells with over-expression of MHC class I chain-related protein A (MICA) affects phagocytic function of dendritic cells. Methods At first a K562 cell line with ectopic MICA expression, called K562-MICA, was generated by gene trans-fection and G418 screen. Both K562 and K562-MICA cells were stained with fluorescent CFSE, treated with mitomycin C, and then co-cultured with THP1 cells or dendritic cells derived from peripheral blood monocytes overnight. Phagocytic activities were evaluated through detection of percentage of ap-optotic cells by flow cytometry. Meanwhile, some activating receptors on THP1 cells and NKG2D expression on DC were measured by flow cytometry. Finally NKG2D neutralizing antibody was added to cell co-culture system to observe whether phagocytosis of DC would be varied correspondingly. Results K562-MICA cells stimulated THP1 cell to enhance expression of CD86 and MICA, but had no effects on HLA-DR and NKG2D expression. Compared with K562 cells, apoptotic bodies from K562-MICA cells were more susceptible to be uptake by DC. Apoptosed K562-MICA cells induced DC to increase NKG2D expression. In addition, NKG2D antibody could significantly inhibit phagocytosis of DC. Conclusion MICA over-expression on K562 cells promoted phagocytic function of DC, and the function depended on NKG2D expression on DC.%目的 观察过表达MHC Ⅰ类相关抗原A(MICA)的K562细胞,体外经诱导凋亡后,对树突状细胞(DC)吞噬功能的影响.方法 首先利用脂质体介导的基因转染技术和G418筛选过程,建立稳定表达MICA的K562细胞(K562-MICA).其次分别取K562、K562-MICA细胞经CFSE标记,并用丝裂霉素C诱导凋亡,与单核细胞系THP1或外周血单核细胞来源的DC共孵育过夜,流式细胞术检测2种细胞吞噬凋亡小体的活性.同时检测THPI细胞表面相关活化性受体的表达,以及DC表面NKG2D受体的情况.最后,在细胞共培养体系中加入NKG2D抗体,观

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

    Science.gov (United States)

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

    2013-06-01

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

  15. The hepatic Raldh1 expression is elevated in Zucker fatty rats and its over-expression introduced the retinal-induced Srebp-1c expression in INS-1 cells.

    Directory of Open Access Journals (Sweden)

    Yang Li

    Full Text Available The roles of vitamin A (VA in the development of metabolic diseases remain unanswered. We have reported that retinoids synergized with insulin to induce the expression of sterol-regulatory element-binding protein 1c gene (Srebp-1c expression in primary rat hepatocytes. Additionally, the hepatic Srebp-1c expression is elevated in Zucker fatty (ZF rats, and reduced in those fed a VA deficient diet. VA is metabolized to retinoic acid (RA for regulating gene expression. We hypothesized that the expression of RA production enzymes contributes to the regulation of the hepatic Srebp-1c expression. Therefore, we analyzed their expression levels in Zucker lean (ZL and ZF rats. The mRNA levels of retinaldehyde dehydrogenase family 1 gene (Raldh1 were found to be higher in the isolated and cultured primary hepatocytes from ZF rats than that from ZL rats. The RALDH1 protein level was elevated in the liver of ZF rats. Retinol and retinal dose- and time-dependently induced the expression of RA responsive Cyp26a1 gene in hepatocytes and hepatoma cells. INS-1 cells were identified as an ideal tool to study the effects of RA production on the regulation of gene expression because only RA, but not retinal, induced Srebp-1c mRNA expression in them. Recombinant adenovirus containing rat Raldh1 cDNA was made and used to infect INS-1 cells. The over-expression of RALDH1 introduced the retinal-mediated induction of Srebp-1c expression in INS-1 cells. We conclude that the expression levels of the enzymes for RA production may contribute to the regulation of RA responsive genes, and determine the responses of the cells to retinoid treatments. The elevated hepatic expression of Raldh1 in ZF rats may cause the excessive RA production from retinol, and in turn, result in higher Srebp-1c expression. This excessive RA production may be one of the factors contributing to the elevated lipogenesis in the liver of ZF rats.

  16. DEK over-expression promotes mitotic defects and micronucleus formation.

    Science.gov (United States)

    Matrka, Marie C; Hennigan, Robert F; Kappes, Ferdinand; DeLay, Monica L; Lambert, Paul F; Aronow, Bruce J; Wells, Susanne I

    2015-01-01

    The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation.

  17. Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响%Effect of Ezrin gene knockdown and over-expression on invasion of glioma U87 cells

    Institute of Scientific and Technical Information of China (English)

    刘乃杰; 秦治刚; 孙利波; 金星一; 叶保国; 张金男; 朱庆三

    2013-01-01

    Objective To study the relationship between Ezrin gene and infiltrative growth of glioma through Ezrin gene knockdown and over-expression. Methods According to Ezrin gene sequence in GenBank, primers were designed using Prime Primer 5. 0 software, with which the gene fragment encoding CDS region of Ezrin gene was amplified from U87 cells and cloned into expression vector pEGFP-1. U87 cells were transfected with the constructed recombinant plasmid pEGFP-C 1/Ezrin as well as plasmids shRNA-EZrin-2 and pEGFP-C 1 in mediation of LipofectimineTM 2000 respectively, then deter-mined for expression of Ezrin protein by Western blot, and for migration by scarification test. Results The homologies of mRNAs of cloned Ezrin gene were 99% to those of homo sapiens ezrin (EZR) and transcript variant 1 reported in Gen-Bank. The homologies of amino acids encoding by the cloned gene was 99% to that of ezrin [Homo sapiens] (Sequence ID: ref-NP_003370. 2-, Length:586), with variations of S66P, K258R, P265L and K577R, while the opening read frame was correct. The relative expression level (1. 17) of Ezrin protein in U87 cells transfected with recombinant plasmid pEGFP-C1 /Ezrin was higher those transfected with shRNA-Ezrin-2 (0. 47) and pEGFP-C1 (0. 82). Scarification test showed migration of a small quantity of U87 cells transfected with shRNA-Ezrin-2 to the wound. However, in pEGFP-C1/ Ezrin transfection group, the wound was almost filled with cells. Conclusion Ezrin gene knockdown blocked , while the over-expression promoted the migration of U87 cells, indicating that Ezrin gene involved in the invasive growth of U87 cells.%目的 分析Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响,以探讨脑胶质瘤浸润性生长的机理.方法 从U87细胞中扩增Ezrin基因CDS区片段,克隆至表达载体pEGFP-C1中,构建Ezrin基因表达质粒pEGFP-C1/Ezrin.将pEGFP-C1/Ezrin、Ezrin基因shRNA质粒shRNA-Ezrin-2和pEGFP-C1以脂质体LipofectimineTM 2000

  18. Generation of a persistently infected MDBK cell line with natural bovine spongiform encephalopathy (BSE.

    Directory of Open Access Journals (Sweden)

    Dongseob Tark

    Full Text Available Bovine spongiform encephalopathy (BSE is a zoonotic transmissible spongiform encephalopathy (TSE thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD. Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrPBSE and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrPBSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.

  19. Generation of a persistently infected MDBK cell line with natural bovine spongiform encephalopathy (BSE).

    Science.gov (United States)

    Tark, Dongseob; Kim, Hyojin; Neale, Michael H; Kim, Minjeong; Sohn, Hyunjoo; Lee, Yoonhee; Cho, Insoo; Joo, Yiseok; Windl, Otto

    2015-01-01

    Bovine spongiform encephalopathy (BSE) is a zoonotic transmissible spongiform encephalopathy (TSE) thought to be caused by the same prion strain as variant Creutzfeldt-Jakob disease (vCJD). Unlike scrapie and chronic wasting disease there is no cell culture model allowing the replication of proteinase K resistant BSE (PrPBSE) and the further in vitro study of this disease. We have generated a cell line based on the Madin-Darby Bovine Kidney (MDBK) cell line over-expressing the bovine prion protein. After exposure to naturally BSE-infected bovine brain homogenate this cell line has shown to replicate and accumulate PrPBSE and maintain infection up to passage 83 after initial challenge. Collectively, we demonstrate, for the first time, that the BSE agent can infect cell lines over-expressing the bovine prion protein similar to other prion diseases. These BSE infected cells will provide a useful tool to facilitate the study of potential therapeutic agents and the diagnosis of BSE.

  20. The GATA1s isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress MYB, CCND2 and SKI during erythroid differentiation of K562 cells

    Directory of Open Access Journals (Sweden)

    Halsey Christina

    2012-08-01

    Full Text Available Abstract Background Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans—particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL where GATA1FL mutations are an essential driver for disease pathogenesis. Methods Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation. Results We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI. Conclusions These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL.

  1. Vascular endothelial growth inhibitor affects the invasion, apoptosis and vascularisation in breast cancer cell line MDA-MB-231

    Institute of Scientific and Technical Information of China (English)

    Gao Yinguang; Ge Zhicheng; Zhang Zhongtao; Bai Zhigang; Ma Xuemei; Wang Yu

    2014-01-01

    Background Breast cancer is one of the most common malignant female diseases worldwide.It is a significant threat to every woman's health.Vascular endothelial growth inhibitor (VEGI) is known to be abundant in endothelial cells.According to previous literature,overexpression of VEGI has been shown to inhibit tumor neovascularisation and progression in cellular and animal models,but there has been limited research on the significance of VEGI in the breast cancer.Methods In our study,cell lines MDA-MB-231 were first constructed in which VEGI mediated by lentivirus over-expressed.The effects of VEGI over-expression on MDA-MB-231 cells were investigated both in vitro and in vivo.The expression of VEGI in the MDA-MB-231 cells after infection of lentivirus was analyzed using real-time PCR and Western blotting.The effect of the biological characteristics of MDA-MB-231 cells was assessed by growth,invasion,adhesion,and migration assay with subcutaneous tumor-bearing nude mice models.Then the growth curves of the subcutaneous tumors were studied.Expressions of VEGI,CD31 and CD34 in the tumors were analyzed by immunohistochemistry and apoptosis was detected by flow cytometry and immunohistochemistry.Results Infection of MDA-MB-231 cells within the lentivirus resulted in approximately a 1 000-fold increase in the expression of VEGI.As can be seen in the invasion,adhesion and migration assay,the over-expression of VEGI can inhibit the ability of MDA-MB-231 cells during migration,adhesion and invasion.The volume of the subcutaneous tumor in the over-expression group was distinctly and significantly less than that of the control groups.Immunohistochemistry analysis of the tumor biopsies cleady showed the expression of VEGI in the over-expression group increased while CD31 and CD34 decreased significantly.In vitro and in vivo,the early apoptosis rate and the apoptosis index were increased within the VEGI over-expression group as compared with the control group.Conclusions Taken

  2. Integrated analysis of breast cancer cell lines reveals unique signaling pathways

    Energy Technology Data Exchange (ETDEWEB)

    Heiser, Laura M.; Wang, Nicholas J.; Talcott, Carolyn L.; Laderoute, Keith R.; Knapp, Merrill; Guan, Yinghui; Hu, Zhi; Ziyad, Safiyyah; Weber, Barbara L.; Laquerre, Sylvie; Jackson, Jeffrey R.; Wooster, Richard F.; Kuo, Wen-Lin; Gray, Joe W.; Spellman, Paul T.

    2009-03-31

    Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EGFR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes. We were interested in identifying subnetworks within the EGFR-MAPK pathway that are similarly deregulated across subsets of breast cancers. To that end, we mapped genomic, transcriptional and proteomic profiles for 30 breast cancer cell lines onto a curated Pathway Logic symbolic systems model of EGFR-MEK signaling. This model was comprised of 539 molecular states and 396 rules governing signaling between active states. We analyzed these models and identified several subtype specific subnetworks, including one that suggested PAK1 is particularly important in regulating the MAPK cascade when it is over-expressed. We hypothesized that PAK1 overexpressing cell lines would have increased sensitivity to MEK inhibitors. We tested this experimentally by measuring quantitative responses of 20 breast cancer cell lines to three MEK inhibitors. We found that PAK1 over-expressing luminal breast cancer cell lines are significantly more sensitive to MEK inhibition as compared to those that express PAK1 at low levels. This indicates that PAK1 over-expression may be a useful clinical marker to identify patient populations that may be sensitive to MEK inhibitors. All together, our results support the utility of symbolic system biology models for identification of therapeutic approaches that will be effective against breast cancer subsets.

  3. EXPRESSION OF EPIDERMAL GROWTH FACTOR RECEPTOR IN DIFFERENT SALIVARY ADENOID CYSTIC CARCINOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    MA Jie; ZONG Zhi-hong; WANG Zhao-yuan

    2005-01-01

    Objective: To investigate the expression of epidermal growth factor receptor, a receptor tyrosine protein kinase, in the subcellular fractions of human salivary adenoid cystic carcinoma cell lines SACC-83 and SACC-LM. Methods: Low metastatic and high metastatic cells of the adenoid cystic carcinoma, SACC-83 and SACC-LM, were cultured. Their subcellular fractions were extracted. The expression of epidermal growth factor receptor was detected with Western blot method, and the results of protein expression were quantitatively analyzed by FluorChem V2.0 software. Results: The results of Western blot analysis indicated that, EGFR expression on the membrane of SACC-83 cells was significantly higher than that of SACC-LM cells, but its expression in cytoplasm was significantly less in the former than the later (P<0.01). In SACC-83 cell line, EGFR was over-expressed in membrane (P<0.01), but in SACC-LM cell line, EGFR was over-expressed in cytoplasm (P<0.01). Conclusion: The results suggest that the obtaining of metastasis ability is related to the high expression of EGFR protein in cytoplasm, so the molecular targeting therapy to EGFR may be an ideal treatment for the invasion and metastasis of salivary adenoid cystic carcinoma.

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

    Science.gov (United States)

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

    2016-08-01

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

  5. A novel neuron-enriched protein SDIM1 is down regulated in Alzheimer's brains and attenuates cell death induced by DNAJB4 over-expression in neuro-progenitor cells

    Directory of Open Access Journals (Sweden)

    Lei Joy X

    2011-01-01

    Full Text Available Abstract Background Molecular changes in multiple biological processes contribute to the development of chronic neurodegeneration such as late onset Alzheimer's disease (LOAD. To discover how these changes are reflected at the level of gene expression, we used a subtractive transcription-based amplification of mRNA procedure to identify novel genes that have altered expression levels in the brains of Alzheimer's disease (AD patients. Among the genes altered in expression level in AD brains was a transcript encoding a novel protein, SDIM1, that contains 146 amino acids, including a typical signal peptide and two transmembrane domains. Here we examined its biochemical properties and putative roles in neuroprotection/neurodegeneration. Results QRT-PCR analysis of additional AD and control post-mortem human brains showed that the SDIM1 transcript was indeed significantly down regulated in all AD brains. SDIM1 is more abundant in NT2 neurons than astrocytes and present throughout the cytoplasm and neural processes, but not in the nuclei. In NT2 neurons, it is highly responsive to stress conditions mimicking insults that may cause neurodegeneration in AD brains. For example, SDIM1 was significantly down regulated 2 h after oxygen-glucose deprivation (OGD, though had recovered 16 h later, and also appeared significantly up regulated compared to untreated NT2 neurons. Overexpression of SDIM1 in neuro-progenitor cells improved cells' ability to survive after injurious insults and its downregulation accelerated cell death induced by OGD. Yeast two-hybrid screening and co-immunoprecipitation approaches revealed, both in vitro and in vivo, an interaction between SDIM1 and DNAJB4, a heat shock protein hsp40 homolog, recently known as an enhancer of apoptosis that also interacts with the mu opioid receptor in human brain. Overexpression of DNAJB4 alone significantly reduced cell viability and SDIM1 co-overexpression was capable of attenuating the cell death

  6. The over expression of thioredoxin during malignancies

    Directory of Open Access Journals (Sweden)

    Shahaboddin Shabani

    2014-09-01

    Full Text Available Thioredoxin system comprised of thiorexin and NADPH dependent thiorexin reductase, is responsible for redox regulation of cells by controlling the apoptosis, proliferation and other vital processes of cells. The efficacy of thioredoxin system has been represented in a wide range of physiological and biological reactions in bacteria, yeast, plants, mammals and etc. including DNA synthesis, regulation of transcription factors, protein repairing, regulating the photosynthesis and controlling the apoptosis and preventing oxidative stresses, filamentous phage assembly, immune-modulating, neuronal survival, pregnancy and birth and many other  physiological and biological functions. The up-regulation of thioredoxin has been observed in various malignancies, which was associated with tumor angiogenesis and development. In this regard, the thiordoxin system has become a putative target in new chemotherapeutic methods. In this study, we mentioned various features of thioredoxin system in malignant cells and reviewed the articles which have evaluated the expression rate of thioredoxin system in malignancies.

  7. False leukemia-lymphoma cell lines: an update on over 500 cell lines.

    Science.gov (United States)

    Drexler, H G; Dirks, W G; Matsuo, Y; MacLeod, R A F

    2003-02-01

    Human leukemia-lymphoma (LL) cell lines represent an extremely important resource for research in a variety of fields and disciplines. As the cell lines are used as in vitro model systems in lieu of primary cell material, it is crucial that the cells in the culture flasks faithfully correspond to the purported objects of study. Obviously, proper authentication of cell line derivation and precise characterization are indispensable requirements to use as model systems. A number of studies has shown an unacceptable level of LL cell lines to be false. We present here the results of authenticating a comprehensively large sample (n = 550) of LL cell lines mainly by DNA fingerprinting and cytogenetic evaluation. Surprisingly, near-identical incidences (ca 15%) of false cell lines were observed among cell lines obtained directly from original investigators (59/395: 14.9%) and from secondary sources (23/155: 14.8%) implying that most cross-contamination is perpetrated by originators, presumably during establishment. By comparing our data with those published, we were further able to subclassify the false cell lines as (1) virtual: cross-contaminated with and unretrievably overgrown by other cell lines during initiation, never enjoying independent existence; (2) misidentified: cross-contaminated subsequent to establishment so that an original prototype may still exist; or (3) misclassified: unwittingly established from an unintended (often normal) cell type. Prolific classic leukemia cell lines were found to account for the majority of cross-contaminations, eg CCRF-CEM, HL-60, JURKAT, K-562 and U-937. We discuss the impact of cross-contaminations on scientific research, the reluctance of scientists to address the problem, and consider possible solutions. These findings provide a rationale for mandating the procurement of reputably sourced LL cell lines and their regular authentication thereafter.

  8. Virus Discovery Using Tick Cell Lines

    Science.gov (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

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

  9. New Synthetic Route of Two Active Isomeric Metabolites of Erlotinib and Their Bioactivity Studies against Several Tumor Cell Lines%New Synthetic Route of Two Active Isomeric Metabolites of Erlotinib and Their Bioactivity Studies against Several Tumor Cell Lines

    Institute of Scientific and Technical Information of China (English)

    李汉青; 李梦瑶; 李载权; 李良; 毕姗姗; 邓晨辉; 陈睿; 周田彦; 卢炜

    2011-01-01

    The synthesis and differential antiproliferative activity of two active isomeric metabolites of erlotinib were in- vestigated. This synthetic process had demonstrated to avoid the unstable 4-chloroquinazoline intermediates and long procedures. New intermediates and final compounds were identified by IH NMR, 13C NMR and ESI-TOF MS, and their purities were determined by high performance liquid chromatography. In vitro proliferative assay indi- cated that these two metabolites possessed antiproliferative activity against some conventional tumor cell lines and EGFR tyrosine kinase over-expression tumor cell lines as compared to erlotinib control, and their antitumor activity in cellular level was first reported here.

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

    Science.gov (United States)

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

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

  11. 白血病细胞中共表达P2X7受体和Notch1胞内区%Dual Over-expression of P2X7 Receptor and Intracellular Domain of Notch1 in Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    冯丽; 杨骁; 廖金凤; 陈莎燕; 冯文利; 林永敏; 任倩; 郑国光

    2013-01-01

    This study aimed to construct the dual expression vectors of wide type or N187D mutant P2X7 receptor and intracellular domain of Notchl (ICN1) linked by 2A peptide to coexpress them in leukemia cells so as to lay a foundation for further investigating the role of P2X7 in development of leukemia.Overlap PCR was used to construct the dual expression vectors encoding wide type or N187D mutant type P2X7 receptor and ICN1 linked by the self-cleaving 2A sequence.The results showed that stable expressing cell lines were obtained by retroviral infection followed by cell sorting after DNA sequence analysis.RT-PCR,Western blot,intracellular free calcium concentration analysis were used to verify the functionally successful construction of K562 cell line expressing P2X7 receptor alone or with ICN1.DNA sequence analysis revealed that all construction were right.The infection efficiency of packaged constructed virus ranged from 40% to 70% for K562 cells.Stable infected cell line was obtained by cell sorting.RT-PCR analysis revealed that P2X7 receptor and/or ICN1 could be detected at high level in their stable infected cell lines,respectively.Western blot analysis also showed that P2X7 receptor was highly expressed in cell line infected by virus with P2X7 receptor.Sustained increase in intracellular free calcium concentration ([Ca2 +] i) could be observed in K562 cells overexpressing either type of P2X7 receptor upon stimulation with BzATP.It is concluded that the wide type or N187D mutant P2X7 receptor and ICN1 are simultaneously and functionally over-express in leukemia cells,which lay a foundation for further studying the role of P2X7 receptor in the development of leukemia.%本研究旨在构建野生型及N187D突变型P2X7与ICN1基因的共表达载体,并在白血病细胞中表达,为进一步探讨P2X7在白血病发展中的作用奠定基础.采用Overlap PCR法,通过2A连接,构建野生型或N187DP2X7与ICN1的共表达载体;经DNA序列分析验

  12. Two Retroviruses Packaged in One Cell Line can Combined Inhibit the Replication of HIV-1 in TZM-bl Cells

    Institute of Scientific and Technical Information of China (English)

    Zhipin Liang; Zhiyuan Guo; Xin Wang; Xiaohong Kong; Chang Liu

    2012-01-01

    The cellular protein tetherin tethers the HIV-1 viral particles on the cellular membrane to inhibit the replication of HIV-1.However,the HIV-1 accessory protein Vpu counteracts the antiviral function of tetherin.In this study,two retroviral vector plasmids were constructed.One inhibited the vpu gene expression; the other one over-expressed the tetherin.Both retroviral vector plasmids could be packaged in the packaging cell line PT67 to obtain the corresponding retroviruses.The retroviral vector plasmids'functions of tetherin over-expression or vpu-RNAi were detected at the cell level.Retroviral vector plasmids were transfected to PT67 cells at different ratios from 0T3V to 3T0V,and then mixed retroviruses were harvested.The antiviral functions of mixed retroviruses were detected in HIV-1 infected TZM-bl cells.The results showed that packaged mixed retroviruses could repress the replication of HIV-1 in TZM-bl cells.

  13. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-07-30

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

  14. Stem cell characteristics in prostate cancer cell lines.

    NARCIS (Netherlands)

    Pfeiffer, M.J.; Schalken, J.A.

    2010-01-01

    BACKGROUND: Recent studies indicate the presence of a small, stem-like cell population in several human cancers that is crucial for the tumour (re)population. OBJECTIVE: Six established prostate cancer (PCa) cell lines-DU145, DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3-were examined for their stem cell pr

  15. Peganum harmala L.’s anti-growth effect on a breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Somayeh Hashemi Sheikh Shabani

    2015-12-01

    Full Text Available This research was done to evaluate the induction of apoptosis in MDA-MB-231 breast cancer cell line by Peganum harmala’s extract, in which a significant amount of ß-carbolines is included. The apoptosis incidence was assessed through Annexin-V-Flous kit. The expressions of genes through which intrinsic apoptosis pathway are involved, Bax, Bcl-2, Bid, and Puma, over the genes the expressions of which are linked to extrinsic apoptosis pathway, TRAIL, Caspase8, p21, and p53, were examined by RT-PCR and Real-time PCR. The results demonstrate that the extract decreases the growth rate of the cancer cell line through inducing apoptosis mechanism. As long as the expression of anti-apoptosis Bcl-2 gen reduced dramatically, an over-expression in Bax and Puma genes was monitored indicating activation of intrinsic apoptosis pathway. A notable over-expression observed with TRAIL and Caspase8 genes as well as Bid gene. The latter is an intermediate for both intrinsic and extrinsic pathways of apoptosis.

  16. A meta-analysis to evaluate the cellular processes regulated by the interactome of endogenous and over-expressed estrogen receptor alpha.

    Science.gov (United States)

    Simões, Joana; Amado, Francisco M; Vitorino, Rui; Helguero, Luisa A

    2015-01-01

    The nature of the proteins complexes that regulate ERα subcellular localization and activity is still an open question in breast cancer biology. Identification of such complexes will help understand development of endocrine resistance in ER+ breast cancer. Mass spectrometry (MS) has allowed comprehensive analysis of the ERα interactome. We have compared six published works analyzing the ERα interactome of MCF-7 and HeLa cells in order to identify a shared or different pathway-related fingerprint. Overall, 806 ERα interacting proteins were identified. The cellular processes were differentially represented according to the ERα purification methodology, indicating that the methodologies used are complementary. While in MCF-7 cells, the interactome of endogenous and over-expressed ERα essentially represents the same biological processes and cellular components, the proteins identified were not over-lapping; thus, suggesting that the biological response may differ as the regulatory/participating proteins in these complexes are different. Interestingly, biological processes uniquely associated to ERα over-expressed in HeLa cell line included L-serine biosynthetic process, cellular amino acid biosynthetic process and cell redox homeostasis. In summary, all the approaches analyzed in this meta-analysis are valid and complementary; in particular, for those cases where the processes occur at low frequency with normal ERα levels, and can be identified when the receptor is over-expressed. However special effort should be put into validating these findings in cells expressing physiological ERα levels.

  17. Activation of STAT3 signaling in human stomach adenocarcinoma drug-resistant cell line and its relationship with expression of vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    Li-Fen Yu; Ying Cheng; Min-Min Qiao; Yong-Ping Zhang; Yun-Lin Wu

    2005-01-01

    AIM: To investigate the difference in activation of STAT3signaling between two human stomach adenocarcinoma cell lines: 5-fluorouracil resistant cell line and its parental cell line, and to evaluate its relationship with the expression of vascular endothelial growth factor (VEGF).METHODS: Western blot and electrophoretic mobility shift assay (EMSA) were used to detect the expression of phospho-STAT3 protein and constitutive activation of STAT3in two human stomach adenocarcinoma cell lines, 5-fluorouracil resistant cell line SGC7901/R and its parental cell line SGC7901, respectively. The mRNA expression of VEGF was analysed by semi-quantitative RT-PCR. The expressive intensity of VEGF protein was measured by immunocytochemistry.RESULTS: The expressions of phospho-STAT3 protein and constitutive activation of ST AT3 between two human stomach adenocarcinoma cell lines were different.Compared with the parental cell line SGC7901, the STAT3-DNA binding activity and the expressive intensity of phospho-STAT3 protein were lower in the drug-resistant cell line SGC7901/R. The expression levels of VEGF mRNA and its encoded protein were also decreased in drugresistant cell line.CONCLUSION: Over-expression of VEGF may be correlated with elevated STAT3 activation in parental cell line. Lower VEGF expression may be correlated with decreased STAT3activation in resistant cell line, which may have resulted from negative feedback regulation of STAT signaling.

  18. Over-expression of XIST, the Master Gene for X Chromosome Inactivation, in Females With Major Affective Disorders

    Science.gov (United States)

    Ji, Baohu; Higa, Kerin K.; Kelsoe, John R.; Zhou, Xianjin

    2015-01-01

    Background Psychiatric disorders are common mental disorders without a pathological biomarker. Classic genetic studies found that an extra X chromosome frequently causes psychiatric symptoms in patients with either Klinefelter syndrome (XXY) or Triple X syndrome (XXX). Over-dosage of some X-linked escapee genes was suggested to cause psychiatric disorders. However, relevance of these rare genetic diseases to the pathogenesis of psychiatric disorders in the general population of psychiatric patients is unknown. Methods XIST and several X-linked genes were studied in 36 lymphoblastoid cell lines from healthy females and 60 lymphoblastoid cell lines from female patients with either bipolar disorder or recurrent major depression. XIST and KDM5C expression was also quantified in 48 RNA samples from postmortem human brains of healthy female controls and female psychiatric patients. Findings We found that the XIST gene, a master in control of X chromosome inactivation (XCI), is significantly over-expressed (p = 1 × 10− 7, corrected after multiple comparisons) in the lymphoblastoid cells of female patients with either bipolar disorder or major depression. The X-linked escapee gene KDM5C also displays significant up-regulation (p = 5.3 × 10− 7, corrected after multiple comparisons) in the patients' cells. Expression of XIST and KDM5C is highly correlated (Pearson's coefficient, r = 0.78, p = 1.3 × 10− 13). Studies on human postmortem brains supported over-expression of the XIST gene in female psychiatric patients. Interpretations We propose that over-expression of XIST may cause or result from subtle alteration of XCI, which up-regulates the expression of some X-linked escapee genes including KDM5C. Over-expression of X-linked genes could be a common mechanism for the development of psychiatric disorders between patients with those rare genetic diseases and the general population of female psychiatric patients with XIST over-expression. Our studies

  19. Activator protein-1 involved in growth inhibition by RASSF1A gene in the human gastric carcinoma cell line SGC7901

    Institute of Scientific and Technical Information of China (English)

    Zheng-Hao Deng; Ji-Fang Wen; Jing-He Li; De-Sheng Xiao; Jian-Hua Zhou

    2008-01-01

    AIM:To investigate the role of Ras association domain family protein 1 isoform A (RASSFIA) in gastric tumorigenesis.METHODS:Through over-expression of RASSFIA gene in the SGC7901 cell line which was induced by a lipofectamine-mediated gene transfer approach.Activator protein-1 (AP-1) DNA binding activity was measured by electrophoretic mobility shift assay (EMSA).RESULTS:Compared with the control clones,cells over expressing RASSF1A exhibited significant inhibition of cell growth with G1 cell cycle arrest in vitro and in vivo.The over-expression of RASSF1A significantly inhibited AP-1activity in SGC7901 cells (0.981 + 0.011 vs 0.354 ± 0.053,P<0.001).In addition,both Western blot analysis and immunocytochemistry demonstrated that RASSF1A down-regulated the expression of c-Fos (0.975±0.02 vs0.095+0.024,P<0.001) but not c-Jun.CONCLUSION:Over-expression of RASSF1A inhibits the growth of SGC7901 cells by negatively regulating the AP-1 activity,the latter in turn negatively signals cell proliferation.

  20. Antineoplastic effects of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Candelaria Myrna

    2006-01-01

    Full Text Available Abstract Background Among the epigenetic alterations occurring in cancer, DNA hypermethylation and histone hypoacetylation are the focus of intense research because their pharmacological inhibition has shown to produce antineoplastic activity in a variety of experimental models. The objective of this study was to evaluate the combined antineoplastic effect of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in a panel of cancer cell lines. Results Hydralazine showed no growth inhibitory effect on cervical, colon, breast, sarcoma, glioma, and head & neck cancer cell lines when used alone. On the contrary, valproic acid showed a strong growth inhibitory effect that is potentiated by hydralazine in some cell lines. Individually, hydralazine and valproic acid displayed distinctive effects upon global gene over-expression but the number of genes over-expressed increased when cells were treated with the combination. Treatment of HeLa cells with hydralazine and valproic acid lead to an increase in the cytotoxicity of gemcitabine, cisplatin and adriamycin. A higher antitumor effect of adriamycin was observed in mice xenografted with human fibrosarcoma cells when the animals were co-treated with hydralazine and valproic acid. Conclusion Hydralazine and valproic acid, two widely used drugs for cardiovascular and neurological conditions respectively have promising antineoplastic effects when used concurrently and may increase the antitumor efficacy of current cytotoxic agents.

  1. NOR1基因转染对肝癌细胞HepG2基因表达谱的影响%Changes in Global Gene Expression Induced by NOR1 Over-expression in HepG2 Cells

    Institute of Scientific and Technical Information of China (English)

    李登清; 唐华; 桂嵘; 聂新民

    2008-01-01

    Previous work from this laboratory has cloned a novel gene NOR1 and showed its extensive expression in normal tissues and down-regulation in carcinomas. To further investigate its downstream target genes and better understand its function, NOR1 was over-expressed in HepG2 hepatoma cells and global changes in gene expressions from a stable line were identified by cDNA microarrays. The results discovered 59 genes up-regulated in these cells compared with the original cells, including Grb2, HBP17,TNFRSF11B genes that have been implicated in tumorigenesis and cancer development. In addition, 103 down-regalated genes were also identified, including genes encoding Bik, MAP2K6 and ZFP95 proteins. The expression patterns of certain genes identified by microarrays were validated by quantitative real-time PCR and the results showed that expression difference were statistically significant (P< 0.05). These data suggest that NOR1 may influence the biology and cancerous behaviors of HepG2 cells by regulating expression of a set of genes involved in signal traasduction, cell cycle regulation, transcription and Wanslation controls.%NOR1基因是一在正常组织中广泛表达且在肿瘤组织中表达下调的新基因.为进一步研究NOR1基因的功能和寻找其下游基因,利用脂质体技术将NOR1基因转染进HepG2细胞,采用cDNA微阵列技术分析其基因表达谱的改变.试验表明NOR1基因的转染能使Grb2,HBP17,TNFRSF11B等59个基因上调,同时也下调Bik,MAp2K6,ZFP95等103个基因.随后用实时荧光定量PCR对cDNA 微阵列结果中上述3个上调表达基因进行验证,结果表明,基因表达差异具有统计学意义(P<0.05),荧光定量PCR结果与微阵列结果相符.这些结果提示,NOR1基因对肝癌HepG2细胞的生物学行为的影响可能与它对细胞信号转导,细胞周期调控,转录、翻译调控相关基因的表达影响有关.

  2. Identification of differentially expressed genes in two new human bladder carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To screen and identify differentially expressed genes in two new human urothelial carcinoma cell lines, BLS-211 and BLX. Methods Suppression subtractive hybridization (SSH) was used to createa subtracted library, and clones were sequenced. Results Totally 13 over-expressed genes in BLX and 9 in BLS-211 cells were obtained, respectively. Among them, 18 were known genes and 4 were new ESTs (Expressed Sequence Tag), and were collected by GenBank dbEST database (The access number was EB390424-7). Conclusion SSH is a powerful method for the identification of differentially expressed genes. The differential expression of some BCG-associated genes in different cells may be related to the different responses to clinical BCG therapy. The identified new ESTs can be cloned for full length to further study their functions.

  3. α-Synuclein Over-Expression Induces Increased Iron Accumulation and Redistribution in Iron-Exposed Neurons.

    Science.gov (United States)

    Ortega, Richard; Carmona, Asuncion; Roudeau, Stéphane; Perrin, Laura; Dučić, Tanja; Carboni, Eleonora; Bohic, Sylvain; Cloetens, Peter; Lingor, Paul

    2016-04-01

    Parkinson's disease is the most common α-synucleinopathy, and increased levels of iron are found in the substantia nigra of Parkinson's disease patients, but the potential interlink between both molecular changes has not been fully understood. Metal to protein binding assays have shown that α-synuclein can bind iron in vitro; therefore, we hypothesized that iron content and iron distribution could be modified in cellulo, in cells over-expressing α-synuclein. Owing to particle-induced X-ray emission and synchrotron X-ray fluorescence chemical nano-imaging, we were able to quantify and describe the iron distribution at the subcellular level. We show that, in neurons exposed to excess iron, the mere over-expression of human α-synuclein results in increased levels of intracellular iron and in iron redistribution from the cytoplasm to the perinuclear region within α-synuclein-rich inclusions. Reproducible results were obtained in two distinct recombinant expression systems, in primary rat midbrain neurons and in a rat neuroblastic cell line (PC12), both infected with viral vectors expressing human α-synuclein. Our results link two characteristic molecular features found in Parkinson's disease, the accumulation of α-synuclein and the increased levels of iron in the substantia nigra.

  4. Cellular radiosensitivity of small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1997-01-01

    PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based...

  5. Ethanol induces mouse spermatogenic cell apoptosis in vivo through over-expression of Fas/Fas-L, p53, and caspase-3 along with cytochrome c translocation and glutathione depletion.

    Science.gov (United States)

    Jana, Kuladip; Jana, Narayan; De, Dipak Kumar; Guha, Sujoy Kumar

    2010-09-01

    Although it has been well established that spermatogenic cells undergo apoptosis when treated with ethanol, the molecular mechanisms behind it remain to be investigated. Adult male mice were given intra-peritoneal injection (IP) of ethanol at a dose of 3 g (15%, v/v) per kg body weight per day during the period of 14 days. Testicular androgenesis and apoptotic germ cell death, along with different interrelated proteins expression, were evaluated. Ethanol treatment induced apoptotic spermatogenic cell death with a decrease in the plasma and intra-testicular testosterone concentration. Western blot analysis revealed that repeated ethanol treatment decreased the expression of steroidogenic acute regulatory protein (StAR), 3 beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17 beta-hydroxysteroid dehydrogenase (17beta-HSD); increased the expression of active caspase-3, p53, Fas and Fas-L; and led to up-regulation of Bax/Bcl-2 ratio and translocation of cytochrome c from mitochondria to cytosol in testis. It has also been shown in our study that repeated ethanol treatment led to up-regulation of caspase-3, p53, Fas, Fas-L transcripts; increase in caspase-3 and caspase-8 activities; diminution of 3beta-HSD, 17beta-HSD, and GPx activities; decrease in the mitochondrial membrane potential along with ROS generation and depletion of glutathione pool in the testicular tissue. The present study has indicated that the ethanol treatment induced apoptosis in the mouse testis through the increased expression of Fas/Fas-L and p53, up-regulation of Bax/Bcl-2 ratio, cytosolic translocation of cytochrome c along with caspase-3 activation and glutathione depletion.

  6. Over-expression of P2X7 receptors in spinal glial cells contributes to the development of chronic postsurgical pain induced by skin/muscle incision and retraction (SMIR) in rats.

    Science.gov (United States)

    Ying, Yan-Lu; Wei, Xu-Hong; Xu, Xue-Bing; She, Shou-Zhang; Zhou, Li-Jun; Lv, Jing; Li, Dai; Zheng, Bin; Liu, Xian-Guo

    2014-11-01

    Many patients suffer from chronic postsurgical pain (CPSP) following surgery, and the underlying mechanisms are poorly understood. In the present work, with use of the skin/muscle incision and retraction (SMIR) model, the role of P2X7 receptors (P2X7Rs) in spinal glial cells in the development of CPSP was evaluated. Consistent with previous reports, we found that SMIR decreased the ipsilateral 50% paw withdrawal threshold (PWT), lasting for at least 2weeks. No injury was done to L3 dorsal root ganglia (DRG) neurons and no axonal or Schwann cell damage at the retraction site in the saphenous nerve was observed 7days after SMIR. The results of immunofluorescence showed that both microglia and astrocytes were activated in the spinal dorsal horn following SMIR. In addition, both P2X7Rs and tumor necrosis factor-alpha (TNF-α) were up-regulated following SMIR. Double immunofluorescence staining revealed that the up-regulated P2X7R immunoreactivity was mainly located in microglia, and to a lesser extent in astrocytes, but not in neurons. Intrathecal delivery of specific P2X7R antagonist BBG (10μM in 10μl volume) or A438079 (10μM in 10μl volume), started 30min before the surgery and once daily thereafter for 7days, prevented the mechanical allodynia. Intrathecal injection of BBG inhibited the activation of microglia and astrocytes, and the up-regulation of TNF-α induced by SMIR. These data suggest that P2X7Rs in the spinal dorsal horn might mediate the development of CPSP via activation of glial cells and up-regulation of TNF-α.

  7. Comparing the epidermal growth factor interaction with four different cell lines: intriguing effects imply strong dependency of cellular context.

    Directory of Open Access Journals (Sweden)

    Hanna Björkelund

    Full Text Available The interaction of the epidermal growth factor (EGF with its receptor (EGFR is known to be complex, and the common over-expression of EGF receptor family members in a multitude of tumors makes it important to decipher this interaction and the following signaling pathways. We have investigated the affinity and kinetics of (125I-EGF binding to EGFR in four human tumor cell lines, each using four culturing conditions, in real time by use of LigandTracer®.Highly repeatable and precise measurements show that the overall apparent affinity of the (125I-EGF - EGFR interaction is greatly dependent on cell line at normal culturing conditions, ranging from K(D ≈ 200 pM on SKBR3 cells to K(D≈8 nM on A431 cells. The (125I-EGF - EGFR binding curves (irrespective of cell line have strong signs of multiple simultaneous interactions. Furthermore, for the cell lines A431 and SKOV3, gefitinib treatment increases the (125I-EGF - EGFR affinity, in particular when the cells are starved. The (125I-EGF - EGFR interaction on cell line U343 is sensitive to starvation while as on SKBR3 it is insensitive to gefitinib and starvation.The intriguing pattern of the binding characteristics proves that the cellular context is important when deciphering how EGF interacts with EGFR. From a general perspective, care is advisable when generalizing ligand-receptor interaction results across multiple cell-lines.

  8. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

    Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

    2009-01-01

    compare susceptibility between cell lines and between lineages within a laboratory and between laboratories (Inter-laboratory Proficiency Test). The objective being that the most sensitive cell line and lineages are routinely selected for diagnostic purposes.In comparing cell lines, we simulated "non......-cell-culture-adapted" virus by propagating the virus in heterologous cell lines to the one tested. A stock of test virus was produced and stored at - 80 °C and tests were conducted biannually. This procedure becomes complicated when several cell lines are in use and does not account for variation among lineages. In comparing...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...

  9. Induced pluripotent stem cell lines derived from human somatic cells.

    Science.gov (United States)

    Yu, Junying; Vodyanik, Maxim A; Smuga-Otto, Kim; Antosiewicz-Bourget, Jessica; Frane, Jennifer L; Tian, Shulan; Nie, Jeff; Jonsdottir, Gudrun A; Ruotti, Victor; Stewart, Ron; Slukvin, Igor I; Thomson, James A

    2007-12-21

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal karyotypes, express telomerase activity, express cell surface markers and genes that characterize human ES cells, and maintain the developmental potential to differentiate into advanced derivatives of all three primary germ layers. Such induced pluripotent human cell lines should be useful in the production of new disease models and in drug development, as well as for applications in transplantation medicine, once technical limitations (for example, mutation through viral integration) are eliminated.

  10. Angiopoietin-like 4 is over-expressed in rheumatoid arthritis patients: association with pathological bone resorption.

    Directory of Open Access Journals (Sweden)

    Catherine Swales

    Full Text Available Osteoclasts are responsible for the bone loss associated with rheumatoid arthritis (RA. The secreted adipokine angiopoietin-like 4 (ANGPTL4 specifically increases osteoclast-mediated bone resorption. We have investigated expression of ANGPTL4 and its regulatory transcription factor, hypoxia-inducible factor-1 alpha (HIF-1α, in osteoclasts and other cells within rheumatoid synovium. We have also examined whether circulating levels of ANGPTL4 differ in RA patients compared with that in normal controls or patients with osteoarthritis (OA.Immunohistochemical analysis revealed that bone-apposing osteoclasts within the rheumatoid synovium express both ANGPTL4 and HIF-1α. ANGPTL4 was also strongly expressed in synovial lining cells, endothelial cells, stromal cells, CD68+ macrophages and plasma cells within RA synovium. Little ANGPTL4 was evident in normal synovial tissue. This reflected the over-expression of HIF-1α in rheumatoid versus normal synovial tissue. The concentration of ANGPTL4 was higher in both the serum and the synovial fluid of RA patients than in patients with OA or normal controls. High serum ANGPTL4 associated with elevated levels of the serum marker of bone resorption, receptor activator for nuclear factor κB ligand (RANKL.Over-expression of ANGPTL4 in multiple cell types within the rheumatoid synovium potentially provides a local pool of ANGPTL4 to stimulate osteoclast-mediated bone resorption in RA. Additionally, correlation of high serum ANGPTL4 with circulating RANKL suggests that ANGPTL4 may represent a novel marker for bone destruction in RA.

  11. Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.

    Science.gov (United States)

    Li, Zhiqian; Zeng, Baosheng; Ling, Lin; Xu, Jun; You, Lang; Aslam, Abu F M; Tan, Anjiang; Huang, Yongping

    2015-01-01

    RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.

  12. Assessment of the Selenoprotein M (SELM over-expression on human hepatocellular carcinoma tissues by immunohistochemistry

    Directory of Open Access Journals (Sweden)

    E. Guerriero

    2014-10-01

    Full Text Available Selenium is an essential trace mineral of fundamental importance to human healthy and exerts its biological function through selenoproteins. In particular, Selenoprotein M (SELM is located in the endoplasmic reticulum and contains the common redox motif of cysteine-X-X-selenocysteine type. It attracts great attention due to its high expression in brain and its potential roles as antioxidant, neuroprotective, and cytosolic calcium regulator. Recently, our group found SELM over-expression  in human hepatocellular carcinoma (HCC cell lines. In this report some paraffin-embedded tissues from liver biopsy of patients with hepatitis C virus (HCV-related cirrhosis and HCC were immunohistochemically stained and SELM expression scoring was evaluated. Our results evidence for the first time an increase of SELM expression in HCC liver tissues, and its gradual expression raise associated with an increased malignancy grade. Therefore, we propose to use i SELM as putative marker for HCC as well as ii simple immunohistochemistry technique to distinguish between the different grades of malignancy. 

  13. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... Genetics Group Web site at http://www.nist.gov/mml/biochemical/genetics/index.cfm . Once the total number... methods for human cell line authentication the identity of a cell line need no longer be in doubt. NIST...

  14. Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma.

    Science.gov (United States)

    Gupta, S; Srivastava, M; Ahmad, N; Bostwick, D G; Mukhtar, H

    2000-01-01

    Aberrant or increased expression of cyclooxygenase (COX)-2 has been implicated in the pathogenesis of many diseases including carcinogenesis. COX-2 has been shown to be over-expressed in some human cancers. Employing semi-quantitative reverse transcription-PCR, immunoblotting, and immunohistochemistry we assessed COX-2 expression in samples of pair-matched benign and cancer tissue obtained from the same prostate cancer patient. Mean levels of COX-2 mRNA were 3.4-fold higher in prostate cancer tissue (n = 12) compared with the paired benign tissue. The immunoblot analysis demonstrated that as compared to benign tissue COX-2 protein was over-expressed in 10 of 12 samples examined. Immunohistochemical analysis also verified COX-2 over-expression in cancer than in benign tissue. To our knowledge, this is the first in vivo study showing an over-expression of COX-2 in prostate cancer. These data suggest that COX-2 inhibitors may be useful for prevention or therapy of prostate cancer in humans.

  15. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  16. Over-expression of mango (Mangifera indica L.) MiARF2 inhibits root and hypocotyl growth of Arabidopsis.

    Science.gov (United States)

    Wu, Bei; Li, Yun-He; Wu, Jian-Yong; Chen, Qi-Zhu; Huang, Xia; Chen, Yun-Feng; Huang, Xue-Lin

    2011-06-01

    An auxin response factor 2 gene, MiARF2, was cloned in our previous study [1] from the cotyledon section of mango (Mangifera indica L. cv. Zihua) during adventitious root formation, which shares an 84% amino acid sequence similarity to Arabidopsis ARF2. This study was to examine the effects of over-expression of the full-length MiARF2 open reading frame on the root and hypocotyl growth in Arabidopsis. Phenotype analysis showed that the T(3) transgenic lines had about 20-30% reduction in the length of hypocotyls and roots of the seedlings in comparison with the wild-type. The transcription levels of ANT and ARGOS genes which play a role in controlling organ size and cell proliferation in the transgenic seedlings also decreased. Therefore, the inhibited root and hypocotyl growth in the transgenic seedlings may be associated with the down-regulated transcription of ANT and ARGOS by the over-expression of MiARF2. This study also suggests that although MiARF2 only has a single DNA-binding domain (DBD), it can function as other ARF-like proteins containing complete DBD, middle region (MR) and carboxy-terminal dimerization domain (CTD).

  17. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Bergoc, R. M.; Medina, V.; Cricco, G.; Mohamed, N.; Martin, G.; Nunez, M.; Croci, M.; Crescenti, E. J.; Rivera, E. S.

    2004-07-01

    Cutaneous melanoma is a skin cancer resulting from the malign transformation of skin-pigment cells, the melanocytes. The radiotherapy, alone or in combination with other treatment, is an important therapy for this disease. the objective of this paper was to determine in vitro the radiosensitivity of two human melanoma cell lines with different metastatic capability: WM35 and MI/15, and to study the effect of drugs on radiobiological parameters. The Survival Curves were adjusted to the mathematical Linear-quadratic model using GrapsPad Prism software. Cells were seeded in RPMI medium (3000-3500 cells/flask), in triplicate and irradiated 24 h later. The irradiation was performed using an IBL 437C H Type equipment (189 TBq, 7.7 Gy/min) calibrated with a TLD 700 dosimeter. The range of Doses covered from 0 to 10 Gy and the colonies formed were counted at day 7th post-irradiation. Results obtained were: for WM35, {alpha}=0.37{+-}0.07 Gy''-1 and {beta}=0.06{+-}0.02 Gy''-2, for M1/15m {alpha}=0.47{+-}0.03 Gy''-1 and {beta}=0.06{+-}0.01 Gy''-2. The {alpha}/{beta} values WM35: {alpha}/{beta} values WM35: {alpha}/{beta}=6.07 Gy and M1/15: {alpha}/{beta}{sub 7}.33 Gy were similar, independently of their metastatic capabillity and indicate that both lines exhibit high radioresistance. Microscopic observation of irradiated cells showed multinuclear cells with few morphologic changes non-compatible with apoptosis. By means of specific fluorescent dyes and flow cytometry analysis we determined the intracellular levels of the radicals superoxide and hydrogen peroxide and their modulation in response to ionizing radiation. The results showed a marked decreased in H{sub 2}O{sub 2} intracellular levels with a simultaneous increase in superoxide that will be part of a mechanism responsible for induction of cell radioresistance. This response triggered by irradiated cells could not be abrogated by different treatments like histamine or the

  18. Effects of over-expressed Smac gene coupling with cisplatin on proliferation and apoptosis of hepatocarcinoma cells%Smac基因过表达联合顺铂对SMMC-7721细胞增殖和凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    郭彩霞; 李艳博; 杜海英; 刘颖; 孙磊; 金明华; 孙志伟

    2008-01-01

    Objective To investigate the effects of over-expressed Smae gene combined with eisplatin (CDDP) on proliferation and apoptosis of hepatic carcinoma cells. Methods The recombinant plasmid pcDNA3.1+-hSmac was introduced into the human hepatic carcinoma SMMC-7721 cells using a lipusome-mediated method. The expression of Smac protein was detected by Western blot and flow eytometry.The cells were treated with three different doses of CDDP, 5, 15 and 25 μg/ml, for 24 hours after the transfection.MTr colorimetry was used to detect the cellular growth-inhibitory effects; acridine orange-ethidium bromide fluorescent staining (AO/EB) and flow cytometry with annexin V-PI double staining methods were used to detect the changes of cell apoptosis. Results Western blot and flow cytometry results demonstrated that the Smac protein level in SMMC-7721 cells was significantly increased after the transfection (P < 0.01). Compared with that of the control group, the over-expressed Smae gene inhibited the cell growth and induced cell apoptosis (P < 0.01). After being treated with CDDP, the inhibitory rates were increased significantly with increasing concentrations of CDDP compared with that of the control group, and the inhibitory rate of the CDDP-treated plus Smae group was significantly higher than that of the CDDP-treated group (P < 0.01). The results detected by AO/EB and flow cytometry demonstrated that the apoptotic rates of CDDP-treated plus Smac group were higher than those of the CDDP-treated group (P < 0.01). The results demonstrated that the Smac over-expression enhanced the effects of cell growth inhibition and apoptotic promotion induced by CDDP. Conclusion The pro-apoptotic Smac gene could be over-expressed in hepatocarcinoma SMMC-7721 cells and inhibit cell growth and induce apoptosis. Moreover the over-expressed Smac could enhance the chemotherapeutic sensitivity of SMMC-7721 to cisplatin. This experimental work may help in further study on the regulatory

  19. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  20. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

    Objective To investigate the biological characteristics of cell lines of healthy and diseased human dental alveoli. Methods Primary cell lines from either healthy or diseased human dental alveoli were obtained. Two cell lines, H-258 and H-171 derived from healthy and diseased human tissues respectively, were selected for morphological study and research on their growth and aging, using cell counting, and histochemical and immunohistochemical staining. Results Primary cell lines were successfully established from innormal dental alveoli. After freezing and thawing for three times, cell growth was continued and no morphological alterations were observed. The doubling time was 53.4 hours and mean division index (MDI) was 4‰. Cells were kept normal after twenty generations with no obvious reduction of doubling time and MDI. Of twenty-six primary cell lines derived from healthy human dental alveoli, only three cell lines achieved generation. After freezing and thawing for twice, cultured cells were still alive at a decreased growth speed, with doubling time of 85.9 hours and MDI of 3‰. Both cell lines, H-171 and H-258, shared the characteristics of osteoblast. Conclusions Primary cell lines of diseased human dental alveoli show greater growth potential. All cell lines of dental alveoli share characteristics of osteoblast. The technique we developed may be put into practice for the treatment of abnormal dental alveoli.

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

    OpenAIRE

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  2. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  3. Over-expressed CmbT multidrug resistance transporter improves the fitness of Lactococcus lactis

    Directory of Open Access Journals (Sweden)

    Filipić Brankica

    2013-01-01

    Full Text Available The influence of the over-expression of CmbT multidrug resistance transporter on the growth rate of Lactococcus lactis NZ9000 was studied. L. lactis is a lactic acid bacteria (LAB widely used as a starter culture in dairy industry. Recently characterized CmbT MDR transporter in L. lactis confers resistance to a wide variety of toxic compounds as well as to some clinically relevant antibiotics. In this study, the cmbT gene was over-expressed in the strain L. lactis NZ9000 in the presence of nisin inducer. Over-expression of the cmbT gene in L. lactis NZ9000 was followed by RT-PCR. The obtained results showed that the cmbT gene was successfully over-expressed by addition of sub-inhibitory amounts of nisin. Growth curves of L. lactis NZ9000/pCT50 over-expressing the cmbT gene and L. lactis NZ9000 control strain were followed in the rich medium as well as in the chemically defined medium in the presence solely of methionine (0.084 mM or mix of methionine and cysteine (8.4 mM and 8.2 mM, respectively. Resulting doubling times revealed that L. lactis NZ9000/pCT50 had higher growth rate comparing to the control strain. This could be a consequence of the CmbT efflux activity, which improves the fitness of the host bacterium through the elimination of toxic compounds from the cell.

  4. Reduced expression of β-catenin inhibitor Chibby in colon carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Marion M Schuierer; Elisabeth Graf; Ken-Ichi Takemaru; Wolfgang Dietmaier; Anja-Katrin Bosserhoff

    2006-01-01

    AIM: To analyse the Chibby expression and its function in colon carcinoma cell lines and colorectal carcinoma (CRC).METHODS: Chibby expression levels were investigated by quantitative RT-PCR in a panel of seven different colon carcinoma cell lines. By sequencing, we analysed mutational status of Chibby. To test whether Chibby exhibited effects on β-catenin signalling in colon carcinoma cells, we transfected SW480 cells with Chibby expression plasmid and, subsequently, analysed activity of β-catenin and tested for alterations in cellular phenotype. In addition, we examined Chibby mRNA levels in samples of colorectal carcinomas and adjacent normal tissues by using quantitative RT-PCR and hybridised gene chips with samples from CRC and normal tissues.RESULTS: Chibby mRNA expression was strongly downregulated in colon carcinoma cell lines in comparison to normal colon epithelial cells and no mutation in any of the examined colon carcinoma cell lines was found. Further, we could show that Chibby inhibited β-catenin activity in TOPflash assays when over-expressed in SW480 cells. Proliferation and invasion assays with Chibby transfected SW480 cells did not reveal profound differences compared to control cells. In contrast to these in vitro data, quantitative RT-PCR analyses of Chibby mRNA levels in CRC tumor samples did not show significant differences to specimens in adjacent non-cancerous tissue.Consistent with these findings, gene chips analysing tissue samples of tumors and corresponding normal tissue did not show altered Chibby expressionCONCLUSION: Altered Chibby expression might beobserved in vitro in different colon carcinoma cell lines.However, this finding could not be confirmed in vitro in CRC tumors, indicating that Chibby is not likely to promote CRC tumor development or progression. As Chibby is an important inhibitor of β-catenin signalling, our data implicate that the usability of colon carcinoma cell lines for in vitro studies analysing the Wnt

  5. Immune responses induced by Hepa1-6 hepotoma cell after over-expression SEA and CD80 in vitro%SEA-CD80基因过表达Hepa1-6肝癌细胞体外诱导的免疫学效应

    Institute of Scientific and Technical Information of China (English)

    司少艳; 史亮; 刘俊丽; 宋淑军; 景青萍; 吴继华; 张建中

    2011-01-01

    This study is aimed to determine whether Hepal-6 hepotoma cells could induce antitumor immune response after over-expression Staphylococcus enterotoxin A (SEA) and CD80. Mouse spleen lymphocytes were cocultured with hepal-6 cells infected with empty adenoviruse vector Ad (empty) or the recombinant aden-oviruses of Ad-MMRE-mTERT-B7, Ad-MMRE-mTERT-SEA or Ad-MMRE-mTERT-BIS. Lymphocyte proliferation was assayed by BrdU incorporation using a commercial kit, flow cytometric analysis was used to detect proliferation of T Lymphocyte subgroups. The production of IL-2, TNF-a and IFN-γ was tested by enzyme-linked immunosorbent assay, and antitumor activity of CTL was determined by lactate dehydrogenase release assay. Compared with Hepa1-6 infected with empty adenovirus or without infection, Hepal-6 infected with recombinant adenoviruses could significantly induce proliferatioin of spleen lymphocytes and T Lymphocyte subgroups, enhance IL-2, TNF-a and IFN-γ production and CTL killing activities. The antitumor response was significantly stronger in Hepal-6 cells after dual-gene over-expression than that in Hepal-6 cells after single-gene over-expression. The results indicate that SEA and CD80 expressed on the membrane of Hepal-6 cells by infection with recombinant adenovirus has immune activities. This study provides some experimental evidence for further tumor immune genetherapy with the recombinant adenovirus.%观察经SEA和CD80重组腺病毒感染Hepa1-6肝癌细胞后体外能否诱导抗肿瘤免疫学反应.方法 Hepa1-6细胞分别经空载体腺病毒Ad(空)和重组腺病毒Ad-MMRE-mTERT-B7、Ad-MMRE-mTERT-SEA、Ad-MMRE -mTERT-BIS感染后,和小鼠脾淋巴细胞共培养,然后采用Brdu酶联免疫法(ELISA)检测淋巴细胞增殖;流式细胞术检测T淋巴细胞亚群增殖;ELISA法检测细胞因子IL-2、TNF-α和IFN-γ的产生;LDH释放法检测CTLs对Hepa1-6的杀伤作用.结果与感染空载体腺病毒Ad(空)和未感染Hepa 1-6细胞相比,

  6. A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

    OpenAIRE

    Martins, Marta; McCusker, Matthew P.; Viveiros, Miguel; Couto, Isabel; Fanning, Séamus; Pagès, Jean-Marie; Amaral, Leonard

    2013-01-01

    It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer...

  7. Cloning of aminopeptidase Npromoter and its activity in hematopoietic cell and different tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expres sion in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may pro vide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radio therapy.

  8. MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

    NARCIS (Netherlands)

    KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

    1995-01-01

    In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

  9. GSL2 over-expression confers resistance to Pectobacterium atrosepticum in potato.

    Science.gov (United States)

    Mohan, Sara; Meiyalaghan, Sathiyamoorthy; Latimer, Julie M; Gatehouse, Michelle L; Monaghan, Katrina S; Vanga, Bhanupratap R; Pitman, Andrew R; Jones, E Eirian; Conner, Anthony J; Jacobs, Jeanne M E

    2014-03-01

    Over-expression of the potato Gibberellin Stimulated-Like 2 ( GSL2 ) gene in transgenic potato confers resistance to blackleg disease incited by Pectobacterium atrosepticum and confirms a role for GSL2 in plant defence. The Gibberellin Stimulated-Like 2 (GSL2) gene (also known as Snakin 2) encodes a cysteine-rich, low-molecular weight antimicrobial peptide produced in potato plants. This protein is thought to play important roles in the innate defence against invading microbes. Over-expression of the GSL2 gene in potato (cultivar Iwa) was achieved using Agrobacterium-mediated gene transfer of a plant expression vector with the potato GSL2 gene under the regulatory control elements of the potato light-inducible Lhca3 gene. The resulting plants were confirmed as being transgenic by PCR, and subsequently analysed for transcriptional expression of the Lhca3-GSL2-Lhca3 chimeric potato gene. Quantitative RT-PCR analysis demonstrated that the majority of the transgenic potato lines over-expressed the GSL2 gene at the mRNA level. Based on qRT-PCR results and evaluation of phenotypic appearance, eight lines were selected for further characterisation and evaluated in bioassays for resistance to Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica), the causal agent of blackleg in potato. Three independent pathogenicity bioassays showed that transgenic lines with significantly increased transcriptional expression of the GSL2 gene exhibit resistance to blackleg disease. This establishes a functional role for GSL2 in plant defence against pathogens in potato.

  10. Over-expression of ST3Gal-I promotes mammary tumorigenesis

    DEFF Research Database (Denmark)

    Picco, Gianfranco; Julien, Sylvain; Brockhausen, Inka;

    2010-01-01

    3Gal-I adds sialic acid to the galactose residue of core 1 (Galbeta1,3GalNAc) O-glycans and this enzyme is over-expressed in breast cancer resulting in the expression of sialylated core 1 glycans. In order to study the role of ST3Gal-I in mammary tumor development, we developed transgenic mice......Changes in glycosylation are common in malignancy, and as almost all surface proteins are glycosylated, this can dramatically affect the behavior of tumor cells. In breast carcinomas, the O-linked glycans are frequently truncated, often as a result of premature sialylation. The sialyltransferase ST...... that over-express the sialyltransferase under the control of the human membrane-bound mucin 1 promoter. These mice were then crossed with PyMT mice that spontaneously develop mammary tumors. As expected, ST3Gal-I transgenic mice showed increased activity and expression of the enzyme in the pregnant...

  11. Over-expression of thymosin beta 4 promotes abnormal tooth development and stimulation of hair growth.

    Science.gov (United States)

    Cha, Hee-Jae; Philp, Deborah; Lee, Soo-Hyun; Moon, Hye-Sung; Kleinman, Hynda K; Nakamura, Takashi

    2010-01-01

    Thymosin beta 4 has multi-functional roles in cell physiology. It accelerates wound healing, hair growth and angiogenesis, and increases laminin-5 expression in corneal epithelium. Furthermore, thymosin beta 4 stimulates tumor growth and metastasis by induction of cell migration and vascular endothelial growth factor-mediated angiogenesis. Using a construct on the skin-specific keratin-5 promoter, we have developed thymosin beta 4 over-expressing transgenic mice to further study its functional roles. Thymosin beta 4 in adult skin and in embryonic stages of the transgenic mouse was analyzed by both Western blot and immunohistochemistry. The over-expression of thymosin beta 4 was observed especially around hair follicles and in the teeth in the transgenic mice. We examined the phenotype of the thymosin beta 4 over-expressing mice. Hair growth was accelerated. In addition, the transgenic mice had abnormally-shaped white teeth and dull incisors. We found that the expression of laminin-5 was up-regulated in the skin of the transgenic mice. We conclude that thymosin beta 4 has an important physiological role in hair growth and in tooth development.

  12. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Mohan Durga

    2012-01-01

    Full Text Available In India, the unprecedented growth rate and urbanization along with the rapid increase in motor vehicle activity and industrialization are contributing to high levels of urban air pollution. The population is mainly exposed to high air pollution concentrations, where motor vehicle emissions constitute the main source of fine and ultrafine particles. Motor exhaust emissions is a mixture of gases and Particulate Matter (PM. Diesel and petrol fuels in vehicles produce combustion-derived particles as a result of combustion. Vehicle exhaust particles are the main constituents of environmental nanoparticles. In the present investigation, environmental nanoparticles such as Diesel Exhaust Particles (DEP and Petrol Exhaust Particles (PEP were collected from on-road vehicles using a specially designed collection chamber. The surface morphology of the collected particles was analyzed through Transmission Electron Microscope (TEM, and the elemental mapping was performed through EDAX analysis. Results indicated the presence of nanometer-size particles in both the categories of vehicle exhaust. These small-size particles of respirable range can enter the respiratory tract of humans and get deposited in the lungs and cause various effects inside the human body. The aim of this study is to assess the cytotoxicity of the collected Diesel Exhaust Nanoparticles (DENPs and Petrol Exhaust Nanoparticles (PENPs. Cytotoxicity endpoint, such as IC50 (50% Inhibitory Concentration, was determined after a 24-h exposure. Results of this study indicated that all five cell lines were sensitive to these vehicle exhaust nanoparticles at varying levels.

  13. Deep sequencing reveals low incidence of endogenous LINE-1 retrotransposition in human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hubert Arokium

    Full Text Available Long interspersed element-1 (LINE-1 or L1 retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs. However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs. Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3' end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.

  14. Deep sequencing reveals low incidence of endogenous LINE-1 retrotransposition in human induced pluripotent stem cells.

    Science.gov (United States)

    Arokium, Hubert; Kamata, Masakazu; Kim, Sanggu; Kim, Namshin; Liang, Min; Presson, Angela P; Chen, Irvin S

    2014-01-01

    Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3' end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.

  15. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines.

    Science.gov (United States)

    Pigni, Matteo; Ashok, Devika; Acha-Orbea, Hans

    2016-01-01

    It is notoriously difficult to obtain large quantities of non-activated dendritic cells ex vivo. For this reason, we produced and characterized a mouse model expressing the large T oncogene under the CD11c promoter (Mushi mice), in which CD8α(+) dendritic cells transform after 4 months. We derived a variety of stable cell lines from these primary lines. These cell lines reproducibly share with freshly isolated dendritic cells most surface markers, mRNA and protein expression, and all tested biological functions. Cell lines can be derived from various strains and knockout mice and can be easily transduced with lentiviruses. In this article, we describe the derivation, culture, and lentiviral transduction of these dendritic cell lines.

  16. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

    2011-01-01

    incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...... except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein......The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...

  17. The tumor-selective over-expression of the human Hsp 70 gene is attributed to the aberrant controls at both initiation and elongation levels of transcription

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors, linked to the poor prognosis, being refractory to chemo- and radio-therapies as well as the advanced stage of tumorous lesions in particular. However, both the nature and details of aberrations in the control of the Hsp70 expression in tumor remain enigmatic. By comparing various upstream segments of the Hsp70gene for each's ability to drive the luciferase reporter genes in the context of the tumor cell lines varying in their p53 status and an immortal normal liver cell line, we demonstrated in a great detail the defects in the control mechanisms at the both initiation and elongation levels of transcription being instrumental to the tumor selective profile of its expression. Our data should not only offer new insights into our understanding of the tumor specific over-expression of the human Hsp70 gene, but also paved the way for the rational utilization of the tumor selective mechanism with the Hsp70 at the central stage for targeting the therapeutic gene expression to human tumors.

  18. Phorbol Esters Isolated from Jatropha Meal Induced Apoptosis-Mediated Inhibition in Proliferation of Chang and Vero Cell Lines

    Directory of Open Access Journals (Sweden)

    Syahida Ahmad

    2012-10-01

    Full Text Available The direct feeding of Jatropha meal containing phorbol esters (PEs indicated mild to severe toxicity symptoms in various organs of different animals. However, limited information is available on cellular and molecular mechanism of toxicity caused by PEs present in Jatropha meal. Thus, the present study was conducted to determine the cytotoxic and mode of action of PEs isolated from Jatropha meal using human hepatocyte (Chang and African green monkey kidney (Vero cell lines. The results showed that isolated PEs inhibited cell proliferation in a dose-dependent manner in both cell lines with the CC50 of 125.9 and 110.3 μg/mL, respectively. These values were compatible to that of phorbol 12-myristate 13-acetate (PMA values as positive control i.e., 124.5 and 106.3 μg/mL respectively. Microscopic examination, flow cytometry and DNA fragmentation results confirmed cell death due to apoptosis upon treatment with PEs and PMA at CC50 concentration for 24 h in both cell lines. The Western blot analysis revealed the overexpression of PKC-δ and activation of caspase-3 proteins which could be involved in the mechanism of action of PEs and PMA. Consequently, the PEs isolated form Jatropha meal caused toxicity and induced apoptosis-mediated proliferation inhibition toward Chang and Vero cell lines involving over-expression of PKC-δ and caspase-3 as their mode of actions.

  19. Authentication of the R06E Fruit Bat Cell Line

    Directory of Open Access Journals (Sweden)

    Ingo Jordan

    2012-05-01

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

  20. Production of transgenic pigs over-expressing the antiviral gene Mx1

    OpenAIRE

    2014-01-01

    The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15–25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated...

  1. CmMYB19 Over-Expression Improves Aphid Tolerance in Chrysanthemum by Promoting Lignin Synthesis

    Science.gov (United States)

    Wang, Yinjie; Sheng, Liping; Zhang, Huanru; Du, Xinping; An, Cong; Xia, Xiaolong; Chen, Fadi; Jiang, Jiafu; Chen, Sumei

    2017-01-01

    The gene encoding the MYB (v-myb avian myeloblastosis vira l oncogene homolog) transcription factor CmMYB19 was isolated from chrysanthemum. It encodes a 200 amino acid protein and belongs to the R2R3-MYB subfamily. CmMYB19 was not transcriptionally activated in yeast, while a transient expression experiment conducted in onion epidermal cells suggested that the CmMYB19 product localized to the nucleus. CmMYB19 transcription was induced by aphid (Macrosiphoniella sanborni) infestation, and the abundance of transcript was higher in the leaf and stem than in the root. The over-expression of CmMYB19 restricted the multiplication of the aphids. A comparison of transcript abundance of the major genes involved in lignin synthesis showed that CmPAL1 (phenylalanine ammonia lyase 1), CmC4H (cinnamate4 hydroxylase), Cm4CL1 (4-hydroxy cinnamoyl CoA ligase 1), CmHCT (hydroxycinnamoyl CoA-shikimate/quinate hydroxycinnamoyl transferase), CmC3H1 (coumarate3 hydroxylase1), CmCCoAOMT1 (caffeoyl CoA O-methyltransferase 1) and CmCCR1 (cinnamyl CoA reductase1) were all upregulated, in agreement with an increase in lignin content in CmMYB19 over-expressing plants. Collectively, the over-expression of CmMYB19 restricted the multiplication of the aphids on the host, mediated by an enhanced accumulation of lignin. PMID:28287502

  2. Effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29

    Institute of Scientific and Technical Information of China (English)

    Chang-Wen Yu; Bi-Sheng Zhu

    2016-01-01

    Objective:To explore effect of leptin on cell proliferation and apoptosis of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Methods: MTT and flow cytometry were adopted for detecting the effect of exogenous leptin on cell cycle of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29.Results: Leptin with mass concentration (0 ng/mL, 5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could stimulate the growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29; exogenous leptin with mass concentration (5 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL) could inhibit cell growth of gastric adenocarcinoma cell line-SGC7901 and colon cancer cell line-HT-29 after 72 h; among which, inhibiting effects of cell line-SGC7901 and cell line-HT-29 were the most significant under the effect of exogenous leptin with mass concentration-200 ng/mL.Conclusion:Within a certain concentration and action time, exogenous leptin can stimulate the growth of gastric adenocarcinoma cell line and colon cancer cell line, and then promot the tumor cell proliferation and/or inhibit the tumor cell apoptosis.

  3. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

    As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties we

  4. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

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

  5. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

    1994-12-31

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

  6. Beryllium-stimulated apoptosis in macrophage cell lines.

    Science.gov (United States)

    Sawyer, R T; Fadok, V A; Kittle, L A; Maier, L A; Newman, L S

    2000-08-21

    In vitro stimulation of bronchoalveolar lavage cells from patients with chronic beryllium disease (CBD) induces the production of TNF-alpha. We tested the hypothesis that beryllium (Be)-stimulated TNF-alpha might induce apoptosis in mouse and human macrophage cell lines. These cell lines were selected because they produce a range of Be-stimulated TNF-alpha. The mouse macrophage cell line H36.12j produces high levels of Be-stimulated TNF-alpha. The mouse macrophage cell line P388D.1 produces low, constitutive, levels of TNF-alpha and does not up-regulate Be-stimulated TNF-alpha production. The DEOHS-1 human CBD macrophage cell line does not produce constitutive or Be-stimulated TNF-alpha. Apoptosis was determined by microscopic observation of propidium iodide stained fragmented nuclei in unstimulated and BeSO(4)-stimulated macrophage cell lines. BeSO(4) induced apoptosis in all macrophage cell lines tested. Beryllium-stimulated apoptosis was dose-responsive and maximal after 24 h of exposure to 100 microM BeSO(4). In contrast, unstimulated and Al(2)(SO(4))(3)-stimulated macrophage cell lines did not undergo apoptosis. The general caspase inhibitor BD-fmk inhibited Be-stimulated macrophage cell line apoptosis at concentrations above 50 microM. Our data show that Be-stimulated macrophage cell line apoptosis was caspase-dependent and not solely dependent on Be-stimulated TNF-alpha levels. We speculate that the release of Be-antigen from apoptotic macrophages may serve to re-introduce Be material back into the lung microenvironment, make it available for uptake by new macrophages, and thereby amplify Be-stimulated cytokine production, promoting ongoing inflammation and granuloma maintenance in CBD.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-11-01

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

  8. Human embryonic stem cell lines derived from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhen Fu FANG; Fan JIN; Hui GAI; Ying CHEN; Li WU; Ai Lian LIU; Bin CHEN; Hui Zhen SHENG

    2005-01-01

    Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF,Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.

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

    Science.gov (United States)

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

    2000-02-01

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

  10. Over-Expression of BnMAPK1 in Brassica napus Enhances Tolerance to Drought Stress

    Institute of Scientific and Technical Information of China (English)

    WENG Chang-mei; LU Jun-xing; WAN Hua-fang; WANG Shu-wen; WANG Zhen; LU Kun; LIANG Ying

    2014-01-01

    Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases widely conserved in all eukaryotes and involved in responses to biotic and abiotic stresses. In this study, two over-expressing BnMAPK1 oilseed rape lines, ov3 and ov11, were used to study the drought-resistant mechanism of BnMAPK1 under natural drought and simulation drought through spraying 10%PEG 8000 in seedlings. Zhongyou 821 (WT) was used as control. Compared with wild type, transgenic seedlings had higher leaf water content, higher root activity, slightly higher peroxidase (POD) and superoxide dismutase (SOD) activity, higher proline content and lower malondialdehyde (MDA) content. The expression of drought-resistant related genes, including P5CSB, PLC, LEA4 and SCE1, have been up-regulated in some degree and the expressed time of transgenic lines were earlier than that of wild type. These results suggested that over-expression of BnMAPK1 can enhance the resistance to drought in oilseed rape (Brassica napus).

  11. An intronic SNP in the thyroid hormone receptor β gene is associated with pituitary cell-specific over-expression of a mutant thyroid hormone receptor β2 (R338W in the index case of pituitary-selective resistance to thyroid hormone

    Directory of Open Access Journals (Sweden)

    Cochran Craig

    2011-08-01

    Full Text Available Abstract Background The syndrome of resistance to thyroid hormone (RTH is caused by mutations in the thyroid hormone receptor β gene (THRB. The syndrome varies from asymptomatic to diffuse hypothyroidism, to pituitary-selective resistance with predominance of hyperthyroid signs and symptoms. The wide spectrum of clinical presentation is not completely attributable to specific THRB mutations. The THRB gene encodes two main isoforms, TR β1 which is widely distributed, and TR β2, whose expression is limited to the cochlea, retina, hypothalamus, and pituitary. Recent data demonstrated that in mice an intron enhancer region plays a critical role in the pituitary expression of the β2 isoform of the receptor. We thus hypothesized that polymorphisms in the human homologous region could modulate the pituitary expression of the mutated gene contributing to the clinical presentation of RTH. Methods Screening and in vitro characterization of polymorphisms of the intron enhancer region of the THRB gene in the index case of pituitary-selective RTH. Results The index case of pituitary-selective resistance is characterized by the missense R338W exon 9 mutation in cis with two common SNPs, rs2596623T and rs2596622C, located in the intron enhancer region of the THRB gene. Reporter gene assay experiments in GH3 pituitary-derived cells indicate that rs2596623T generates an increased pituitary cell-specific activity of the TR β2 promoter suggesting that rs2596623T leads to pituitary over-expression of the mutant allele. Conclusions The combined coding mutation and non-coding SNP therefore generate a tissue-specific dominant-negative condition recapitulating the patient's peculiar phenotype. This case illustrates the role of regulatory regions in modifying the clinical presentation of genetic diseases.

  12. Derivation of human embryonic stem cell line Genea022

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    Biljana Dumevska

    2016-03-01

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

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

    Science.gov (United States)

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

    2008-03-01

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

  14. Establishment of Germ-line Competent C57BL/6J Embryonic Stem Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Gui-jun YAN; Zheng GU; Jian WANG; Jia-ke TSO

    2004-01-01

    Objective To establish C57BL/6J embryonic stem (ES) cell lines with potential germline contribution Methods ES cells were isolated from blastocyst inner cell mass of C57BL/6J mice, and cultured for 15 passages, and then injected into blastococels of lCR mice blastocysts to establish chimeric mice.Results Three ES cell lines (mC57ESl,mC57ES3, mC57ES7) derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker "stage-specific embryonic antigen-1" and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC5 7ES1 cells consis tently differentiated into derivatives of all three embryonic germ layers. When mC57ES1cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained.One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.

  15. Cytotoxinic Mechanism of Hydroxyapatite Nanoparticles on Human Hepatoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    CAO Xian-ying; QI Zhi-tao; DAI Hong-lian; YAN Yu-hua; LI Shi-pu

    2003-01-01

    Stable and single-dispersed HAP nanoparticles were synthesized with chemical method assisted by ultrasonic treatment.HAP nanoparticles were surveyed by AFM and Zataplus.The effect on the Bel-7402 human hepatoma cell lines treated with HAP nanoparticles was investigated by the MTT methods and observation of morphology,and the mechanism was studied in changes of cell cycle and ultrastructure.The result shows that inhibition of HAP nanoparticles on the Bel-7402 human hepatoma cell lines is obviously in vitro.HAP nanoparticles the entered cancer cytoplasm,and cell proliferation is stopped at G1 phase of cell cycle,thus,cancer cells die directly.

  16. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

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

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

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

  18. Establishment of human embryonic stem cell line from gamete donors

    Institute of Scientific and Technical Information of China (English)

    LI Tao; ZHOU Can-quan; MAI Qing-yun; ZHUANG Guang-lun

    2005-01-01

    Background Human embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study. Methods Three oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.Results Four ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.Conclusions HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

  19. Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

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    Khordadmehr, M.,

    2014-05-01

    Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

  20. 抑癌基因蛋白p16在宫颈癌中过度表达对其结合Cdk4功能的影响%Effects of over-expressed p16 on its binding capacity with Cdk4 in cervical squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    庞天云; 邓飞; 郑晓娟; 邹琳; 李飞虹; 胡新荣

    2011-01-01

    目的 观察在宫颈癌中过度表达的p16是否仍然具有正常结合Cdk4的功能,进一步阐明过表达的p16不能抑制宫颈癌发生发展的原因.方法 对142例宫颈标本,包括慢性宫颈炎19例,CIN Ⅰ级(LCIN)15例,CIN Ⅱ/Ⅲ级(HCIN)42例,宫颈鳞状细胞癌(SCC)66例进行p16和Cdk4免疫组化染色分析.选取1例p16高表达SCC(22C)和1例p16低表达SCC(43C)的新鲜标本进行免疫沉淀和Western blot分析.结果 按照慢性宫颈炎、LCIN、HCIN和SCC的顺序,p16阳性率分别是0、60.0%、78.6%和92.4%;Cdk4 阳性率分别是21.1%、46.7%、73.2%和92.4%,表明p16和CDK4的表达随宫颈肿瘤的进展而协调性升高.在同一例标本中,83.9%的p16高表达SCC的Cdk4同时呈高表达;86.7%的p16低表达SCC的Cdk4呈高表达或平行低表达,表明大多数SCC含有足够的与p16结合的Cdk4.结合Cdk4的p16与总p16的比例在p16高表达SCC(22C)中是66.9%,在p16低表达SCC(43C)中是42.2%,表明在SCC中过表达p16的近半左右与Cdk4结合,且p16表达越高,其与Cdk4结合的总量也越多.结论 在宫颈癌中过度表达的p16 仍然具有结合Cdk4 的能力.本研究首次从宫颈癌组织水平证实过表达的p16可以结合Cdk4,为研究p16对宫颈癌的作用向前推进了一步.%Objective To observe the hinding capacity of over - expressed p16 with Cdk4 in cervical carcinoma.Methods Immunohistochemiscal staining of p16 and Cdk4 was performed on 142 samples of cervical tissues, including 19, 15 , 42 and 66 cases of cervicitis, low cervical intraepithelial neoplasm ( LCIN ) , high CIN ( HCIN ) and invasive cervical squamous cell carcinoma ( SCC ), respectively. Co - immunoprecipitation ( Co - IP ) and Western blot were used to test the activity of p16 binding Cdk4 in high p16 expression and low p16 expression SCC. Results In the lesions of cervicitis, LCIN, HCIN and SCC, the p16 positive rates were 0, 60. 0% , 78. 6% and 92. 4% , respectively, with Cdk4 positive rates of 21. 1

  1. Over-expression of StAPX in tobacco improves seed germination and increases early seedling tolerance to salinity and osmotic stresses.

    Science.gov (United States)

    Sun, Wei-Hong; Duan, Ming; Shu, De-Feng; Yang, Sha; Meng, Qing-Wei

    2010-08-01

    Ascorbate peroxidase plays a key role in scavenging reactive oxygen species under environmental stresses and in protecting plant cells against toxic effects. The Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that StAPX was transferred into the tobacco genome and StAPX was induced by salt and osmotic stresses in tomato leaves. Over-expression of StAPX in tobacco improved seed germination rate and elevated stress tolerance during post-germination development. Two transgenic lines showed higher APX activity and accumulated less hydrogen peroxide than wild-type plants after stress treatments. The photosynthetic rates, the root lengths, the fresh and dry weights of the transgenic lines were distinctly higher than those of wild-type plants under stress conditions. Results indicated that the over-expression of StAPX had enhanced tolerance to salt stress and osmotic stress in transgenic tobacco plants.

  2. Multidrug resistance modulators PSC 833 and CsA show differential capacity to induce apoptosis in lymphoid leukemia cell lines independently of their MDR phenotype.

    Science.gov (United States)

    Lopes, Eloisi C; Garcia, Mariana; Benavides, Fernando; Shen, Jianjun; Conti, Claudio J; Alvarez, Elida; Hajos, Silvia E

    2003-05-01

    Among the mechanisms that induce multidrug resistance (MDR), one of those most frequent is over-expression of a phosphoglycoprotein (Pgp) encoded in the mouse by the mdr-1 and mdr-3 genes. We have demonstrated that cyclosporin-A (CsA) as well as its analogue PSC 833 were able to revert the MDR phenotype in murine cell lines resistant to vincristine (LBR-V160) or doxorubicin (LBR-D160). The aim of this work was to evaluate the ability of PSC 833 and CsA to modulate mdr-1, mdr-3 and mrp-1 genes as well as to induce apoptosis analyzing the mechanism involved in the above tumor cell lines. By semi-quantitative RT-PCR, we demonstrated that mdr-3 was over-expressed in both resistant lines while mdr-1 was over-expressed only in LBR-V160; in contrast, mrp-1 expression was not evidenced in any of the cell lines. After treatment with 0.1 microg ml(-1) of either PSC 833 or CsA, LBR-V160 showed no changes in mdr-1 but decreased mdr-3 expression, while LBR-D160 failed to display any modification in the expression of these genes. Apoptosis was evidenced by fluorescence microscopy, S minuscule accumulation and agarose gel electrophoresis. Our results demonstrated that CsA (1 microg ml(-1)) was able to induce apoptosis in all cell lines: 18.31% (+/-4.46) for LBR-, 25.96% (+/-5.24) for LBR-V160 and 27.36% (+/-4.12) for LBR-D160, while PSC 833 (1 microg ml(-1)) only induced apoptosis 21.51% (+/-5.73) in LBR-V160 cell line. The expression of Bcl-2 family proteins (Bcl-2, Bax and Bcl-x(L)) was analyzed by flow cytometry showing high expression of the three proteins which was not significantly modified after treatment with either PSC 833 or CsA on the sensitive as well as on the resistant cell lines. Single stranded conformation polymorphisms analysis of p53 (Trp53) gene in the cell lines showed no mutation in exons 5-8 of the tumor suppressor gene. We conclude that depending on the concentration used, PSC 833 and CsA may act either by modulating the mdr-3 gene (0.1 microg ml(-1)) or

  3. Over-Expression of SlSHN1 Gene Improves Drought Tolerance by Increasing Cuticular Wax Accumulation in Tomato

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    Ayed M. Al-Abdallat

    2014-10-01

    Full Text Available Increasing cuticular wax accumulation in plants has been associated with improving drought tolerance in plants. In this study, a cDNA clone encoding the SlSHN1 transcription factor, the closest ortholog to WIN/SHN1 gene in Arabidopsis, was isolated from tomato plant. Expression analysis of SlSHN1 indicated that it is induced in response to drought conditions. The over-expression of SlSHN1 in tomato under the control of the constitutive CaMV 35S promoter produced plants that showed mild growth retardation phenotype with shiny and dark green leaves. Scanning electron microscopy showed that the over-expression of SlSHN1 in tomato resulted in higher cuticular wax deposition on leaf epidermial tissue when compared to non-transformed plants. Expression analysis in transgenic lines over-expressing SlSHN1 indicated that several wax-related synthesis genes were induced. Transgenic tomato plants over-expressing SlSHN1 showed higher drought tolerance when compared with wild type plants; this was reflected in delayed wilting of transgenic lines, improved water status and reduced water loss rate when compared with wild type plants. In conclusion, the SlSHN1 gene can modulate wax accumulation and could be utilized to enhance drought tolerance in tomato plant.

  4. JKT-1 is not a human seminoma cell line.

    Science.gov (United States)

    de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; van Gurp, Ruud J H L M; van Drunen, Ellen; Beverloo, H Berna; Lau, Yun-Fai Chris; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Hatakeyama, Shingo; Ohyama, Chikara; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2007-08-01

    The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded.

  5. Investigation of radiosensitivity gene signatures in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    John S Hall

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

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

    Science.gov (United States)

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

    2013-10-01

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

  7. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Tet-control system is developed to tightly control target gene expression in mammalian cells by using the regulatory elements of tetracycline-repressor of the transposor Tn10 from E.Coli.We have transfected reverse tetracycline-controlled transactivator gene (rtTA) into genome of Jurkat cells and established two Jurkat tet-on cell lines.Induction of luciferase reporter activity with doxycycline,a tetracycline derivative,is dose-dependent with a peak value of 32-fold increment.Establishment of Jurkat tet-on cell lines greatly facilitates quantitative studies on target gene functions in the cells.

  8. Establishment and characterization of rat portal myofibroblast cell lines.

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    Michel Fausther

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

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

  11. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

    Verdijk, R.; Franke, H.R.; Wolbers, F.; Vermes, I.

    2007-01-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancers cells in virtro. In this study, six bisphosphonates were administered to three breast caner cell lines. Cell proliferation was measured by quantification of th expressio of Cyclin D1 mRNA. Apoptosis was determined by flow cytome

  12. Establishment of FGFR4 over-expression cell line and analysis of its biological activity%高表达FGFR4细胞株的建立及其生物活性分析

    Institute of Scientific and Technical Information of China (English)

    黄静; 安红丽; 张涛

    2014-01-01

    目的 构建FGFR4高表达细胞,研究FGFR4对细胞增殖能力的影响及相关蛋白活化机制,为研究以FGFR4为靶点的药物提供细胞模型.方法构建FGFR4真核表达载体并转染HEK293细胞,筛选稳定表达FGFR4细胞株,免疫印迹和免疫荧光分析FGFR4表达水平,细胞计数检测FGFR4高表达细胞的增殖情况,流式细胞仪检测FGFR4对细胞周期的影响,同时对ERK1/2磷酸化水平进行免疫印迹分析.结果 在相同培养环境下,HEK293-FGFR4细胞同HEK293-pcDNA细胞相比,其增殖能力提高.周期分析表明,FGFR4使G0/G1期降低,促进细胞的增殖.同时,对MAPK信号通路进行了初步分析,发现FGFR4蛋白可以引起ERK1/2的磷酸化,从而导致MAPK/ERK1/2通路的激活,说明FGFR4可激活MAPK信号通路.结论 成功构建了FGFR4高表达细胞株,为筛选以FGFR4为靶标的抗肿瘤药物奠定了前期实验基础.

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

    Science.gov (United States)

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

    2011-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

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

  15. Phenotypes and karyotypes of human malignant mesothelioma cell lines.

    Directory of Open Access Journals (Sweden)

    Vandana Relan

    Full Text Available BACKGROUND: Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. METHODS: Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. RESULTS: Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30-72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5-17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. CONCLUSION: These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of

  16. Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

    Institute of Scientific and Technical Information of China (English)

    Zhi-Gang Zhao; Wen-Lu Shen

    2005-01-01

    AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer.METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting.RESULTS: The number of viable cells decreased in a doseand time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h.CONCLUSION: Antisense HSP70 oligomers

  17. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

    Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

  18. Determinants of intrinsic radiosensitivity of mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Radford, I.R. [Peter MacCallum Cancer Institute, East Melbourne, VIC (Australia). Research Division

    1998-12-31

    Differences in the radiosensitivity of normal and cancerous cells could arise in various ways. Although there is no compelling data to support the view, the currently prevailing opinion is that differences in radiosensitivity are related to differences in some aspect of enzymatic DNA repair. A test of the importance of possible differences in enzymatic DNA repair in determining relative radiosensitivity would be to compare lethality in cells containing equivalent numbers of DNA lesions. Six cell lines were used in these studies: two Chinese hamster (CHO and V79) and a monkey (Vero) fibroblast-like line, a mouse melanoma line (B16-F1), and a rat (RUC-2) and a human (SQ-20B) carcinoma line. This group of cell lines displays a wide range of sensitivities to external beam low-LET radiation, ranging from the relatively radiosensitive B16-F1 and Vero lines through to the highly radioresistant RUC-2 line. However, it is important to note that none of the lines has a demonstrated defect in enzymatic DNA repair and that all appear to die by necrosis following a lethal radiation insult. Despite having significantly different radiosensitivities, CHO and V79 cells showed comparable responses to DNA-associated {sup 125}I-decays with D{sub o} values of around 65. More surprisingly, the radiosensitive B16-F1 line and the radioresistant RUC-2 line both had responses with D{sub o} values of around 133 {sup 125}I-decays. The factor of two difference between the D{sub o} values for these two pairs of cell lines is probably attributable to CHO and V79 cells being pseudo-diploid whereas B 16-F1 and RUC2 appear to have derived from tetraploid cells. The generality of the above result, for DNA lesions of different quality, was tested by comparing the sensitivities of CHO and V79 cells to DNA-associated {sup 3}H-decays. Again, consistent with the {sup 125}I-decay data, there was no significant difference in the D{sub o} values for these lines. Our {sup 3}H- and {sup 125}I-decay data are

  19. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    Stem cell banks curating and distributing human embryonic stem cells have been established in a number of countries and by a number of private institutions. This paper identifies and critically discusses a number of arguments that are used to justify the importance of such banks in policy...... are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  20. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

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

  1. 维生素E琥珀酸酯联合紫杉醇对Her-2过表达乳腺癌细胞的诱导凋亡研究%Vitamin E succinate combined with paclitaxel on the apoptosis of Her-2 over-expressing breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    赵妍; 李里; 姜秋颖; 申维喜; 武露艳; 阎婷婷

    2011-01-01

    Background and purpose: Vitamin E succinate (VES) is the esterification of natural vitamin E derivative. Studies have confirmed that VES could induce the apoptosis of breast cancer, prostate cancer, tongue cancer, kidney cancer, but has no toxicity to normal cells and tissues. This experiment detected the apoptosis rates of Her-2 over-expressing breast cancer cells which were administered with VES and paclitaxel at different dosages,alone or together. Methods: Immunocytochemical method was used to detect Her-2 expression of MDA-MB-453 cells. TUNEL assay was used to detect apoptosis rates of MDA-MB-453 cells treated with VES at the concentration of 10, 20 mg/L and paclitaxel of 50 and 100 nmol/L, alone or together, for 24 or 48 h. Then apoptosis action of various combinations was compared. Results: The expression rates of 95% Her-2 were 63.32%-69.60%; VES and paclitaxel both induced apoptosis of MDA-MB-453 cells, and it was dose-and time-dependent. It was strongest in apoptosis at the combination treatment of 10 mg/L VES and 100 nmol/L paclitaxel in MDA-MB-453 cells 48 h later. Conclusion:VES and paclitaxel both can induce the apoptosis of MDA-MB-453 cells. It is stronger when the two drugs are administered together.%背景与目的:维生素E琥珀酸酯(vitamin E succinate,VES)是天然维生素E的酯化衍生物,研究已证实其可诱导乳腺癌、前列腺癌、舌癌及肾癌等恶性肿瘤凋亡,却对正常细胞组织没有毒性作用.本实验以VES、紫杉醇单独及联合作用于Her-2过表达乳腺癌细胞,检测各组药物对乳腺癌细胞的诱导凋亡率.方法:免疫细胞化学法检测MDA-MB-453细胞Her-2蛋白表达水平.以VES、紫杉醇单独作用于MDA-MB-453细胞24、48 h,用TUNEL法检测细胞凋亡率,以浓度为10、20 mg/L的VES与浓度为50和100 nmol/L的紫杉醇组合,作用于MDA-MB-453细胞24、48 h,用TUNEL法检测不同组合的细胞凋亡率,比较各种组合对Her-2过表达乳腺癌细胞的

  2. Cardiac-specific over-expression of epidermal growth factor receptor 2 (ErbB2 induces pro-survival pathways and hypertrophic cardiomyopathy in mice.

    Directory of Open Access Journals (Sweden)

    Polina Sysa-Shah

    Full Text Available BACKGROUND: Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored. METHODOLOGY/PRINCIPAL FINDINGS: We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2-3 times larger than those of control littermates, express increased atrial natriuretic peptide and β-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib. CONCLUSIONS/SIGNIFICANCE: These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling

  3. Establishment of a new bovine leukosis virus producing cell line.

    Science.gov (United States)

    Beier, D; Riebe, R; Blankenstein, P; Starick, E; Bondzio, A; Marquardt, O

    2004-11-01

    Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

  4. Expressional patterns of chaperones in ten human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Slavc Irene

    2004-12-01

    Full Text Available Abstract Background Chaperones (CH play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity.

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

    Science.gov (United States)

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

    2013-10-01

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

  6. CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models.

    Science.gov (United States)

    Xue, Fuying; Wu, Yanping; Zhao, Xinghui; Zhao, Taoran; Meng, Ying; Zhao, Zhanzhong; Guo, Junwei; Chen, Wei

    2016-08-01

    Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca(2+) ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1.

  7. Over-expression of ST3Gal-I promotes mammary tumorigenesis.

    Science.gov (United States)

    Picco, Gianfranco; Julien, Sylvain; Brockhausen, Inka; Beatson, Richard; Antonopoulos, Aristotelis; Haslam, Stuart; Mandel, Ulla; Dell, Anne; Pinder, Sarah; Taylor-Papadimitriou, Joyce; Burchell, Joy

    2010-10-01

    Changes in glycosylation are common in malignancy, and as almost all surface proteins are glycosylated, this can dramatically affect the behavior of tumor cells. In breast carcinomas, the O-linked glycans are frequently truncated, often as a result of premature sialylation. The sialyltransferase ST3Gal-I adds sialic acid to the galactose residue of core 1 (Galbeta1,3GalNAc) O-glycans and this enzyme is over-expressed in breast cancer resulting in the expression of sialylated core 1 glycans. In order to study the role of ST3Gal-I in mammary tumor development, we developed transgenic mice that over-express the sialyltransferase under the control of the human membrane-bound mucin 1 promoter. These mice were then crossed with PyMT mice that spontaneously develop mammary tumors. As expected, ST3Gal-I transgenic mice showed increased activity and expression of the enzyme in the pregnant and lactating mammary glands, the stomach, lungs and intestine. Although no obvious defects were observed in the fully developed mammary gland, when these mice were crossed with PyMT mice, a highly significant decrease in tumor latency was observed compared to the PyMT mice on an identical background. These results indicate that ST3Gal-I is acting as a tumor promoter in this model of breast cancer. This, we believe, is the first demonstration that over-expression of a glycosyltransferase involved in mucin-type O-linked glycosylation can promote tumorigenesis.

  8. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad(1.17 Gy)), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad(1.65 Gy)), erythroleukemia; 45 (n . 1.1, D0 . 147 rad(1.47 Gy)), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad(0.76 Gy)), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

  9. Effect of failures and repairs on multiple cell production lines

    Energy Technology Data Exchange (ETDEWEB)

    Legato, P.; Bobbio, A.; Roberti, L.

    1989-01-01

    This paper examines a production line composed of multiple stages, or cells, which are passed in sequential order to arrive to the final product. Two possible coordination disciplines are considered, namely: the classical tandem arrangement of sequential working centers with input buffer and the kanban scheme, considered the Japanese shop floor realization of the Just-In-Time (JIT) manifacturing approach. The production line is modelled and analysed by means of Stochastic Petri Nets (SPN). Finally an analysis is made of the possibility that the working cells can incur failure/repair cycles perturbing the production flow of the line and thus reduce performance indices.

  10. Efficacy of ribavirin against malignant glioma cell lines

    Science.gov (United States)

    OGINO, AKIYOSHI; SANO, EMIKO; OCHIAI, YUSHI; YAMAMURO, SHUN; TASHIRO, SHINYA; YACHI, KAZUNARI; OHTA, TAKASHI; FUKUSHIMA, TAKAO; OKAMOTO, YUTAKA; TSUMOTO, KOUHEI; UEDA, TAKUYA; YOSHINO, ATSUO; KATAYAMA, YOICHI

    2014-01-01

    Ribavirin (1-β-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0–1,000 μM) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant

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

    Directory of Open Access Journals (Sweden)

    Ritter Peter R

    2010-03-01

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

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

    Science.gov (United States)

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

    2015-08-21

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

  13. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

    are curiously absent from the particular stem cell banking policy discourse. This to some extent artificially isolates this discourse from the broader discussions about the flows of reproductive materials and tissues in modern society, and such isolation may lead to the interests of important actors being...

  14. MSLN Gene Silencing Has an Anti-Malignant Effect on Cell Lines Overexpressing Mesothelin Deriving from Malignant Pleural Mesothelioma

    Science.gov (United States)

    Melaiu, Ombretta; Stebbing, Justin; Lombardo, Ylenia; Bracci, Elisa; Uehara, Norihisa; Bonotti, Alessandra; Cristaudo, Alfonso; Foddis, Rudy; Mutti, Luciano; Barale, Roberto; Gemignani, Federica

    2014-01-01

    Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM) are still poorly characterized. So far, mesothelin (MSLN) has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373) has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e. NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A). MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA) to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer. PMID:24465798

  15. MSLN gene silencing has an anti-malignant effect on cell lines overexpressing mesothelin deriving from malignant pleural mesothelioma.

    Directory of Open Access Journals (Sweden)

    Ombretta Melaiu

    Full Text Available Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM are still poorly characterized. So far, mesothelin (MSLN has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373 has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e., NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A. MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer.

  16. Characterisation of thyroid medullary carcinoma TT cell line.

    Science.gov (United States)

    Zabel, M; Grzeszkowiak, J

    1997-01-01

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

  17. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  18. Over-expression of an Arabidopsis δ-OAT gene enhances salt and drought tolerance in transgenic rice

    Institute of Scientific and Technical Information of China (English)

    WU Liangqi; FAN Zhanmin; GUO Lei; LI Yongqing; ZHANG Wenjing; QU Li-Jia; CHEN Zhangliang

    2003-01-01

    δ-OAT, ornithine-δ-aminotransferase, is the key enzyme involved in proline biosynthesis. In this study the Arabidopsisδ-OAT gene was transferred into rice (Oryza sativa L. ssp japonica cv. Zhongzuo 321), whose successful integration was demonstrated by PCR and Southern blot analysis. The over-expression of the gene in transgenic rice was also confirmed. Biochemical analysis showed that, under salt or drought stress conditions, proline contents in the leaves and roots in transgenic rice plants were 5- to 15-fold of those in non-transgenic controls. Under stress conditions, germinating rate of transgenic lines is higher than that of controls. Although the growth of rice plants tested were more and more retarded with the increasing of NaCl concentration, the transgenic plants grow faster compared to the controls under the same stress condition. Meanwhile, the resistance to KCl and MgSO4 stresses was also found enhanced in transgenic rice. Furthermore, the over-expression ofδ-OAT also improved the yield of transgenic plants under stress conditions. The average yield per plant of transgenic lines increases about 12%-41% more than that of control lines under 0.1 mol/L NaCl stress. These data indicated that the over-expression of δ-OAT, with the accumulation of proline, resulted in the enhancement of salt and drought tolerance and an increase of rice yield, which is of significance in agriculture.

  19. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  20. Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers.

    Science.gov (United States)

    Wen, Xiao-Peng; Pang, Xiao-Ming; Matsuda, Narumi; Kita, Masayuki; Inoue, Hiromichi; Hao, Yu-Jin; Honda, Chikako; Moriguchi, Takaya

    2008-04-01

    An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. 'Ballad') plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 microM CuSO4) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments.

  1. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    Science.gov (United States)

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

    2008-03-01

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

  2. Transportation characteristics of nolatrexed in three tumor cell lines

    Institute of Scientific and Technical Information of China (English)

    LI Yi-lei; ZHAO Ai-guo; WU Shu-guang

    2002-01-01

    Objective:To investigate the association of the transportation characteristics of nolatrexed in tumor cells with its drug sensitivity. Methods: The sensitivity of 3 tumor cell lines, C6, SRS82 and LoVo, to nolatrexed were determined by growth inhibition study. After exposure to 20 μmol/L nolatrexed at different time intervals ranging from 0 to 30 min, or to nolatrexed at different concentrations ranging from 0 to 40μmol/L for 10 min, the intracellular drug concentration was measured using high-performance liquid chromatography. Results: C6 was the most sensitive cell line among the three, with sensitivity 6. 8-fold and 13.8-fold those of SRS-82 and LoVo cells respectively. Transportation of nolatrexed in the 3 cell lines were qualitatively similar, which rapidly achieved steady-state within 5 min, and linear relationship between the intracellular and extracellular drug concentration was observed. The intracellular steady-state level achieved in C6 was significantly higher than those in the other two cell lines, the latter having comparable levels. Conclusion: Nolatrexed enters the cell very quickly and different transport capacities are involved in the generation of varied sensitivity to nolatrexed in tumor cells.

  3. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    Energy Technology Data Exchange (ETDEWEB)

    Sherwood, J.B.; Shouval, D.

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.

  4. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

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

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

    Science.gov (United States)

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

    2014-09-09

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

  6. SU-E-T-320: The Effect of Survivin Perturbation On the Radiation Response of Breast Cancer Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Smith, D; Debeb, B; Woodward, W [Department of Radiation Oncology, MD Anderson Cancer Center, Houston, TX (United States)

    2014-06-01

    Purpose: Survivin is the smallest member of the inhibitor of apoptosis protein family and is well-known for its universal over-expression in human cancers. Due to its role in apoptosis and cellular proliferation, survivin is implicated in the radiation response in several cancer types, and antisurvivin treatments have had success as a radiation sensitizer in many preclinical cancer models. As no studies to date have reported survivin as a factor affecting radiation resistance in breast cancer models, we sought to evaluate the synergistic relationship between survivin function and irradiation in breast cancer cell lines. Methods: Information regarding survivin protein expression in breast cancer was retrieved from three public databases: Oncomine, Kaplan-Meier Plotter, and GOBO. For the in vitro studies, survivin function was compromised by transducing a non-functional mutant form (survivin-DN) into two breast cancer cell lines, the estrogen receptor-positive MCF7 and the triple-negative, inflammatory SUM149. Cell growth was compared in the survivin-DN and control populations with colony-formation assays. To assess how survivin affects radiation response, clonogenic assays were performed by irradiating the cell lines up to 6 Gy. Results: From the public databases, survivin is more highly expressed in triple-negative breast cancer compared to all other subtypes, and is prognostic of poor survival in all breast cancer patients. In MCF7, the survivin-DN population had decreased colony-formation potential; the opposite was true in SUM149. In the clonogenic assays, abrogation of survivin function radio-protected MCF7 cells in monolayer and 3D growth conditions, while SUM149 survivin-DN cells were radiosensitized in monolayer conditions. Conclusion: We observed synergy between survivin function and radiation, although the results between the two cell lines were disparate. Further investigation is required to identify the mechanism of this discrepancy, including evaluation

  7. RNF13: a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Qiang Zhang; Yunxiao Meng; Lei Zhang; Jie Chen; Dahai Zhu

    2009-01-01

    Protein ubiquitination by E3 ubiquitin ligases plays an important role in cancer development. In this study, we provide experimental evidence that a RING-finger-containing protein RNF13 is an ER/Golgi membrane-associated E3 ubiquitin ligase and its RING finger domain is required for the ubiquitin iigase activity, lmmunohistochemical analysis of pancreatic ductal adenocarcinoma (PDAC) and paracancerous normal tissues from 72 patients documented RNF13 over-expression in 30 tumor samples (41.7%, 30/72), and its expression was significantly associated with histological grading (P= 0.024). In addition, RNFI3 was detected in precancerous lesions: tubular complexes in chronic pancreatitis (CP) and pancreatic intraepithelial neoplasia (PanlN) (79.3%, 23/29 and 62.8%, 22/35, respectively). Moreover, RNF13 staining was significantly correlated with Tenascin-C expression (P = 0.004) in PDAC samples, further supporting the role of RNF13 in cancer progression. Over-expression of wild type but not RING domain-mutant RNF13 in pancreatic MiaPaca-2 cancer cells increased invasive potential and gelatinolytic activity by matrix metalloproteinase-9. Taken together, these findings reveal that RNF13 is a novel E3 ubiquitin ligase involved in pancreatic carcinogenesis; ubiqui-tin-mediated modification of proteins by RNF13 may participate in pancreatic cancer development.

  8. Functional Expression of Voltage-Gated Sodium Channels Navl.5 in Human Breast Caner Cell Line MDA-MB-231

    Institute of Scientific and Technical Information of China (English)

    Rui GAO; Jing WANG; Yi SHEN; Ming LEI; Zehua WANG

    2009-01-01

    Voltage-gated sodium channels (VGSCs) are known to be involved in the initiation and progression of many malignancies,and the different subtypes of VGSCs play important roles in the metastasis cascade of many tumors.This study investigated the functional expression of Nav 1.5 and its effect on invasion behavior of human breast cancer cell line MDA-MB-231.The mRNA and pro-tein expression of Navl.5 was detected by real time PCR,Western Blot and immunofluorescence.The effects of Navl.5 on cell proliferation,migration and invasion were respectively assessed by MTT and Transwell.The effects of Nav1.5 on the secretion of matrix metalloproteases (MMPs) by MDA-MB-231 were analyzed by RT-PCR.The over-expressed Navl.5 was present on the membrane of MDA-MB-231 cells.The invasion ability in vitro and the MMP-9 mRNA expression were respec-tively decreased to (47.82±0.53)% and (43.97±0.64)% (P<0.05) respectively in MDA-MB-231 cells treated with VGSCs specific inhibitor tetrodotoxin (TTX) by blocking Navl.5 activity.It was con-eluded that Nav1.5 functional expression potentiated the invasive behavior of human breast cancer cell line MDA-MB-231 by increasing the secretion of MMP-9.

  9. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

  10. Reconstruction of endometrium from human endometrial side population cell lines.

    Directory of Open Access Journals (Sweden)

    Irene Cervelló

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

  11. Establishment, immortalisation and characterisation of pteropid bat cell lines.

    Directory of Open Access Journals (Sweden)

    Gary Crameri

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

  12. Monoclonal antibodies against the human leukemia cell line K 562.

    Science.gov (United States)

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  13. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    Science.gov (United States)

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

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

    OpenAIRE

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-01-01

    Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members o...

  15. Skin Biopsy and Patient-Specific Stem Cell Lines

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Medina, D.; Oborn, C.J. (Baylor College of Medicine, Houston, TX (USA)); Li, M.L.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1987-09-01

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

  19. Analysis of LINE-1 expression in human pluripotent cells.

    Science.gov (United States)

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  20. Production of transgenic pigs over-expressing the antiviral gene Mx1.

    Science.gov (United States)

    Yan, Quanmei; Yang, Huaqiang; Yang, Dongshan; Zhao, Bentian; Ouyang, Zhen; Liu, Zhaoming; Fan, Nana; Ouyang, Hongsheng; Gu, Weiwang; Lai, Liangxue

    2014-01-01

    The myxovirus resistance gene (Mx1) has a broad spectrum of antiviral activities. It is therefore an interesting candidate gene to improve disease resistance in farm animals. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the Mx1 gene. These transgenic pigs express approximately 15-25 times more Mx1 mRNA than non-transgenic pigs, and the protein level of Mx1 was also markedly enhanced. We challenged fibroblast cells isolated from the ear skin of transgenic and control pigs with influenza A virus and classical swine fever virus (CFSV). Indirect immunofluorescence assay (IFA) revealed a profound decrease of influenza A proliferation in Mx1 transgenic cells. Growth kinetics showed an approximately 10-fold reduction of viral copies in the transgenic cells compared to non-transgenic controls. Additionally, we found that the Mx1 transgenic cells were more resistant to CSFV infection in comparison to non-transgenic cells. These results demonstrate that the Mx1 transgene can protect against viral infection in cells of transgenic pigs and indicate that the Mx1 transgene can be harnessed to develop disease-resistant pigs.

  1. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

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

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

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

  3. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  4. Oral bioavailability of glyphosate: studies using two intestinal cell lines.

    Science.gov (United States)

    Vasiluk, Luba; Pinto, Linda J; Moore, Margo M

    2005-01-01

    Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

  5. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    Science.gov (United States)

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

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

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M

    2010-09-01

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

  7. Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Valentina Pozzi

    2015-05-01

    Full Text Available Background/Aims: Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs, has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT, an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

  8. HNF1α inhibition triggers epithelial-mesenchymal transition in human liver cancer cell lines

    Directory of Open Access Journals (Sweden)

    Vignjevic Danijela

    2011-10-01

    Full Text Available Abstract Background Hepatocyte Nuclear Factor 1α (HNF1α is an atypical homeodomain-containing transcription factor that transactivates liver-specific genes including albumin, α-1-antitrypsin and α- and β-fibrinogen. Biallelic inactivating mutations of HNF1A have been frequently identified in hepatocellular adenomas (HCA, rare benign liver tumors usually developed in women under oral contraceptives, and in rare cases of hepatocellular carcinomas developed in non-cirrhotic liver. HNF1α-mutated HCA (H-HCA are characterized by a marked steatosis and show activation of glycolysis, lipogenesis, translational machinery and mTOR pathway. We studied the consequences of HNF1α silencing in hepatic cell lines, HepG2 and Hep3B and we reproduced most of the deregulations identified in H-HCA. Methods We transfected hepatoma cell lines HepG2 and Hep3B with siRNA targeting HNF1α and obtained a strong inhibition of HNF1α expression. We then looked at the phenotypic changes by microscopy and studied changes in gene expression using qRT-PCR and Western Blot. Results Hepatocytes transfected with HNF1α siRNA underwent severe phenotypic changes with loss of cell-cell contacts and development of migration structures. In HNF1α-inhibited cells, hepatocyte and epithelial markers were diminished and mesenchymal markers were over-expressed. This epithelial-mesenchymal transition (EMT was related to the up regulation of several EMT transcription factors, in particular SNAIL and SLUG. We also found an overexpression of TGFβ1, an EMT initiator, in both cells transfected with HNF1α siRNA and H-HCA. Moreover, TGFβ1 expression is strongly correlated to HNF1α expression in cell models, suggesting regulation of TGFβ1 expression by HNF1α. Conclusion Our results suggest that HNF1α is not only important for hepatocyte differentiation, but has also a role in the maintenance of epithelial phenotype in hepatocytes.

  9. Fatty acid binding proteins (FABPs in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Jung Klaus

    2011-07-01

    Full Text Available Abstract Background Fatty acid binding proteins (FABP play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines. Methods Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome. Results General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome. Conclusions Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the

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

    Science.gov (United States)

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

    2009-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-12-01

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

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

    Science.gov (United States)

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

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

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

    Science.gov (United States)

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-07-01

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

  14. Over-expression of OsPTR6 in rice increased plant growth at different nitrogen supplies but decreased nitrogen use efficiency at high ammonium supply.

    Science.gov (United States)

    Fan, Xiaorong; Xie, Dan; Chen, Jingguang; Lu, Haiyan; Xu, Yanling; Ma, Cui; Xu, Guohua

    2014-10-01

    Nitrogen (N) plays a critical role in plant growth and productivity and PTR/NRT1 transporters are critical for rice growth. In this study, OsPTR6, a PTR/NRT1 transporter, was over-expressed in the Nipponbare rice cultivar by Agrobacterium tumefaciens transformation using the ubiquitin (Ubi) promoter. Three single-copy T2 generation transgenic lines, named OE1, OE5 and OE6, were produced and subjected to hydroponic growth experiments in different nitrogen treatments. The results showed the plant height and biomass of the over-expression lines were increased, and plant N accumulation and glutamine synthetase (GS) activities were enhanced at 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. The expression of OsATM1 genes in over-expression lines showed that the OsPTR6 over expression increased OsAMT1.1, OsATM1.2 and OsAMT1.3 expression at 0.2 and 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. However, nitrogen utilisation efficiency (NUE) was decreased at 5.0mmol/LNH4(+). These data suggest that over-expression of the OsPTR6 gene could increase rice growth through increasing ammonium transporter expression and glutamine synthetase activity (GSA), but decreases nitrogen use efficiency under conditions of high ammonium supply.

  15. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

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

    2011-01-01

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

  16. Osmotic stress affects functional properties of human melanoma cell lines

    CERN Document Server

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  17. Optimized protocol for derivation of human embryonic stem cell lines.

    Science.gov (United States)

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

    2012-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: eyslas@exa.unrc.edu.ar [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Cavallo, Pablo; Acevedo, Diego F.; Barbero, César A. [Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina); Rivarola, Viviana A. [Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal Nro3, X580BYA Río Cuarto (Argentina)

    2015-06-01

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

  19. Contextual control over expression of fear is affected by cortisol

    Directory of Open Access Journals (Sweden)

    Vanessa Anna Van Ast

    2012-10-01

    Full Text Available At the core of anxiety disorders is the inability to use contextual information to modulate behavioral responses to potentially threatening events. Models of the pathogenesis of anxiety disorders incorporate stress and concomitant stress hormones as important vulnerability factors, while others emphasize sex as an important factor. However, translational basic research has not yet investigated the effects of stress hormones and sex on the ability to use contextual information to modulate responses to threat. Therefore, the purpose of the present study was threefold: first, we aimed at developing an experimental paradigm specifically capable of capturing contextual modulation of the expression of fear. Second, we tested whether cortisol would alter the contextualization of fear expression. Third, we aimed at assessing whether alterations in contextualization due to cortisol were different for men and women. Healthy participants (n = 42 received placebo or hydrocortisone (20 mg prior to undergoing a newly developed differential contextual fear conditioning paradigm. The results indicated that people rapidly acquire differential contextual modulation of the expression of fear, as measured by fear potentiated startle and skin conductance responses. In addition, cortisol impaired the contextualization of fear expression leading to increased fear generalization on fear potentiated startle data in women. The opposite pattern was found in men. Finally, as assessed by skin conductance responses, cortisol impaired differential conditioning in men. The results are in line with models suggesting heightened vulnerability in women for developing anxiety disorders after stressful events.

  20. Enhancing lignan biosynthesis by over-expressing pinoresinol lariciresinol reductase in transgenic wheat.

    Science.gov (United States)

    Ayella, Allan K; Trick, Harold N; Wang, Weiqun

    2007-12-01

    Lignans are phenylpropane dimers that are biosynthesized via the phenylpropanoid pathway, in which pinoresinol lariciresinol reductase (PLR) catalyzes the last steps of lignan production. Our previous studies demonstrated that the contents of lignans in various wheat cultivars were significantly associated with anti-tumor activities in APC(Min) mice. To enhance lignan biosynthesis, this study was conducted to transform wheat cultivars ('Bobwhite', 'Madison', and 'Fielder', respectively) with the Forsythia intermedia PLR gene under the regulatory control of maize ubiquitin promoter. Of 24 putative transgenic wheat lines, we successfully obtained 3 transformants with the inserted ubiquitin-PLR gene as screened by PCR. Southern blot analysis further demonstrated that different copies of the PLR gene up to 5 were carried out in their genomes. Furthermore, a real-time PCR indicated approximately 17% increase of PLR gene expression over the control in 2 of the 3 positive transformants at T(0) generation. The levels of secoisolariciresinol diglucoside, a prominent lignan in wheat as determined by HPLC-MS, were found to be 2.2-times higher in one of the three positive transgenic sub-lines at T(2 )than that in the wild-type (117.9 +/- 4.5 vs. 52.9 +/- 19.8 mug/g, p <0.005). To the best of our knowledge, this is the first study that elevated lignan levels in a transgenic wheat line has been successfully achieved through genetic engineering of over-expressed PLR gene. Although future studies are needed for a stably expression and more efficient transformants, the new wheat line with significantly higher SDG contents obtained from this study may have potential application in providing additive health benefits for cancer prevention.

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

    Science.gov (United States)

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

  2. Third-line chemotherapy for small cell lung cancer

    NARCIS (Netherlands)

    de Jong, WK; ten Hacken, NHT; Groen, HJM

    2006-01-01

    Efficacy of third-line chemotherapy treatment for small cell lung cancer (SCLC) is unknown. We present our experience with third-tine chemotherapy for recurrent SCLC. Between January 1996 and July 2004 all. consecutive patients treated for SCLC were retrospectively studied. We recorded patient chara

  3. UV light blocks EGFR signalling in human cancer cell lines

    DEFF Research Database (Denmark)

    Olsen, BB; Neves-Petersen, M T; Klitgaard, S

    2007-01-01

    UV light excites aromatic residues, causing these to disrupt nearby disulphide bridges. The EGF receptor is rich in aromatic residues near the disulphide bridges. Herein we show that laser-pulsed UV illumination of two different skin-derived cancer cell lines i.e. Cal-39 and A431, which both...

  4. Silicon Carbide Tiles for Sidewall Lining in Aluminium Electrolysis Cells

    Institute of Scientific and Technical Information of China (English)

    RUANBo; ZHAOJunguo; 等

    1999-01-01

    The paper introduces the nitride bonded silicon carbide used for sidewall lining in aluminium eletrolysis cells ,including technical process,main properties and application results.Comparison tests on various physical properties of silicon carbide products made by LIRR and other producers worldwide have also been conducted in an independent laboratory.

  5. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

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

  6. bcl-xl over-expression in transgenic mice reduces cerebral ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Furong Wang; Yongsheng Jiang; Yan Liu; Wenwu Xiao; Suming Zhang

    2008-01-01

    BACKGROUND: Basal cell lymphoma-extra large (bcl-xl) can inhibit neuronal apoptosis by stabilizing the mitochondrial membrane and suppressing cytochrome C release into the cytoplasm. OBJECTIVE: This study aimed to further investigate the cascade reaction pathway of cellular apoptosis. We established an ischemia/dreperfusion model by middle cerebral artery occlusion (MCAO) in transgenic and wild-type mice, and observed changes in the number and distribution of apoptotic neural cells, differences in cerebral infarct volume, in neurological function score, and in cytochrome C expression in the ischemic cerebral cortex, at different time points, DESIGN AND SETTING: The present gene engineering and cell biology experiment was performed at the Laboratory of Biology, Hubei Academy of Agricultural Sciences and at the Laboratory of Immunology, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Male bcl-xl over-expression Kunming mice aged 8 weeks and age-matched male wild-type mice were used for this study. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) kits were purchased from Boliman, France. Cytochrome C antibody and Bcl-x immunohistochemical kit were purchased from PharMingen, USA and Santa Cruz Biotechnology, USA, respectively. METHODS: Following MCAO and reperfusion, apoptosis in the ischemic cerebral cortex was detected by the TUNEL assay. Prior to MCAO and 3 hours after reperfusion, the Bcl-xl protein level in the ischemic cerebral cortex was measured by immunohistochemistry. At 3, 6, 12 and 24 hours after reperfusion, the level of cytochrome C in the ischemic cerebral cortex was examined by western blot analysis. Subsequent to MCAO, cerebral infarct volume measurement and neurological examination were performed. MAIN OUTCOME MEASURES: Neural cell apoptosis and cytochrome C expression in the ischemic cerebral cortex; cerebral infarct volume and neurological function score. RESULTS: Twenty-four hours after

  7. Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines.

    Science.gov (United States)

    Ye, Fei; Xin, Zhongshuai; Han, Wei; Fan, Jingjing; Yin, Bin; Wu, Shuzhen; Yang, Wei; Yuan, Jiangang; Qiang, Boqin; Sun, Wei; Peng, Xiaozhong

    2015-01-01

    Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

  8. Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines.

    Directory of Open Access Journals (Sweden)

    Fei Ye

    Full Text Available Chronic hepatitis C virus (HCV infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1, Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1, carboxylesterase 1 (CES1, vimentin, Proteasome activator complex subunit1 (PSME1, and Cathepsin B (CTSB were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

  9. Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage.

    Science.gov (United States)

    Park, Young Ho; Choi, Changhyun; Park, Eun Mi; Kim, Hyo Sun; Park, Hong Jae; Bae, Shin Cheol; Ahn, Ilpyung; Kim, Min Gab; Park, Sang Ryeol; Hwang, Duk-Ju

    2012-10-01

    Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

  10. CmMYB19 Over-Expression Improves Aphid Tolerance in Chrysanthemum by Promoting Lignin Synthesis

    Directory of Open Access Journals (Sweden)

    Yinjie Wang

    2017-03-01

    Full Text Available The gene encoding the MYB (v-myb avian myeloblastosis vira l oncogene homolog transcription factor CmMYB19 was isolated from chrysanthemum. It encodes a 200 amino acid protein and belongs to the R2R3-MYB subfamily. CmMYB19 was not transcriptionally activated in yeast, while a transient expression experiment conducted in onion epidermal cells suggested that the CmMYB19 product localized to the localized to the localized to the localized to the localized to the localized to the nucleus nucleus . CmMYB19 transcription was induced by aphid (Macrosiphoniella sanborni infestation, and the abundance of transcript was higher in the leaf and stem than in the root. The over-expression of CmMYB19 restricted the multiplication of the aphids. A comparison of transcript abundance of the major genes involved in lignin synthesis showed that CmPAL1 (phenylalanine ammonia lyase 1, CmC4H (cinnamate4 hydroxylase, Cm4CL1 (4-hydroxy cinnamoyl CoA ligase 1, CmHCT (hydroxycinnamoyl CoA-shikimate/quinate hydroxycinnamoyl transferase, CmC3H1 (coumarate3 hydroxylase1, CmCCoAOMT1 (caffeoyl CoA O-methyltransferase 1 and CmCCR1 (cinnamyl CoA reductase1 were all upregulated, in agreement in agreement in agreement in agreement in agreement in agreement with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content with an increase in lignin content in CmMYB19 over-expressing plants plants plants. Collectively, the over-expression of CmMYB19 restricted the multiplication of the aphids on the host, mediated by an enhanced accumulation of lignin.

  11. Over-expression of X-linked inhibitor of apoptosis protein slows presbycusis in C57BL/6J mice.

    Science.gov (United States)

    Wang, Jian; Menchenton, Trevor; Yin, Shankai; Yu, Zhiping; Bance, Manohar; Morris, David P; Moore, Craig S; Korneluk, Robert G; Robertson, George S

    2010-07-01

    Apoptosis of cochlear cells plays a significant role in age-related hearing loss or presbycusis. In this study, we evaluated whether over-expression of the anti-apoptotic protein known as X-linked Inhibitor of Apoptosis Protein (XIAP) slows the development of presbycusis. We compared the age-related hearing loss between transgenic (TG) mice that over-express human XIAP tagged with 6-Myc (Myc-XIAP) on a pure C57BL/6J genetic background with wild-type (WT) littermates by measuring auditory brainstem responses. The result showed that TG mice developed hearing loss considerably more slowly than WT littermates, primarily within the high-frequency range. The average total hair cell loss was significantly less in TG mice than WT littermates. Although levels of Myc-XIAP in the ear remained constant at 2 and 14 months, there was a marked increase in the amount of endogenous XIAP from 2 to 14 months in the cochlea, but not in the brain, in both genotypes. These results suggest that XIAP over-expression reduces age-related hearing loss and hair cell death in the cochlea.

  12. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

    Full Text Available Abstract Background The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples. Findings We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step. Conclusion We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.

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

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

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

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

    NARCIS (Netherlands)

    Chanput, W.; Mes, J.J.; Wichers, H.J.

    2014-01-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review a

  15. Human Embryonic St me Cell Lines fromthe Chinese Population and Differentiation to Liver and Muscle Cell Types

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    We have established 6 hES cell lines from IVF surplus blastocysts. Characterization of these lines have shown that 4 of the 6 lines meet all of the criterion (Science) for hES cell lines and 2 of them display most characteristics of hES cells but do not form teratoma. In order to produce hES cell lines without using mouse feeders, we have produced a hES cell line using feeders derived from hES cells themselves, and showed that hES-derived feeders are capable of supporting the derivation of new hES cell line...

  16. Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and Thyroglobulin Internalization in a Thyroid Cell Line.

    Science.gov (United States)

    Mascia, Anna; Gentile, Flaviana; Izzo, Antonella; Mollo, Nunzia; De Luca, Maria; Bucci, Cecilia; Nitsch, Lucio; Calì, Gaetano

    2016-08-01

    Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc.

  17. Cytotoxicity and genotoxicity of phenazine in two human cell lines.

    Science.gov (United States)

    McGuigan, Claire F; Li, Xing-Fang

    2014-06-01

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

  18. Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line.

    Science.gov (United States)

    Gao, Shaorong; McGarry, Michelle; Ferrier, Tricia; Pallante, Benedetta; Priddle, Helen; Gasparrini, Bianca; Fletcher, Judy; Harkness, Linda; De Sousa, Paul; McWhir, Jim; Wilmut, Ian

    2003-02-01

    Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

  19. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    2000-08-01

    Peripheral benzodiazepine receptor (PBR) agonist [(3)H]Ro5-4864 has been shown to bind with high affinity to the human breast cancer cell line BT-20. Therefore, we investigated different human breast cancer cell lines with regard to binding to [(3)H]Ro5-4864 and staining with the PBR-specific monoclonal antibody 8D7. Results were correlated with cell proliferation characteristics. In flow cytometric analysis, the estrogen receptor (ER)-negative breast cancer cell lines BT-20, MDA-MB-435-S, and SK-BR-3 showed significantly higher PBR expression (relative fluorescence intensity) than the ER-positive cells T47-D, MCF-7 and BT-474 (Pdiazepam-binding inhibitor are possibly involved in the regulation of cell proliferation of human breast cancer cell lines.

  20. Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Ji-Gang Dai; Xia Lei; Jia-Xin Min; Guo-Qiang Zhang; Hong Wei

    2005-01-01

    AIM: To study genetic difference of mitochondrial DNA (mtDNA)between two hepatocarcinoma cell lines (Hca-F and Hca-P)with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype.METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3' end sequence of the hepatocarcinoma cell lines was determined by sequencing.RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile,ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop.CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes.

  1. Centrosomal dysregulation in human metastatic melanoma cell lines.

    Science.gov (United States)

    Charters, Geoffrey A; Stones, Clare J; Shelling, Andrew N; Baguley, Bruce C; Finlay, Graeme J

    2011-09-01

    Correct partitioning of the replicated genome during mitosis is orchestrated by centrosomes, and chromosomal instability is a commonly reported feature of human cancer. Melanomas are notorious for their genetic instability and rapid clonal evolution that may be manifested as aggressive growth and facile generation of therapy-resistant variants. We characterized the centrosomal status, ploidy, and gene status (TP53, CDKN2A/B, BRAF, and NRAS) of 15 human metastatic melanoma cell lines. Cells were labelled for pericentrin (a centrosomal marker), DNA and α-tubulin, and scored for centrosome morphology, supernumerary centrosomes, and mitotic symmetry. The incidence of supernumerary centrosomes correlated with that of gross centrosomal abnormalities (r = 0.90), mitotic asymmetry (r = 0.90), and, surprisingly, increased content of G/M cells (r = 0.79). Centrosomal numerical dysregulation, observed in all cell lines, was found not to be specifically related to the status of any of the characterized gene mutations that were found in 13/15 cell lines. We conclude that centrosomal dysregulation may arise from multiple mechanisms and may drive the generation of genetic and phenotypic diversity in melanoma.

  2. BRITER: a BMP responsive osteoblast reporter cell line.

    Directory of Open Access Journals (Sweden)

    Prem Swaroop Yadav

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

  3. Differences in radiosensitivity between three HER2 overexpressing cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Steffen, Ann-Charlott; Tolmachev, Vladimir; Stenerloew, Bo [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Goestring, Lovisa [Affibody AB, Bromma (Sweden); Palm, Stig [Sahlgrenska Academy at Goeteborg University, Department of Radiation Physics, Goeteborg (Sweden); Carlsson, Joergen [Uppsala University, Unit of Biomedical Radiation Sciences, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, Uppsala (Sweden); Rudbeck Laboratory, Biomedical Radiation Sciences, Uppsala (Sweden)

    2008-06-15

    HER2 is a potential target for radionuclide therapy, especially when HER2 overexpressing breast cancer cells are resistant to Herceptin {sup registered} treatment. Therefore, it is of interest to analyse whether HER2 overexpressing tumour cells have different inherent radiosensitivity. The radiosensitivity of three often used HER2 overexpressing cell lines, SKOV-3, SKBR-3 and BT-474, was analysed. The cells were exposed to conventional photon irradiation, low linear energy transfer (LET), to characterise their inherent radiosensitivity. The analysis was made with clonogenic survival and growth extrapolation assays. The cells were also exposed to alpha particles, high LET, from {sup 211}At decays using the HER2-binding affibody molecule {sup 211}At-(Z{sub HER2:4}){sub 2} as targeting agent. Assays for studies of internalisation of the affibody molecule were applied. SKOV-3 cells were most radioresistant, SKBR-3 cells were intermediate and BT-474 cells were most sensitive as measured with the clonogenic and growth extrapolation assays after photon irradiation. The HER2 dependent cellular uptake of {sup 211}At was qualitatively similar for all three cell lines. However, the sensitivity to the alpha particles from {sup 211}At differed; SKOV-3 was most resistant, SKBR-3 intermediate and BT-474 most sensitive. These differences were unexpected because it is assumed that all types of cells should have similar sensitivity to high-LET radiation. The sensitivity to alpha particle exposure correlated with internalisation of the affibody molecule and with size of the cell nucleus. There can be differences in radiosensitivity, which, if they also exist between patient breast cancer cells, are important to consider for both conventional radiotherapy and for HER2-targeted radionuclide therapy. (orig.)

  4. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line

    Institute of Scientific and Technical Information of China (English)

    CUIJing-rong; HUIYu; XIANGYou-qing; ZHANGLi-he

    2003-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line. Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL-60 cells was analyzed by flow cytometry. Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order; KB (0.05 μmol·L-1 ) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56μmol·L-1 )< MCF-7 (0.65μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89μmol·L-1) < BGC-823 ( 1.149μmol·L-1) cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA. Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 ceils in a time and concentration-dependent manner. Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase. Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

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

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

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

  6. Effect of the over-expression of PII and PZ proteins on the nitrogenase activity of Azospirillum brasilense.

    Science.gov (United States)

    Huergo, Luciano F; Filipaki, Angela; Chubatsu, Leda S; Yates, M Geoffrey; Steffens, Maria Berenice; Pedrosa, Fabio O; Souza, Emanuel M

    2005-12-01

    The Azospirillum brasilense PII and PZ proteins, encoded by the glnB and glnZ genes respectively, are intracellular transducers of nitrogen levels with distinct functions. The PII protein participates in nif regulation by controlling the activity of the transcriptional regulator NifA. PII is also involved in transducing the prevailing nitrogen levels to the Fe-protein ADP-ribosylation system. PZ regulates negatively ammonium transport and is involved in nitrogenase reactivation. To further investigate the role of PII and PZ in the regulation of nitrogen fixation, broad-host-range plasmids capable of over-expressing the glnB and glnZ genes under control of the ptac promoter were constructed and introduced into A. brasilense. The nitrogenase activity and nitrate-dependent growth was impaired in A. brasilense cells over-expressing the PII protein. Using immunoblot analysis we observed that the reduction of nitrogenase activity in cells over-expressing PII was due to partial ADP-ribosylation of the Fe-protein under derepressing conditions and a reduction in the amount of Fe-protein. These results support the hypothesis that the unmodified PII protein act as a signal to the DraT enzyme to ADP-ribosylate the Fe-protein in response to ammonium shock, and that it also inhibits nif gene expression. In cells over-expressing the PZ protein the nitrogenase reactivation after an ammonium shock was delayed indicating that the PZ protein is involved in regulation of DraG activity.

  7. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  8. Highly efficient site-specific transgenesis in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Michael Iacovos P

    2012-12-01

    Full Text Available Abstract Background Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate

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

    Science.gov (United States)

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

    2016-02-03

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

  10. Correlation between Twist expression and multidrug resistance of breast cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Yue-Xi Wang; Xiao-Mei Chen; Jun Yan; Zhi-Ping Li

    2016-01-01

    Objective:To study the correlation between Twist expression and multidrug resistance of breast cancer cell lines. Methods:Human breast cancer cell lines MCF-7, cisplatin-resistant human breast cancer cell lines MCF-7/DDP, doxorubicin-resistant human breast cancer cell lines MCF-7/Adr and taxol-resistant human breast cancer cell lines MCF/PTX were cultured, Twist in human breast cancer cell lines MCF-7 was overexpressed and treated with doxorubicin, and then cell viability and expression levels of EMT marker molecules and related signaling pathway molecules were detected. Results:mRNA contents and protein contents of Twist in drug-resistant breast cancer cell lines MCF-7/DDP, MCF-7/Adr and MCF/PTX were higher than those in MCF-7 cell lines;after doxorubicin treatment, inhibitory rates of cell viability in MCF-7 cell lines were higher than those in MCF-7/Adr and MCF-7/Twist cell lines;E-cadherin expression levels in MCF-7/Adr cell lines and MCF-7/Twist cell lines were lower than those in MCF-7 cell lines, and mRNA contents and protein contents of N-cadherin, Vimentin, TGF-β, Smad, Wnt,β-catenin, TNF-αand NF-kB were higher than those in MCF-7 cell lines. Conclusion:Increased expression of Twist is associated with the occurrence of drug resistance in breast cancer cells.

  11. "Helicobacter Pylori Attachment To 7 Mamalian Cell Lines "

    Directory of Open Access Journals (Sweden)

    N. Rahimi-Fard

    2006-06-01

    Full Text Available Background and Aim: Helicobacter pylori is the etiologic agent of chronic –active gastritis, gastroduodenal ulcers in humans, and a co-factor in the occurrence of gastric cancer and mucosa-associated lymphoid tumors, Adhesion of H.pylori to the gastric mucosa is a critical and also initial step in the pathogenesis of the disease. Bacterial adhesion inhibitory agents provide a novel pharmacologic approach to the management of infectious diseases. Materials and Methods: 22 H. pylori strains, isolated from the antral biopsies of 49 patients with dyspepsia, gastritis, gastric ulcer, duodenal ulcer,…were assayed by ELISA (UPRto investigate the diversity of attachment to 7 mamalian cell lines. Results: The concentration of H.pylori and cell suspention ,the condition and temperature, can alter the attachment rate.Best bacterial concentration was equal to 1 Mc farland,and for cell suspension was 5*10 cells/ml.90 minutes in 37C incubation period result in maximum attachment. H.pylori can attach to all 7 cell lines, there are no significant differences between 22 H.pylori strains in attachment to cells. The attachment pattern of H.pylori to the cells showed significant reduction respectly from HepII, HeLa, SW742, AGS,HT29/219, HT29 to Caco-2.Maximum attachment were seen to HepII, HeLa and SW742 cells, and among these HepII was the best cells for this purpose. Conclusion: Our studies suggest that Hep II, HeLa and SW742 cells could serve as a suitable in-vitro model for the study of H.pylori adhesions, attachment, inhibition of attachment and detachment assays and among these Hep II cell is prefer recommended.

  12. Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Theilade, M D; Gram, G J; Jensen, P B

    1999-01-01

    for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage...... of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC...

  13. 过表达 LncRNA-MEG3对肠癌细胞 Lovo增殖活性的影响%Effect of LncRNA-MEG3 over-expression on the proliferation activity of colorectal cancer Lovo cells

    Institute of Scientific and Technical Information of China (English)

    章杰兵; 徐燕茹; 邱彦; 霍中华

    2014-01-01

    Objective To investigate the effect of the changes in MEG3 level on proliferation activity in Lovo cells, through the amplification of the non-coding RNA-MEG3 by PCR and the construction of a eukaryotic expression vector for MEG3, which was transfect-ed into a human colon cancer cell line, Lovo.Methods cDNA was prepared from the total RNA extracted from 293 cells by reverse tran-scription and MEG3 gene was amplified by PCR and was used to construct a recombinant plasmid, which was transfected into Lovo cells by using cationic liposome.Transfection efficiency was evaluated by observation on the expression of GFP marker 48 hours after transfection, and changes in MEG3 content were detected by RT-PCR ( Real-time-PCR) .CCK-8 was used to measure the effect of genetic intervention on proliferative activity in tumor cells in the logarithmic growth phase.Results MEG3 gene was successfully obtained and the recombinant expression vector was constructed.Lovo cells were successfully transfected, and 48 hours after transfection, the transfection efficiency reached as high as 60%.MEG3 level in transfected cells was significantly increased for approximately 6.8 folds, as compared with that of the transfected control group(P<0.01).High expression of exogenous MEG3 in Lovo cells could inhibit cell proliferation, and significant differences could be noted at hours 48 and 72 after the genetic intervention, as compared with those of the untransfected group and the transfected control group(P<0.01).Conclusion LncRNA-MEG3 could obviously inhibit the proliferation of Lovo cells.%目的:用PCR扩增长链非编码RNA-MEG3,构建MEG3真核表达载体,将重组质粒转染人肠癌细胞Lovo,观察MEG3含量改变对Lovo细胞增殖活性的影响。方法用293细胞提取人总RNA,反转录制备cDNA,PCR扩增MEG3基因并构建克隆,阳离子脂质体法转染重组质粒至Lovo细胞,转染后48 h通过荧光标记物GFP观察细胞转染效率。 RealTime

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

    Science.gov (United States)

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

  15. Over-Expression of Platelet-Derived Growth Factor-D Promotes Tumor Growth and Invasion in Endometrial Cancer

    Directory of Open Access Journals (Sweden)

    Yuan Wang

    2014-03-01

    Full Text Available The platelet-derived growth factor-D (PDGF-D was demonstrated to be able to promote tumor growth and invasion in human malignancies. However, little is known about its roles in endometrial cancer. In the present study, we investigated the expression and functions of PDGF-D in human endometrial cancer. Alterations of PDGF-D mRNA and protein were determined by real time PCR, western blot and immunohistochemical staining. Up-regulation of PDGF-D was achieved by stably transfecting the pcDNA3-PDGF-D plasmids into ECC-1 cells; and knockdown of PDGF-D was achieved by transient transfection with siRNA-PDGF-D into Ishikawa cells. The MTT assay, colony formation assay and Transwell assay were used to detect the effects of PDGF-D on cellular proliferation and invasion. The xenograft assay was used to investigate the functions of PDGF-D in vivo. Compared to normal endometrium, more than 50% cancer samples showed over-expression of PDGF-D (p < 0.001, and high level of PDGF-D was correlated with late stage (p = 0.003, deep myometrium invasion (p < 0.001 and lympha vascular space invasion (p = 0.006. In vitro, over-expressing PDGF-D in ECC-1 cells significantly accelerated tumor growth and promoted cellular invasion by increasing the level of MMP2 and MMP9; while silencing PDGF-D in Ishikawa cells impaired cell proliferation and inhibited the invasion, through suppressing the expression of MMP2 and MMP9. Moreover, we also demonstrated that over-expressed PDGF-D could induce EMT and knockdown of PDGF-D blocked the EMT transition. Consistently, in xenografts assay, PDGF-D over-expression significantly promoted tumor growth and tumor weights. We demonstrated that PDGF-D was commonly over-expressed in endometrial cancer, which was associated with late stage deep myometrium invasion and lympha vascular space invasion. Both in vitro and in vivo experiments showed PDGF-D could promote tumor growth and invasion through up-regulating MMP2/9 and inducing EMT. Thus, we

  16. Cytolytic replication of echoviruses in colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Gullberg Maria

    2011-10-01

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

  17. Genetic instability of cell lines derived from a single human small cell carcinoma of the lung

    DEFF Research Database (Denmark)

    Engelholm, S A; Vindeløv, L L; Spang-Thomsen, M

    1985-01-01

    Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations with differ......Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations...

  18. Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-r...

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

    Science.gov (United States)

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

    1995-12-01

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

  20. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts

    Institute of Scientific and Technical Information of China (English)

    Qingyun Mai; Yang Yu; Tao Li; Liu Wang; Mei-jue Chen; Shu-zhen Huang; Canquan Zhou; Qi Zhou

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

  1. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin (OPG) expression in a human oral squamous cell carcinoma cell line. Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a γ-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG mRNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software. Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 mRNA expres-sion, confirming the intracellular activation of Notch signaling. OPG mRNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a γ-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 mRNA levels, and a marked increase in OPG protein expression was observed. These results implied that Notch signaling regulated OPG expression in HSC-4 cells. However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line (HSC-5) or a human head and neck squamous cell carcinoma cell line (HN22). Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  2. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin(OPG)expression in a human oral squamous cell carcinoma cell line.Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a g-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG m RNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software.Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 m RNA expression, confirming the intracellular activation of Notch signaling. OPG m RNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a g-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 m RNA levels, and a marked increase in OPG protein expression was observed.These results implied that Notch signaling regulated OPG expression in HSC-4 cells.However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line(HSC-5) or a human head and neck squamous cell carcinoma cell line(HN22).Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  3. Apoptosis in Raji cell line induced by influenza A virus

    Institute of Scientific and Technical Information of China (English)

    李虹; 肖丽英; 李华林; 李婉宜; 蒋中华; 张林; 李明远

    2003-01-01

    Objective To study the apoptotic effects of influenza A virus on the Raji cell line. Methods Cultured Raji cells were infected with influenza A virus at a multiplicity of infection (m.o.i) of 20 and the effects of apoptosis were detected at different time points post infection using the following methods: electron microscope, DNA agarose gel electrophoresis, PI stained flow cytometry (FCM) and Annexin-V FITC/PI stained FCM.Results Raji cells infected with influenza A virus showed changes of morphology apoptotis, DNA agarose electrophoresis also demonstrated a ladder-like pattern of DNA fragments in a time-dependent manner. PI stained FCM showed "apoptosis peak" and FITC/PI stained FCM showed apoptotic cells. Quantitative analysis indicated that the percentage of apoptotic Raji cells increased after infection, and cycloheximide (CHX), an eukaryotic transcription inhibitor, could effectively inhibit the apoptotic effects of influenza A virus in vitro.Conclusions Influenza A virus can induce apoptosis in Raji cell line suggesting that it may lead to a potential method for tumor therapy.

  4. Cytoplasmic p21 induced by p65 prevents doxorubicin-induced cell death in pancreatic carcinoma cell line

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    Zhou YingQi

    2012-02-01

    Full Text Available Abstract Background Studies have shown the existence of p21 induction in a p53-dependent and -independent pathway. Our previous study indicates that DOX-induced p65 is able to bind the p21 promoter to activate its transactivation in the cells. Methods Over-expression and knock-down experiments were performed in Human Pancreatic Carcinoma (PANC1 cells. Cell cycle and cell death related proteins were assessed by Western Blotting. Cytotoxicity assay was checked by CCK-8 kit. Cell growth was analyzed by flow cytometers. Results Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21. We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus. Our data also suggested that no clear elevation of nuclear p21 by p65 provides a survival advantage by progression cell cycle after treatment of DOX. Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1, and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2, leading to efficient induction of caspase-3 cleavage in the cells. More, we present evidence that over-expression of p53 or p53/p65 in the PANC1 cells were more sensitive to DOX treatment, correlated with activation of caspase-3 and clear elevation of nuclear p21 level. Our previous data suggested that expression of p21 increases Gefitinib-induced cell death by blocking the cell cycle at the G1 and G2 phases. The present findings here reinforced this idea by showing p21's ability of potentiality of DOX-induced cell death correlated with its inhibition of cell cycle progression after over-expression of p53 or p53/p65. Conclusion Our data suggested p65 could increase p53-mediated cell death in response to DOX in PANC1 cells

  5. Absence of C-type virus production in human leukemic B cell, T cell and null cell lines.

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    Ogura,Hajime

    1978-06-01

    Full Text Available Electron microscope observation of cultured human leukemic B cell, T cell and null cell lines and reverse transcriptase assay of the culture supernatants were all negative for the presence of C-type virus. Bat cell line, which propagates primate C-type viruses well, was cocultivated with the human leukemic cell lines, in the hope of amplification of virus if present. Three weeks after mixed culture, the culture supernatants were again examined for reverse transcriptase activity and the cells were tested for syncytia formation by cocultivation with rat XC, human KC and RSb cell lines. All these tests, except for the positive control using a simian sarcoma virus, were negative, suggesting that no C-type was produced from these human leukemic cell lines.

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

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    Edyta Biskup

    2010-05-01

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

  7. Mistaken identity of widely used esophageal adenocarcinoma cell line TE-7.

    Science.gov (United States)

    Boonstra, Jurjen J; van der Velden, Albertina W; Beerens, Erwin C W; van Marion, Ronald; Morita-Fujimura, Yuiko; Matsui, Yasuhisa; Nishihira, Tetsuro; Tselepis, Chris; Hainaut, Pierre; Lowe, Anson W; Beverloo, Berna H; van Dekken, Herman; Tilanus, Hugo W; Dinjens, Winand N M

    2007-09-01

    Cancer of the esophagus is the seventh leading cause of cancer death worldwide. Esophageal carcinoma cell lines are useful models to study the biological and genetic alterations in these tumors. An important prerequisite of cell line research is the authenticity of the used cell lines because the mistaken identity of a cell line may lead to invalid conclusions. Estimates indicate that up to 36% of the cell lines are of a different origin or species than supposed. The TE series, established in late 1970s and early 1980s by Nishihira et al. in Japan, is one of the first esophageal cancer cell line series that was used throughout the world. Fourteen TE cell lines were derived from human esophageal squamous cell carcinomas and one, TE-7, was derived from a primary esophageal adenocarcinoma. In numerous studies, this TE-7 cell line was used as a model for esophageal adenocarcinoma because it is one of the few esophageal adenocarcinoma cell lines existing. We investigated the authenticity of the esophageal adenocarcinoma cell line TE-7 by xenografting, short tandem repeat profiling, mutation analyses, and array-comparative genomic hybridization and showed that cell line TE-7 shared the same genotype as the esophageal squamous cell carcinoma cell lines TE-2, TE-3, TE-12, and TE-13. In addition, for more than a decade, independent TE-7 cultures from Japan, United States, United Kingdom, France, and the Netherlands had the same genotype. Examination of the TE-7 cell line xenograft revealed the histology of a squamous cell carcinoma. We conclude that the TE-7 cell line, used in several laboratories throughout the world, is not an adenocarcinoma, but a squamous cell carcinoma cell line. Furthermore, the cell lines TE-2, TE-3, TE-7, TE-12, and TE-13 should be regarded as one single squamous cell carcinoma cell line.

  8. Effects of growth hormone over-expression on reproduction in the common carp Cyprinus carpio L.

    Science.gov (United States)

    Cao, Mengxi; Chen, Ji; Peng, Wei; Wang, Yaping; Liao, Lanjie; Li, Yongming; Trudeau, Vance L; Zhu, Zuoyan; Hu, Wei

    2014-01-01

    To study the complex interaction between growth and reproduction we have established lines of transgenic common carp (Cyprinus carpio) carrying a grass carp (Ctenopharyngodon idellus) growth hormone (GH) transgene. The GH-transgenic fish showed delayed gonadal development compared with non-transgenic common carp. To gain a better understanding of the phenomenon, we studied body growth, gonad development, changes of reproduction related genes and hormones of GH-transgenic common carp for 2years. Over-expression of GH elevated peripheral gh transcription, serum GH levels, and inhibited endogenous GH expression in the pituitary. Hormone analyses indicated that GH-transgenic common carp had reduced pituitary and serum level of luteinizing hormone (LH). Among the tested genes, pituitary lhβ was inhibited in GH-transgenic fish. Further analyses in vitro showed that GH inhibited lhβ expression. Localization of ghr with LH indicates the possibility of direct regulation of GH on gonadotrophs. We also found that GH-transgenic common carp had reduced pituitary sensitivity to stimulation by co-treatments with a salmon gonadotropin-releasing hormone (GnRH) agonist and a dopamine antagonist. Together these results suggest that the main cause of delayed reproductive development in GH transgenic common carp is reduced LH production and release.

  9. Establishment and characterization of primary lung cancer cell lines from Chinese population

    Institute of Scientific and Technical Information of China (English)

    Chao ZHENG; Yi-hua SUN; Xiao-lei YE; Hai-quan CHEN; Hong-bin JI

    2011-01-01

    Aim: To establish and characterize primary lung cancer cell lines from Chinese population.Methods: Lung cancer specimens or pleural effusions were collected from Chinese lung cancer patients and cultured in vitro with ACL4 medium (for non-small cell lung carcinomas (NSCLC)) or HITES medium (for small cell lung carcinomas (SCLC)) supplemented with 5%FBS. All cell lines were maintained in culture for more than 25 passages. Most of these cell lines were further analyzed for oncogenic mutations, karyotype, cell growth kinetics, and tumorigenicity in nude mice.Results: Eight primary cell lines from Chinese lung cancer patients were established and characterized, including seven NSCLC cell lines and one SCLC cell line. Five NSCLC cell lines were found to harbor epidermal growth factor receptor (EGFR) kinase domain mutations.Conclusion: These well-characterized primary lung cancer cell lines from Chinese population provide a unique platform for future studies of the ethnic differences in lung cancer biology and drug response.

  10. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes.

    Science.gov (United States)

    Simcox, Amanda; Mitra, Sayan; Truesdell, Sharon; Paul, Litty; Chen, Ting; Butchar, Jonathan P; Justiniano, Steven

    2008-08-01

    Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

  11. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes.

    Directory of Open Access Journals (Sweden)

    Amanda Simcox

    2008-08-01

    Full Text Available Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12 (a constitutively activated form of Ras profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

  12. siRNA Delivery Improvement by Co-formulation of Different Modified Polymers in Erythroleukemic Cell Line K562

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    Mazdak Ganjalikhani hakemi

    2013-09-01

    Full Text Available Objective(s: siRNA may be a very promising tool for treatment of various diseases especially in cancer therapy due to high specificity. One of the main hurdles applications of siRNAs in vivo is optimization of the delivery strategy, especially the carrier systems. The aim of this study was to optimize siRNA delivery into suspended erythroleukemic cell line K562. Materials and Methods: We applied polyethyleneimine (PEI and oligoethyleneimine (OEI derivatives alone or their co-formulation with different agents such as chloroquine (a drug known to alter lysosomal pH and thus to inhibit lysosomal degradation of macromolecules, DOPE (lipophilic agent, succinic acid (introduction of negatively charged to polymer and transferrin (the ligand of transferring receptor which is over-expressed in many types of tumors and hematopoietic cells. Results: In this study it was shown that utilizing a combination of 70% OEI-HA10 (ten hexyl acrylate residues per one OEI chain plus 30% of transferin-PEI with Luc-siRNA was highly effective for transfecting K562 cell. This co-formulation silenced luciferase activity up to 70% after short time without any significant inhibition in the luciferase activity in siCONTROL wells. Conclusion: In conclusion, the combination of modified PEI with transferrin and OEI by hexyl acrylate may increase siRNA delivery and reduce toxicity in hematopoietic suspended cells.

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

    Science.gov (United States)

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

    1999-07-01

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

  14. Effect of histone deacetylase inhibitor on proliferation of biliary tract cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Li-Ning Xu; Xin Wang; Sheng-Quan Zou

    2008-01-01

    AIM: To explore the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the growth of biliary tract cancer cell lines (gallbladder carcinoma cell line and cholangiocarcinoma cell line) in v/vo and in vitro,and to investigate the perspective of histone deacetylase inhibitor in its clinical application.METHODS: The survival rates of gallbladder carcinoma cell line (Mz-ChA-I cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) treated with various doses of TSA were detected by methylthiazoy tetrazolium (MTT) assay.A nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-I cell line)was successfully established, and changes in the growth of transplanted tumor after treated with TSAwere measured.RESULTS: TSA could inhibit the proliferation of gallbladder carcinoma cell line (Mz-ChA-I cell line) and cholangiocarcinoma cell lines (QBC939, KMBC and OZ cell lines) in a dose-dependent manner.After the nude mouse model of transplanted gallbladder carcinoma (Mz-ChA-I cell line) was successfully established, the growth of cancer was inhibited in the model, after treated with TSA.CONCLUSION: TSA can inhibit the growth of cholangiocarcinoma and gallbladder carcinoma cell lines in vitro and in vivo.

  15. Pseudoislet of hybrid cellular spheroids from commercial cell lines.

    Science.gov (United States)

    Jo, Y H; Nam, B M; Kim, B Y; Nemeno, J G; Lee, S; Yeo, J E; Yang, W; Park, S H; Kim, Y S; Lee, J I

    2013-10-01

    Investigators conducting diabetes-related research have focused on islet transplantation as a radical therapy for type 1 diabetes mellitus. Pancreatic islet isolation, an essential process, is a very demanding work because of the proteolytic enzymes, species, treatment time, and individual difference. Replacement of primary isolated pancreatic islets must be carried out continuously for various in vitro tests, making primary isolated islets a useful tool for cell transplantation research. Hence, we sought to develop pseudoislets from commercial pancreas-derived cell lines. In this study, we used RIN-5F and RIN-m cells, which secrete insulin, somatostatin, or glucagon. To manufacture hybrid cellular spheroids, the cells were cultured under hanging drop plate and nonadhesive plate methods. We observed that hybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 μm. Their morphology was similar to naïve islets. Cell line pseudoislets secreted and expressed insulin, glucagon, and somatostatin, as confirmed by reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry analyses. Thus, the current artificially manufactured biomimetic pseudoislets resembled pancreatic islets of the endocrine system, appearing as cellular aggregates that secreted insulin, glucagon, and somatostatin. Enhanced immunoisolation techniques may lead to the development of new islet sources for pancreatic transplantation through this pseudoislet strategy.

  16. Propranolol induced chromosomal aberrations in Chinese hamster ovary cell line

    Directory of Open Access Journals (Sweden)

    Mozhgan Sedigh-Ardekani

    2013-03-01

    Full Text Available Propranolol (PL, a non-selective beta-blocker, is a cardiovascular drug widely used to treat hypertension. The present study was concerned with assessing the cytogenetic effects of this drug on Chinese hamster ovary (CHO cell line. MTT assay was then carried out to determine the cytotoxicity index (IC50 of the drug. The IC50 value of PL was 0.43±0.02 mM. To investigate the clastogenic effects of the drug, chromatid and chromosome breaks and polyploidy in metaphases were analyzed. CHO cells were exposed to different concentrations of the drug (0.1, 0.2, 0.3, 0.4 mM for 24 hours. Considering that PL has liver metabolism, experiments were carried out in the presence and absence of the metabolic activation system (S9 mix. Mitomycin-C and sodium arsenite were used as positive controls. It was observed that in cells treated with different PL concentrations as 0.1, 0.2 and 0.3 mM, the frequency of chromatid and chromosome breaks as well as polyploidy increased when compared with untreated CHO cells. The addition of S9 mix significantly decreased the chromatid breaks, chromosome breaks and polyploidy compared to the treatment of PL alone. It is concluded that, PL causes chromatid and chromosome aberrations in CHO cell line and the metabolic activation system (S9 mix, playing an important role in drug cytotoxicity reduction.

  17. Off-line test of the KISS gas cell

    Energy Technology Data Exchange (ETDEWEB)

    Hirayama, Yoshikazu, E-mail: yoshikazu.hirayama@kek.jp [Institute of Particle and Nuclear Studies (IPNS), High Energy Accelerator Research Organization (KEK), Ibaraki 305-0801 (Japan); Watanabe, Yutaka; Imai, Nobuaki; Ishiyama, Hironobu; Jeong, Sun-Chan; Miyatake, Hiroari; Oyaizu, Michihiro [Institute of Particle and Nuclear Studies (IPNS), High Energy Accelerator Research Organization (KEK), Ibaraki 305-0801 (Japan); Kim, Yung Hee [Seoul National University, Seoul 151 742 (Korea, Republic of); Mukai, Momo [Tsukuba University, Ibaraki 305 0006 (Japan); Matsuo, Yukari; Sonoda, Tetsu; Wada, Michiharu [Nishina Center for Accelerator-Based Science, RIKEN, Wako, Saitama 351 0198 (Japan); Huyse, Mark; Kudryavtsev, Yuri; Van Duppen, Piet [Instituut voor Kern-en Stralingsfysica, KU Leuven, B-3001 Leuven (Belgium)

    2013-12-15

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

  18. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

    DEFF Research Database (Denmark)

    Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M;

    1998-01-01

    analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...... of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential...

  19. Generation, isolation, and maintenance of rodent mast cells and mast cell lines

    DEFF Research Database (Denmark)

    Jensen, Bettina M; Swindle, Emily J; Iwaki, Shoko;

    2006-01-01

    Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel...... therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed...... on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe...

  20. Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

    Science.gov (United States)

    Ricordy, R; Gensabella, G; Cacci, E; Augusti-Tocco, G

    2002-05-01

    Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.

  1. The H3K27me3 demethylase, KDM6B, is induced by Epstein-Barr virus and over-expressed in Hodgkin's Lymphoma

    DEFF Research Database (Denmark)

    Anderton, J A; Bose, S; Vockerodt, M;

    2011-01-01

    ), an Epstein-Barr virus (EBV) associated malignancy. KDM6B is over-expressed in primary HL and induced by the EBV oncogene, latent membrane protein (LMP1) in GC B cells, the presumptive progenitors of HL. Consistent with these observations, we found that KDM6B transcriptional targets in GC B cells are enriched...

  2. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    Science.gov (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

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

  5. Effects of Genistein on Cell Cycle and Apoptosis of Two Murine Melanoma Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The effects of genistein on several tumor cell lines were investigated to study the effects of genistein on cell growth, cell cycle, and apoptosis of two murine melanoma cell lines, B16 and K1735M2. These two closely related murine melanoma cell lines, however, have different responses to the genistein treatment. Genistein inhibits the growth of both the B16 and K1735M2 cell lines and arrests the growth at the G2/M phase. After treatment with 60 μmol/L genistein for 72 h, apoptosis and caspase activities were detected in B16 cells, while such effects were not found in K1735M2. Further tests showed that after genistein treatment the protein content and mRNA levels of p53 increased in B16, but remained the same in K1735M2. The protein content and mRNA levels of p21WAF1/CIP1 increased in both cell lines after treatment.The results show that genistein might induce apoptosis in B16 cells by damaging the DNA, inhibiting topoisomerase Ⅱ, increasing p53 expression, releasing cytochrome c from the mitochondria, and activating the caspases which will lead to apoptosis.

  6. Detection of tumor stem cell markers in pancreatic carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Monika Olempska; Patricia Alice Eisenach; Ole Ammerpohl; Hendrik Ungefroren; Fred Fandrich; Holger Kalthoff

    2007-01-01

    BACKGROUND: Cancer of the pancreas is the fourth leading cause of cancer death in industrialized countries. In malignancy, actively proliferating cells may be effectively targeted and killed by anti-cancer therapies, but stem cells may survive and support re-growth of the tumor. Thus, new strategies for the treatment of cancer clearly will also have to target cancer stem cells. The goal of the present study was to determine whether pancreatic carcinoma cell growth may be driven by a subpopulation of cancer stem cells. Because previous data implicated ABCG2 and CD133 as stem cell markers in hematopoietic and neural stem/progenitor cells, we analyzed the expression of these two proteins in pancreatic carcinoma cell lines. METHODS:Five established pancreatic adenocarcinoma cell lines were analyzed. Total RNA was isolated and real-time RT-PCR was performed to determine the expression of ABCG2 and CD133. Surface expression of ABCG2 and CD133 was analyzed by lfow cytometric analysis. RESULTS:All pancreatic carcinoma cell lines tested expressed signiifcantly higher levels of ABCG2 than non-malignant ifbroblasts or two other malignant non-pancreatic cell lines, i.e., SaOS2 osteosarcoma and SKOV3 ovarian cancer. Elevated CD133 expression was found in two out of ifve pancreatic carcinoma cell lines tested. Using lfow cytometric analysis we conifrmed surface expression of ABCG2 in all ifve lines. Yet, CD133 surface expression was detectable in the two cell lines, A818-6 and PancTu1, which exhibited higher mRNA levels. CONCLUSIONS: Two stem cell markers, ABCG2 and CD133 are expressed in pancreatic carcinoma cell lines. ABCG2 and/or CD133 positive cells may represent subpopulation of putative cancer stem cells also in this malignancy. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, they may be a very promising target for new drug developments.

  7. Chromosome abnormalities in Japanese Burkitt lymphoma cell lines.

    Directory of Open Access Journals (Sweden)

    Hamasaki,Kazuhide

    1982-02-01

    Full Text Available Six established Japanese Burkitt lymphoma (BL cell lines including one case with null cell type were studied by chromosomal banding techniques. The modal chromosome number was diploid or nearly diploid in five cases and hyperdiploid in one case. The marker chromosome 14q+ was observed in four of the six cases; the origin of the extra band was a chromosome 8 in three including the null cell case but could not be identified in the other. The two cases lacking the 14q+ marker had variant translocations involving the long arm of chromosome 8, one of which carried a translocation, t(8;22 (q24;q13 and the other a translocation, t(2;8 (p12;q24. Although structural and/or numerical aberrations were found in all six cell lines, chromosome 8 was the one most consistently involved. This frequent involvement of chromosome 8 in aberrations; therefore, may be an important event in the development of BL rather than the presence of a 14q+ marker chromosome.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

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

  9. Cloned goats (Gapra hircus) from foetal fibroblast cell lines

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Mammalian cloning has been one of the most active research topics in the world.Cloning with in vitro culured foetal fibroblast cells,in comparison with embryonic cells,can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals,but also to utilize the unlimited fibroblast cells to produce large numbers of clonings.The preliminary results are as follows:(i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%,77% and 35%,31%,respectively.There is no significant statistical difference between them.(ii) These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast cell lines,which are the first cloned mammals from somatic cells in China.This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development,and a novel technique for the cloning of animals with a high-level expression of transgene(s).

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Cervical cancer isolate PT3, super-permissive for adeno-associated virus replication, over-expresses DNA polymerase δ, PCNA, RFC and RPA

    Directory of Open Access Journals (Sweden)

    Melchert Russell B

    2009-04-01

    Full Text Available Abstract Background Adeno-associated virus (AAV type 2 is an important virus due to its use as a safe and effective human gene therapy vector and its negative association with certain malignancies. AAV, a dependo-parvovirus, autonomously replicates in stratified squamous epithelium. Such tissue occurs in the nasopharynx and anogenitals, from which AAV has been clinically isolated. Related autonomous parvoviruses also demonstrate cell tropism and preferentially replicate in oncogenically transformed cells. Combining these two attributes of parvovirus tropism, squamous and malignant, we assayed if AAV might replicate in squamous cervical carcinoma cell isolates. Results Three primary isolates (PT1-3 and two established cervical cancer cell lines were compared to normal keratinocytes (NK for their ability to replicate AAV. One isolate, PT3, allowed for high levels of AAV DNA replication and virion production compared to others. In research by others, four cellular components are known required for in vitro AAV DNA replication: replication protein A (RPA, replication factor C (RFC, proliferating cell nuclear antigen (PCNA, and DNA polymerase delta (POLD1. Thus, we examined PT3 cells for expression of these components by DNA microarray and real-time quantitative PCR. All four components were over-expressed in PT3 over two representative low-permissive cell isolates (NK and PT1. However, this super-permissiveness did not result in PT3 cell death by AAV infection. Conclusion These data, for the first time, provide evidence that these four cellular components are likely important for AAV in vivo DNA replication as well as in vitro. These data also suggest that PT3 will be a useful reagent for investigating the AAV-permissive transcriptome and AAV anti-cancer effect.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Hwang, Meeyul [Research Center for Biomedical Resource of Oriental Medicine, Daegu Haany University (Korea, Republic of); Kim, Ji-Hyun [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Han, Bok-Ghee, E-mail: bokghee@nih.go.kr [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of); Jeon, Jae-Pil, E-mail: jpjeon@cdc.go.kr [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of)

    2012-10-19

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

  13. Internalization of cystatin C in human cell lines.

    Science.gov (United States)

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

    2008-09-01

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

  14. Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Møller, C; Bock, E

    1992-01-01

    characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were...... embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression....

  15. Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection

    Science.gov (United States)

    Chen, Zhilei; Simeon, Rudo; Chockalingam, Karuppiah; Rice, Charles M.

    2010-01-01

    The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10–20 fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2 fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors. PMID:20188762

  16. Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines.

    Science.gov (United States)

    Chinchar, V G; Floyd, A D; Chinchar, G D; Taylor, M W

    1979-02-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.

  17. Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

    Energy Technology Data Exchange (ETDEWEB)

    Zampol, Mariana A.; Busso, Cleverson; Gomes, Fernando [Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo (Brazil); Ferreira-Junior, Jose Ribamar [Escola de Artes, Ciencias e Humanidades, Universidade de Sao Paulo, Sao Paulo (Brazil); Tzagoloff, Alexander [Department of Biological Sciences, Columbia University, NY (United States); Barros, Mario H., E-mail: mariohb@usp.br [Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo (Brazil)

    2010-11-05

    Research highlights: {yields} COQ10 deletion elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}, a synthetic diffusible ubiquinone. {yields} The significance that purified Coq10p contains bound Q{sub 6} was examined by testing over-expression of Coq10p on respiration. {yields} Inhibition of CoQ function due to Coq10p excess strength our hypothesis of Coq10p function in CoQ delivery. {yields} Respiratory deficiency caused by more Coq10p was specific and restored by Q{sub 2} in mitochondria or by Coq8p in cells. {yields} Coq8p over-production on other coq mutants revealed a surprisingly higher stability of other Coq proteins. -- Abstract: COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}. Rescue of respiration by Q{sub 2} is a characteristic of mutants blocked in coenzyme Q{sub 6} synthesis. Unlike Q{sub 6} deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q{sub 6}. The physiological significance of earlier observations that purified Coq10p contains bound Q{sub 6} was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q{sub 2}. This suggests that in vivo binding of Q{sub 6} by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains

  18. New model for gastroenteropancreatic large-cell neuroendocrine carcinoma: establishment of two clinically relevant cell lines.

    Directory of Open Access Journals (Sweden)

    Andreas Krieg

    Full Text Available Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN according to their proliferation index into G1- or G2-neuroendocrine tumors (NET and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC. Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1 or lymph node metastases (NEC-DUE2 from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup.

  19. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines.

    Science.gov (United States)

    Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D; Ziller, Michael; Croft, Gist F; Amoroso, Mackenzie W; Oakley, Derek H; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

    2011-02-04

    The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

  20. Fluorouracil selectively enriches stem-like cells in the lung adenocarcinoma cell line SPC.

    Science.gov (United States)

    Shi, Mu-mu; Xiong, Yan-lei; Jia, Xin-shan; Li, Xin; Zhang, Li; Li, Xiao-lei; Wang, En-Hua

    2013-06-01

    Most adult stem cells are in the G0 or quiescent phase of the cell cycle and account for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. This study sought to enrich cancer stem cells and explore cancer stem-like cell clones using 5-fluorouracil (5-FU) in the lung adenocarcinoma cell line, SPC. Proliferation inhibition was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, according to which half maximal inhibitory concentration values were calculated. Expression levels of stem cell markers after treatment with 5-FU were examined using immunofluorescence and Western blotting. Additionally, side population (SP) cells were sorted using FACS. Properties of SP cells were evaluated by using Transwell, colony-forming assays, and tumor formation experiments. 5-FU greatly inhibits proliferation, especially of cells in S phase. SP cells possess greater invasive potential, higher clone-forming potential, and greater tumor-forming ability than non-SP cells. Treatment with 5-FU enriches the SP cells with stem cell properties in human lung adenocarcinoma cell lines.

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

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Shanyong Yi; Kejun Nan; Aihua Yuan; Chuangxin Lu

    2009-01-01

    Objective:To identify and isolate CD133 positive cancer stem-like cells (CD133+ cells) from the highly invasive human hepatocellular carcinoma cell line(MHCC97H), and examine their potential for clonogenicity and tumorigenicity. Methods: CD133+ and CD133- cells were isolated from MHCC97H cell line by magnetic bead cell sorting(MACS), and the potentials of CD133+ cells for colony formation and tumorigenicity were evaluated by soft agar cloning and tumor formation following nude mice inoculation. Results:CD133+ cells represent a minority(0.5-2.0%) of the tumor cell population with a greater colony-forming efficiency and greater tumor production ability. The colony-forming efficiency of CD133+ cells in soft agar was significantly higher than CD133- cells(36.8±1.4 vs 12.9±0.8, P<0.05).After 6 weeks, 3/5 mice inoculated with 1 × 103 CD133+ cells, 4/5 with 1 × 104 CD133+ cells and 5/5 with 1 × 105 CD133+ cells developed detectable tumors at the injection site, while only one tumor was found in mice treated with same numbers of CD133- cells. Conclusion: CD133 may be a hallmark of liver cancer stem cells (CSC) in human hepatocellular carcinoma(HCC), because the CD133+ cells identified and isolated with anti-CD133 labeled magnetic beads from MHCC97H cell line exhibit high potentials for clonogenicity and tumorigenicity. These CD133+ cells might contribute to hepatocarcinogenesis, as well as the growth and recurrence of human HCC, and therefore may be a useful target for anti-cancer therapy.

  3. Effects of small interfering RNAs targeting fascin on human esophageal squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Garcia Jose

    2010-06-01

    Full Text Available Abstract Background Fascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC. Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation. Methods We examined whether fascin is a potential target in ESCC using in vitro and in vivo studies utilizing a specific siRNA. We established a stable transfectant with downregulated fascin from KYSE170 cell line. Results The fascin downregulated cell lines showed a slower growth pattern by 40.3% (p In vivo, the tumor size was significantly smaller in the tumor with fascin knockdown cells than in mock cells by 95% at 30 days after inoculation. Conclusions These findings suggest that fascin overexpression plays a role in tumor growth and progression in ESCC and that cell death caused by its downregulation might be induced by cell adhesion loss. This indicates that targeting fascin pathway could be a novel therapeutic strategy for the human ESCC.

  4. MicroRNA-181b expression in prostate cancer tissues and its influence on the biological behavior of the prostate cancer cell line PC-3.

    Science.gov (United States)

    He, L; Yao, H; Fan, L H; Liu, L; Qiu, S; Li, X; Gao, J P; Hao, C Q

    2013-04-02

    We examined microRNA-181b (miRNA) expression in prostate cancer tissues and its effect on the prostate cancer cell line PC-3. Tissues from 27 cases of prostate cancer and 30 samples of normal human prostate were collected by surgical removal. Total miRNA was extracted, and the relative expression of miR-181b was quantified using RT-PCR. miR-181b ASO was transfected into prostate cancer PC-3 cells. miR-181b expression in transfected and non-transfected cells was measured using RT-PCR. Changes in cell apoptosis were measured using flow cytometry. MTT and cell growth curve methods were used to assess the influence of miR-181b expression on cell proliferation. The changes in cell invasive ability in vitro were detected using the Transwell chamber method. miR-181b was up-regulated in the prostate cancer tissues compared with the normal prostate samples. It was down-regulated after miR-181b ASO transfection into the prostate cancer PC-3 cells. Down-regulation of miR-181b in the PC-3 cell induced apoptosis, inhibited proliferation, and depressed invasion of PC-3 cells in vitro. As miR-181b is over-expressed in prostate cancer, its down-regulation could have potential as gene therapy for prostate cancer by inducing apoptosis, inhibiting proliferation and depressing invasion by cancer cells.

  5. Generation and Characterization of a MYF5 Reporter Human iPS Cell Line Using CRISPR/Cas9 Mediated Homologous Recombination.

    Science.gov (United States)

    Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

    2016-01-05

    Human iPS cells hold great promise for disease modeling and treatment of degenerative disorders including muscular dystrophies. Although a few research groups have used them for skeletal muscle differentiation, most were based on gene over-expression or long-term mesenchymal differentiation and retrospective identification of myogenic cells. Therefore, this study was aimed to generate a knock-in reporter human iPS cell line for MYF5, as an early myogenic specification gene, to allow prospective identification and purification of myogenic progenitors from human iPS cells. By using a CRISPR/Cas9 double nickase strategy, a 2A-GFP reporter was inserted before the stop codon of the MYF5 gene using homologous recombination. This approach allowed for highly efficient in-frame targeting of MYF5 in human iPS cells. Furthermore, in order to prove the reporter function, endogenous MYF5 expression was induced using a novel dead Cas9-VP160 transcriptional activator. Induced clones demonstrated appropriate MYF5-GFP co-expression. Finally, to confirm the differentiation potential, reporter human iPS clones were differentiated through embryoid body method and MYF5-GFP(+) myogenic cells were sorted and characterized. These data provides valuable guidelines for generation of knock-in reporter human iPS cell lines for myogenic genes which can be used for disease modeling, drug screening, gene correction and future in vivo applications.

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

    Science.gov (United States)

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

    2011-09-01

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

  7. Specific binding of benzodiazepines to human breast cancer cell lines.

    Science.gov (United States)

    Beinlich, A; Strohmeier, R; Kaufmann, M; Kuhl, H

    1999-01-01

    Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.

  8. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  9. Masitinib combined with standard gemcitabine chemotherapy: in vitro and in vivo studies in human pancreatic tumour cell lines and ectopic mouse model.

    Directory of Open Access Journals (Sweden)

    Martine Humbert

    Full Text Available BACKGROUND: Tyrosine kinases are attractive targets for pancreatic cancer therapy because several are over-expressed, including PDGFRalpha/beta, FAK, Src and Lyn. A critical role of mast cells in the development of pancreatic cancer has also been reported. Masitinib is a tyrosine kinase inhibitor that selectively targets c-Kit, PDGFRalpha/beta, Lyn, and to a lesser extent the FAK pathway, without inhibiting kinases of known toxicities. Masitinib is particularly efficient in controlling the proliferation, differentiation and degranulation of mast cells. This study evaluates the therapeutic potential of masitinib in pancreatic cancer, as a single agent and in combination with gemcitabine. METHODOLOGY/FINDINGS: Proof-of-concept studies were performed in vitro on human pancreatic tumour cell lines and then in vivo using a mouse model of human pancreatic cancer. Molecular mechanisms were investigated via gene expression profiling. Masitinib as a single agent had no significant antiproliferative activity while the masitinib/gemcitabine combination showed synergy in vitro on proliferation of gemcitabine-refractory cell lines Mia Paca2 and Panc1, and to a lesser extent in vivo on Mia Paca2 cell tumour growth. Specifically, masitinib at 10 microM strongly sensitised Mia Paca2 cells to gemcitabine (>400-fold reduction in IC(50; and moderately sensitised Panc1 cells (10-fold reduction. Transcriptional analysis identified the Wnt/beta-catenin signalling pathway as down-regulated in the cell lines resensitised by the masitinib/gemcitabine combination. CONCLUSIONS: These data establish proof-of-concept that masitinib can sensitise gemcitabine-refractory pancreatic cancer cell lines and warrant further in vivo investigation. Indeed, such an effect has been recently observed in a phase 2 clinical study of patients with pancreatic cancer who received a masitinib/gemcitabine combination.

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  1. File list: Unc.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  13. File list: Oth.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

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  14. Oxidative stress induces hypomethylation of LINE-1 and hypermethylation of the RUNX3 promoter in a bladder cancer cell line.

    Science.gov (United States)

    Wongpaiboonwattana, Wikrom; Tosukhowong, Piyaratana; Dissayabutra, Thasinas; Mutirangura, Apiwat; Boonla, Chanchai

    2013-01-01

    Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients. We previously demonstrated a relationship between increased oxidative stress and hypomethylation of the transposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressor gene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In this study, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell (UM-UC-3) under oxidative stress conditions, stimulated by challenge with H2O2 for 72 h. Cells were pretreated with an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 and RUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR, respectively. Levels of LINE-1 methylation were significantly decreased in H2O2-treated cells, and reestablished after pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cells exposed to H2O2. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylation of LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally induced by reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotes urothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanistic pathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promoter might differ.

  15. Analysis of G-banding in tumor cell lines derived from human neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Junhua Zou; Yanhui Li

    2006-01-01

    BACKGROUND: The application of neural stem cell (NSC) is restricted because of its tumorigenesis, and the possible pathogenesis needs investigation.OBJECTIVE: To compare the differences of chromosomal G-banding between human NSCs (hNSCs) derived tumor cell line and hNSCs derived normal cell lines.DESIGN: A randomized controlled observation.SETTING: Building of Anatomy, Peking University Health Science Center.MATERIALS: The hNSC lines and hNSC-derived tumor cell lines were provided by the Research Center of Stem Cells, Peking University; DMEM/F12 (1:1) medium, N2 additive, B27 additive epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were produced by GIBCO BRL Company (USA); fetal bovine serum by HYCLONE Company (USA).METHODS: The experiments were carried out in the Department of Genetics, Peking University Health Science Center from February 2003 to July 2004. Human fetal striatal NSCs were inoculated hypodermically on the right scapular of nude mice; Normal human fetal striatal NSCs were cultured to 5-8 passages as controls. Karyotyping was performed on the 5th passage of hNSC-derived tumor cells at 6 weeks after hN-SC transplantation into nude mice (T1) and tumor cells at 15 weeks after transplantation (T2). Metaphase chromosomes were examined with microscope, G-banding cytogenetic analysis and karyotyping were performed according to the Cytoscan Karyotyping FISH and CGH software system (United biotechnology USA Corporation).MAIN OUTCOME MEASURES: G-banded analytical results of human fetal striatal nerve stem cells derived tumor cell lines (T1 and T2) of metaphase chromosomes were observed.RESULTS: ① Chromosome analysis of hNSC-derived tumor cell lines 1 (T1): Twenty-five well-spread metaphases were randomly selected for analysis. The karyotypes were 64, XX (8, 32%); 65, XX (1, 4%); 67,XX (5, 20%); 68, XX (11, 44%). The modal number of chromosomes in this cell lines was 68, which were all hypotriploid. The analysis of 8 G

  16. Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Zhang Ping

    2006-09-01

    Full Text Available Abstract Background Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. Methods A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated. Results Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells. Conclusion The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis

  17. A vertically integrated dynamic RAM-cell: Buried bit line memory cell with floating transfer layer

    NARCIS (Netherlands)

    Mouthaan, Ton; Vertregt, Maarten

    1986-01-01

    A charge injection device has been realized in which charge can be injected on to an MOS-capacitor from a buried layer via an isolated transfer layer. The cell is positioned vertically between word and bit line. LOCOS (local oxidation) is used to isolate the cells and (deep) ion implantation to real

  18. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

    Science.gov (United States)

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

    2010-05-28

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

  19. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Qin, J.-Z.; Xin, H. [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States); Nickoloff, B.J., E-mail: bnickol@lumc.edu [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States)

    2010-05-28

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

  20. Characterisation and Manipulation of Docetaxel Resistant Prostate Cancer Cell Lines

    LENUS (Irish Health Repository)

    O'Neill, Amanda J

    2011-10-07

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

  1. Isolation and Enrichment of Mouse Female Germ Line Stem Cells

    Directory of Open Access Journals (Sweden)

    Somayeh Khosravi-Farsani

    2015-01-01

    Full Text Available Objective: The existence of female germ-line stem cells (FGSCs has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. Materials and Methods: In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH and stage-specific embryonic antigen-1 (SSEA1 markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3, alkaline phosphatase (AP activity test and immunocytochemistry. Results: Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 ± 0.49% (Mean ± SDV positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. Conclusion: We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries.

  2. Characterization of cancer stem-like cells in the side population cells of human gastric cancer cell line MKN-45

    Institute of Scientific and Technical Information of China (English)

    Hai-hong ZHANG; Ai-zhen CAI; Xue-ming WEI; Li DING; Feng-zhi LI; Ai-ming ZHENG; Da-jiang DAI

    2013-01-01

    Objective:Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells,including several gastric cancer cell lines.This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45.Methods:We used fluorescence activated cell sorting (FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45 (cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells.Results:This study found that the SP cells had higher clone formation efficiency than major population (MP) cells.Five stemness-related gene expression profiles,including OCT-4,SOX-2,NANOG,CD44,and adenosine triphosphate (ATP)-binding cassette transporters gene ABCG2,were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).Western blot was used to show the difference of protein expression between SP and MP cells.Both results show that there was significantly higher protein expression in SP cells than in MP cells.When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice,SP cells show higher tumorigenesis tendency than MP cells.Conclusions:These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.

  3. 过表达巨噬细胞移动抑制因子对子宫颈癌SiHa细胞中白细胞介素8及基质金属蛋白酶9表达的影响%Effects of over-expression of macrophage migration inhibitory factor on the expression of interleukin-8 and martix metalloproteinase-9 of human cervical cancer SiHa cells

    Institute of Scientific and Technical Information of China (English)

    郭红霞; 吴素慧; 贾睿; 尚海霞

    2013-01-01

    Objective To investigate the effects of macrophage migration inhibitory factor (MIF) overexpression on the expression of interleukin-8 (IL-8),martix metalloproteinase-9 (MMP-9) and invasion of human cervical cancer SiHa cells.Methods Chemical synthesis MIF eDNA gene,designed primer sequence including XhoI and BamHI enzyme sites,MIF gene was amplified by polymerase chain reaction (PCR),constructed eukaryotic expression vector pEGFP-N1/MIF and transfected into SiHa cells using Lipofectamine and won over-expression of MIF.The expression of MIF in supernatant fluid was detected by ELISA,the expression of MIF,IL-8,MMP-9 in both mRNA and protein levels were detected by real-time fluorescence quantitative-PCR and immunocytochemistry respectively.The effect of over-expressed MIF on migration was detected by Boyden small chamber.Results The expression of protein in supernatant fluid transfected with pEGFP-N1/MIF was significantly increased (Fgroup =8267.564,P < 0.01),the expression of MIF,IL-8,MMP-9 in both mRNA and protein in SiHa cells transfected with pEGFP-N1/MIF were significantly increased (F values were 7019.619,2148.094,3303.540,1565.114,2807.300,523.466,P < 0.01),and there was a positive correlation among MIF,IL-8,MMP-9 expression in both mRNA and protein (r values were 0.865,0.895,0.934,0.908,P < 0.01).Invasion ability in SiHa cells transfected with pEGFP-N1/MIF was obviously increased (F=3430.898,P< 0.01).Conclusion The over-expression MIF gene in SiHa cells can promote cervical cancer cell invasion and metastasis of ability,which could be associated with the upregulation of IL-8 and MMP-9 expression.%目的 研究过表达巨噬细胞移动抑制因子(MIF)对子宫颈癌SiHa细胞中白细胞介素8(IL-8)、基质金属蛋白酶9(MMP-9)表达及细胞侵袭迁移能力的影响.方法 化学合成MIF cDNA,设计含Xhol和BamHI酶切位点的引物序列,利用聚合酶链反应(PCR)方法 扩增MIF基因片段,构建人pEGFP-N1/MIF真核表达载体,

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

    Science.gov (United States)

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

    2008-11-01

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

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

    Science.gov (United States)

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

    2016-08-31

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

  6. Identification of a secreted casein kinase 1 in Leishmania donovani: effect of protein over expression on parasite growth and virulence.

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    Mary Dan-Goor

    Full Text Available Casein kinase 1 (CK1 plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780 is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001. Analysis by flow cytometry showed a higher percentage, ∼4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005. These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.

  7. OVER-EXPRESSION OF A TRUNCATED TYPEⅡ TGF-β RECEPTOR IN NORMAL DERMAL FIBROBLASTS DECREASES TGF-β1 AUTOPRODUCTION

    Institute of Scientific and Technical Information of China (English)

    刘伟; 蔡泽浩; 商庆新; 钱云良; 曹谊林

    2003-01-01

    Objective To determine over expression of a truncated type Ⅱ TGF-β receptor in down regulating TGF-β1 autoproduction in normal dermal fibroblasts.MethodsIn vitro cultured dermal fibroblasts were treated with rhTGF β1(5ng/ml) or recombinant adenovirus containing a truncated type Ⅱ TGF-β receptor gene(50pfu/cell). Their effects on regulating gene expression of TGF β1 were observed with Northern Blot.Resultsrh TGF β1 up regulated the gene expression of TGF-β1(34%~150%) and type Ⅰ procollagen(13%~190%).Over expression of a truncated receptor Ⅱ decreased the gene expression of TGF-β1(53%~66%).ConclusionOver expression of the truncated TGF-β receptor Ⅱ down regulated TGF-β1 autoproduction via blocking signal transduction of TGF-β. This study may provide a new strategy for scar gene therapy.

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

    Science.gov (United States)

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

    2017-01-01

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

  9. Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

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    Yonatan Y Mahller

    Full Text Available BACKGROUND: Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers. METHODOLOGY/PRINCIPAL FINDINGS: Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice. CONCLUSIONS/SIGNIFICANCE: These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

  10. HER3 over-expression and overall survival in gastrointestinal cancers.

    Science.gov (United States)

    Wang, Yadong; Yang, Haiyan; Duan, Guangcai

    2015-12-15

    Published studies on the association between human epidermal factor receptor 3 (HER3) expression and overall survival (OS) in gastrointestinal cancers have yielded conflicting results. The aim of this study was to explore the association of HER3 over-expression with OS in gastrointestinal cancers. A systematic search was performed through Medline/PubMed, Embase, Science Direct and Elsevier. The summary odds ratio (OR) with 95% confidence interval (CI) was calculated to estimate the strength of the association. Overall, we observed that HER3 over-expression was associated with worse OS at five years (OR = 1.38, 95% CI: 1.04-1.82); however, HER3 over-expression was not associated with worse OS at three years (OR = 1.33, 95% CI: 0.97-1.84). The cumulative meta-analysis showed similar results. In subgroup analyses by tumor type, HER3 over-expression in gastric cancers was associated with worse OS at both three years (OR = 1.69, 95% CI: 1.28-2.25) and five years (OR = 1.74, 95% CI: 1.26-2.41). In conclusion, our results suggest that HER3 over-expression may be associated with worse overall survival in gastric cancers. Well-designed studies with a large sample size are required to further confirm our findings.

  11. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M;

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...... of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell...

  12. Cell Motility and Invasiveness of Neurofibromin-Deficient Neural Crest Cells and Malignant Triton Tumor Lines

    Science.gov (United States)

    2005-06-01

    immunoblotting techniques to characterize signaling pathways activated by TGF-beta and PDGF-BB in MPNST -like sarcoma cell lines isolated from cisNfl+/-;p53...mouse model to include characterizations of genomic instability in the context of malignant transformation, and to test possible modifiers of MPNST ...growth and invasiveness. 15. SUBJECT TERMS neurofibromatosis type 1; neural crest cells; cell motility and Migration; PDGF; TGF-beta; MPNST

  13. HLXB9 gene expression, and nuclear location during in vitro neuronal differentiation in the SK-N-BE neuroblastoma cell line.

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    Claudia Giovanna Leotta

    Full Text Available Different parts of the genome occupy specific compartments of the cell nucleus based on the gene content and the transcriptional activity. An example of this is the altered nuclear positioning of the HLXB9 gene in leukaemia cells observed in association with its over-expression. This phenomenon was attributed to the presence of a chromosomal translocation with breakpoint proximal to the HLXB9 gene. Before becoming an interesting gene in cancer biology, HLXB9 was studied as a developmental gene. This homeobox gene is also known as MNX1 (motor neuron and pancreas homeobox 1 and it is relevant for both motor neuronal and pancreatic beta cells development. A spectrum of mutations in this gene are causative of sacral agenesis and more broadly, of what is known as the Currarino Syndrome, a constitutional autosomal dominant disorder. Experimental work on animal models has shown that HLXB9 has an essential role in motor neuronal differentiation. Here we present data to show that, upon treatment with retinoic acid, the HLXB9 gene becomes over-expressed during the early stages of neuronal differentiation and that this corresponds to a reposition of the gene in the nucleus. More precisely, we used the SK-N-BE human neuroblastoma cell line as an in vitro model and we demonstrated a transient transcription of HLXB9 at the 4th and 5th days of differentiation that corresponded to the presence, predominantly in the cell nuclei, of the encoded protein HB9. The nuclear positioning of the HLXB9 gene was monitored at different stages: a peripheral location was noted in the proliferating cells whereas a more internal position was noted during differentiation, that is while HLXB9 was transcriptionally active. Our findings suggest that HLXB9 can be considered a marker of early neuronal differentiation, possibly involving chromatin remodeling pathways.

  14. Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth.

    Science.gov (United States)

    Kanjanaphachoat, Parawee; Wei, Bi-Yin; Lo, Shuen-Fang; Wang, I-Wen; Wang, Chang-Sheng; Yu, Su-May; Yen, Ming-Liang; Chiu, Sheng-Hsien; Lai, Chien-Chen; Chen, Liang-Jwu

    2012-04-01

    A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, darkbrown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the darkbrown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.

  15. Over Expression of Voltage Dependent Anion Channel 2 (VDAC2 in Muscles of Electrically Stunned Chickens

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    Norshahida Abu Samah, Azura Amid, and Faridah Yusof

    2011-12-01

    Full Text Available Water bath stunning is a common practice in commercial slaughterhouses. Such treatment is economic and in line with animal welfare practice. However, the conditions applied for the stunning process may vary from a slaughterhouse to another slaughterhouse. Such a loose regulation on the stunning procedure has opened up doors for food adulteration such as over dose stunning. In this study, a simple and reliable approach using proteomics have been developed to study the effect of different currents and voltages in stunning on the protein expression of the chickens. Protein profiles of the chickens were constructed in order to detect any differences in protein expression and modifications. The different voltage studied were 10 V, 40 V and 70 V while the values for current studied were 0.25 A, 0.5 A, and 0.75 A. After the proteomics analyses using 2D Platinum ImageMaster 6.0 and Matrix-assisted laser desorption ionization- time of flight (MALDI TOF spectrometry identification, Voltage dependent anion channel 2 (VDAC2 was identified to be over expressed in the muscle sample of over stunned chicken. The over expression of VDAC2 was confirmed at the transcriptional level of RNA expression. Real Time PCR showed that all over stunned samples contained higher mRNA expression level for VDAC2 genes. The mRNA level of VDAC2 was up-regulated by 59.87 fold change when normalized with housekeeping gene. In conclusion, VDAC2 could serve as potential biomarkers for identification of electrically stimulated chickens. The existence of these biomarkers will help to monitor the slaughtering and stunning process in the future. It will revolutionize the food authentication field and give a new breathe to the meat industry.ABSTRAK: Kaedah "waterbath stunning" merupakan amalan biasa di pusat-pusat penyembelihan. Kaedah ini adalah ekonomik dan selari dengan amalan kebajikan haiwan. Walaubagaimanapun, syarat-syarat yang digunakan untuk proses kejutan tersebut mungkin

  16. Over-expressed copper/zinc superoxide dismutase localizes to mitochondria in neurons inhibiting the angiotensin II-mediated increase in mitochondrial superoxide

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    Shumin Li

    2014-01-01

    Full Text Available Angiotensin II (AngII is the main effector peptide of the renin–angiotensin system (RAS, and contributes to the pathogenesis of cardiovascular disease by exerting its effects on an array of different cell types, including central neurons. AngII intra-neuronal signaling is mediated, at least in part, by reactive oxygen species, particularly superoxide (O2·−. Recently, it has been discovered that mitochondria are a major subcellular source of AngII-induced O2·−. We have previously reported that over-expression of manganese superoxide dismutase (MnSOD, a mitochondrial matrix-localized O2·− scavenging enzyme, inhibits AngII intra-neuronal signaling. Interestingly, over-expression of copper/zinc superoxide dismutase (CuZnSOD, which is believed to be primarily localized to the cytoplasm, similarly inhibits AngII intra-neuronal signaling and provides protection against AngII-mediated neurogenic hypertension. Herein, we tested the hypothesis that CuZnSOD over-expression in central neurons localizes to mitochondria and inhibits AngII intra-neuronal signaling by scavenging mitochondrial O2·−. Using a neuronal cell culture model (CATH.a neurons, we demonstrate that both endogenous and adenovirus-mediated over-expressed CuZnSOD (AdCuZnSOD are present in mitochondria. Furthermore, we show that over-expression of CuZnSOD attenuates the AngII-mediated increase in mitochondrial O2·− levels and the AngII-induced inhibition of neuronal potassium current. Taken together, these data clearly show that over-expressed CuZnSOD in neurons localizes in mitochondria, scavenges AngII-induced mitochondrial O2·−, and inhibits AngII intra-neuronal signaling.

  17. Over-expressed copper/zinc superoxide dismutase localizes to mitochondria in neurons inhibiting the angiotensin II-mediated increase in mitochondrial superoxide.

    Science.gov (United States)

    Li, Shumin; Case, Adam J; Yang, Rui-Fang; Schultz, Harold D; Zimmerman, Matthew C

    2013-01-01

    Angiotensin II (AngII) is the main effector peptide of the renin-angiotensin system (RAS), and contributes to the pathogenesis of cardiovascular disease by exerting its effects on an array of different cell types, including central neurons. AngII intra-neuronal signaling is mediated, at least in part, by reactive oxygen species, particularly superoxide (O2 (•-)). Recently, it has been discovered that mitochondria are a major subcellular source of AngII-induced O2 (•-). We have previously reported that over-expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix-localized O2 (•-) scavenging enzyme, inhibits AngII intra-neuronal signaling. Interestingly, over-expression of copper/zinc superoxide dismutase (CuZnSOD), which is believed to be primarily localized to the cytoplasm, similarly inhibits AngII intra-neuronal signaling and provides protection against AngII-mediated neurogenic hypertension. Herein, we tested the hypothesis that CuZnSOD over-expression in central neurons localizes to mitochondria and inhibits AngII intra-neuronal signaling by scavenging mitochondrial O2 (•-). Using a neuronal cell culture model (CATH.a neurons), we demonstrate that both endogenous and adenovirus-mediated over-expressed CuZnSOD (AdCuZnSOD) are present in mitochondria. Furthermore, we show that over-expression of CuZnSOD attenuates the AngII-mediated increase in mitochondrial O2 (•-) levels and the AngII-induced inhibition of neuronal potassium current. Taken together, these data clearly show that over-expressed CuZnSOD in neurons localizes in mitochondria, scavenges AngII-induced mitochondrial O2 (•-), and inhibits AngII intra-neuronal signaling.

  18. Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

    Directory of Open Access Journals (Sweden)

    Weyhe Dirk

    2010-10-01

    Full Text Available Abstract Background The anti-infective agent Taurolidine (TRD has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Methods Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3 were incubated with TRD (100 μM, 250 μM and 1000 μM. Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. Results TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3 as well as genes involved in the ER stress response (PPP1R15A, in ubiquitination (TRAF6 and mitochondrial apoptotic pathways (PMAIP1. Conclusions This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis.

  19. In vitro Acute Cytotoxicity of Abamectin to the Gill Cell Line of Flounder Paralichthy olivaceus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  20. Tooth regeneration from newly established cell lines from a molar tooth germ epithelium.

    Science.gov (United States)

    Komine, Akihiko; Suenaga, Momoko; Nakao, Kazuhisa; Tsuji, Takashi; Tomooka, Yasuhiro

    2007-04-13

    In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.

  1. Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

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    Tobias Hannes

    2015-01-01

    Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

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

    Directory of Open Access Journals (Sweden)

    Behra Martine

    2012-01-01

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

  3. Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Jie Du; Xiaoqing Gao; Li Deng; Nengbin Chang; Huailin Xiong; Yu Zheng

    2014-01-01

    Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro-tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su-pernatant were signiifcantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes-enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen-chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

  4. In vitro platinum drug chemosensitivity of human cervical squamous cell carcinoma cell lines with intrinsic and acquired resistance to cisplatin.

    OpenAIRE

    Mellish, K. J.; Kelland, L R; Harrap, K. R.

    1993-01-01

    The platinum drug chemosensitivity of five human cervical squamous cell carcinoma cell lines (HX/151, HX/155, HX/156, HX/160 and HX/171) derived from previously untreated patients has been determined. Compared to our data obtained previously using human ovarian carcinoma cell lines, all five lines were relatively resistant to cisplatin, carboplatin, iproplatin and tetraplatin. One of the lines (HX/156) was exceptionally sensitive to the novel platinum (IV) ammine/amine dicarboxylates JM216 [b...

  5. Reptin is required for the transcription of telomerase reverse transcriptase and over-expressed in gastric cancer

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    Liu Tiantian

    2010-05-01

    Full Text Available Abstract Background Telomerase is activated in oncogenesis, which confers an immortal phenotype to cancer cells. The AAA + ATPase Reptin is required for telomerase biogenesis by maintaining telomerase RNA (hTER stability and is aberrantly expressed in certain cancers. Given its role in chromatin remodeling and transcription regulation, we determined the effect of Reptin on the transcription of the telomerase reverse transcriptase (hTERT gene, a key component of the telomerase complex and its expression in gastric cancer. Results Knocking down Reptin or its partner Pontin using small interfering RNA in gastric and cervical cancer cells led to significant decreases in hTERT mRNA, but hTERT promoter activity was inhibited in only Reptin-depleted cells. Reptin interacted with the c-MYC oncoprotein and its stimulatory effect on the hTERTpromoter was significantly dependent on functional E-boxes in the promoter. Moreover, Reptin bound to the hTERT proximal promoter and the loss of the Reptin occupancy led to dissociation of c-MYC from the hTERT promoter in Reptin-depleted cells. Reptin inhibition dramatically impaired clonogenic potential of gastric cancer cells by inducing cell growtharrest and over-expression of Reptin was observed in primary gastric cancer specimens. Conclusions The hTERT gene is a direct target of Reptin, and hTERT transcription requires constitutive expression of Reptin and its cooperation with c-MYC. Thus, Reptin regulates telomerase at two different levels. This finding, together with the requirementof Reptin for the clonogenic potential of cancer cells and its over-expression in gastriccancer and other solid tumors, suggests that Reptin may be a putative therapeutic target.

  6. MicroRNA-155 targets the SKI gene in human melanoma cell lines.

    Science.gov (United States)

    Levati, Lauretta; Pagani, Elena; Romani, Sveva; Castiglia, Daniele; Piccinni, Eugenia; Covaciu, Claudia; Caporaso, Patrizia; Bondanza, Sergio; Antonetti, Francesca R; Bonmassar, Enzo; Martelli, Fabio; Alvino, Ester; D'Atri, Stefania

    2011-06-01

    The SKI protein is a transcriptional coregulator over-expressed in melanoma. Experimentally induced down-regulation of SKI inhibits melanoma cell growth in vitro and in vivo. MicroRNAs (miRNAs) negatively modulate gene expression and have been implicated in oncogenesis. We previously showed that microRNA-155 (miR-155) is down-regulated in melanoma cells as compared with normal melanocytes and that its ectopic expression impairs proliferation and induces apoptosis. Here, we investigated whether miR-155 could mediate melanoma growth inhibition via SKI gene silencing. Luciferase reporter assays demonstrated that miR-155 interacted with SKI 3'UTR and impaired gene expression. Transfection of melanoma cells with miR-155 reduced SKI levels, while inhibition of endogenous miR-155 up-regulated SKI expression. Specifically designed small interfering RNAs reduced SKI expression and inhibited proliferation. However, melanoma cells over-expressing a 3'UTR-deleted SKI were still susceptible to the antiproliferative effect of miR-155. Our data demonstrate for the first time that SKI is a target of miR-155 in melanoma. However, impairment of SKI expression is not the leading mechanism involved in the growth-suppressive effect of miR-155 found in this malignancy.

  7. Expression of tumor antigens on primary ovarian cancer cells compared to established ovarian cancer cell lines

    Science.gov (United States)

    Kloudová, Kamila; Hromádková, Hana; Partlová, Simona; Brtnický, Tomáš; Rob, Lukáš; Bartůňková, Jiřina; Hensler, Michal; Halaška, Michael J.; Špíšek, Radek; Fialová, Anna

    2016-01-01

    In order to select a suitable combination of cancer cell lines as an appropriate source of antigens for dendritic cell-based immunotherapy of ovarian cancer, we analyzed the expression level of 21 tumor associated antigens (BIRC5, CA125, CEA, DDX43, EPCAM, FOLR1, Her-2/neu, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MUC-1, NY-ESO-1, PRAME, p53, TPBG, TRT, WT1) in 4 established ovarian cancer cell lines and in primary tumor cells isolated from the high-grade serous epithelial ovarian cancer tissue. More than 90% of tumor samples expressed very high levels of CA125, FOLR1, EPCAM and MUC-1 and elevated levels of Her-2/neu, similarly to OVCAR-3 cell line. The combination of OV-90 and OVCAR-3 cell lines showed the highest overlap with patients' samples in the TAA expression profile. PMID:27323861

  8. HER2 over-expression and response to different chemotherapy regimens in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Jin ZHANG; Yan LIU

    2008-01-01

    Purpose: To exam the relationship between HER2 over-expression and different adjuvant chemotherapies in breast cancer. Patients and Methods: A total of 1625 primary breast cancer patients who received post-surgery adjuvant chemotherapy in Tianjin Cancer Hospital, China, from July 2002 to November 2005 were included in the study. Among them, 600 patients were given CMF (CTX+MTX+5-Fu) regimen, 600 given CEF (CTX+E-ADM+5-Fu) regimen, and 425 given anthracyclines plus taxanes regimen, with mean follow-up time of 42 months. Results: In CMF treatment group, the 3-year disease free survival (DFS)in HER2 over-expressed patients was lower than that of the HER2-negative ones (89.80% vs 91.24%, P=0.0348); in node-positive subgroup, the 3-year DFS was 84.72% in HER2 over-expressed patients, and 90.18% in the HER-2-negative ones (P=0.0271).Compared to CMF regimen, anthracyclines and anthracyclines plus taxanes regimens are more effective (P<0.05) in node-positive HER2 over-expression than those in the node-negative. Conclusion: HER2 over-expression is an independent index for predicting poor prognosis and short DFS for breast cancer patients. HER2 over-expressed patients are resistant to CMF regimen chemotherapy, but sensitive to anthracyclines-based or anthracyclines plus taxanes regimen. HER2 expression can be taken as a marker for therapies in breast cancer.

  9. Development of improved vaccine cell lines against rotavirus

    Science.gov (United States)

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

    2017-01-01

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

  10. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  11. Comprehensive characterization of genomic instability in pluripotent stem cells and their derived neuroprogenitor cell lines

    Directory of Open Access Journals (Sweden)

    Nestor Luis Lopez Corrales

    2012-12-01

    Full Text Available The genomic integrity of two human pluripotent stem cells and their derived neuroprogenitor cell lines was studied, applying a combination of high-resolution genetic methodologies. The usefulness of combining array-comparative genomic hybridization (aCGH and multiplex fluorescence in situ hybridization (M-FISH techniques should be delineated to exclude/detect a maximum of possible genomic structural aberrations. Interestingly, in parts different genomic imbalances at chromosomal and subchromosomal levels were detected in pluripotent stem cells and their derivatives. Some of the copy number variations were inherited from the original cell line, whereas other modifications were presumably acquired during the differentiation and manipulation procedures. These results underline the necessity to study both pluripotent stem cells and their differentiated progeny by as many approaches as possible in order to assess their genomic stability before using them in clinical therapies.

  12. Lipid analysis of eight human breast cancer cell lines with ToF-SIMS.

    Science.gov (United States)

    Robinson, Michael A; Graham, Daniel J; Morrish, Fionnuala; Hockenbery, David; Gamble, Lara J

    2015-06-28

    In this work, four triple negative (TN) cell lines, three ER+ and PR+ receptor positive (RP) cell lines, and one ER+, PR+, and HER2+ cell line were chemically distinguished from one another using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA). PCA scores separation was observed between the individual cell lines within a given classification (TN and RP) and there were distinctly different trends found in the fatty acid and lipid compositions of the two different classifications. These trends indicated that the RP cell lines separated out based on the carbon chain length of the lipids while the TN cell lines showed separation based on cholesterol-related peaks (in the positive ion data). Both cell types separated out by trends in fatty acid chain length and saturation in the negative ions. These chemical differences may be manifestations of unique metabolic processes within each of the different cell lines. Additionally, the HER2+ cell line was distinguished from three other RP cell types as having a unique distribution of fatty acids including anticorrelation to 18-carbon chain fatty acids. As these cell lines could not be grown in the same growth media, a combination of chemical fixation, rinsing, C60 (+) presputtering, and selection of cellular regions-of-interest is also presented as a successful method to acquire ToF-SIMS data from cell lines grown in different media.

  13. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines.

    Science.gov (United States)

    Varamini, P; Soltani, M; Ghaderi, A

    2009-01-01

    There are reports on the presence of various compounds exerting different biological activities in Ruta graveolens, a plant of Rutaceae family. The aim of the present study was to evaluate in vitro cytotoxicity of the total extract of R. graveolens against tumor cell lines of different origin. Aerial parts of the plant was extracted with 70% ethanol by sonication method and cytotoxic activity was examined on RAJI, RAMOS, RPMI8866, U937, Jurkat, MDA-MB-453, MCF-7, LNCap-FGC-10, 5637, HeLa, SK-OV-3, A549, Mehr-80 and also peripheral blood mononuclear cells (PBMC) by the use of WST-1 assay. Results were expressed as IC(50) values. R. graveolens extract showed high cytotoxic activity against RAJI and RAMOS, two Burkitt's lymphoma cell lines, with an IC(50) equal to 24.3 microg/ml and 35.2 microg/ml respectively and LNCap-FGC-10, a prostate adenocarcinoma cell line with an IC(50) equal to 27.6 microg/ml as well as Mehr-80, a newly established Large Cell Lung Carcinoma (IC(50)=46.2 microg/ml). No significant anti-proliferative activity was observed on other cell lines including MCF-7, MDA-MB-453, SK-OV-3, HeLa, 5637, JURKAT and RPMI8866. Adverse cytotoxic effect of R. graveolens was investigated against PBMCs and a significantly lower effect of this extract (IC(50)=104 microg/ml) was seen on normal cells compared with RAJI and RAMOS, two haematopoietic cell lines.

  14. Single-cell printing to form three-dimensional lines of olfactory ensheathing cells

    Energy Technology Data Exchange (ETDEWEB)

    Othon, Christina M; Ringeisen, Bradley R [Naval Research Laboratory/Code 6113, 4555 Overlook Ave. SW, Washington, DC 20375 (United States); Wu Xingjia; Anders, Juanita J [Department of Anatomy, Physiology and Genetics, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 (United States)], E-mail: ringeisen@nrl.navy.mil

    2008-09-01

    Biological laser printing (BioLP(TM)) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes ({approx}{mu}Ls) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 {mu}m, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth.

  15. Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

    Directory of Open Access Journals (Sweden)

    Létourneau Isabelle J

    2012-08-01

    Full Text Available Abstract Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133 were derived from solid tumor (TOV and ascites (OV, at specific time points at diagnosis and relapse (R. Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133, or cisplatin/topotecan (2295. Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2, the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R and OV2295(R2 cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian

  16. Influences of the interferon induced transmembrane protein I on the proliferation, invasion, and metastasis of the colorectal cancer SW480 cell lines

    Institute of Scientific and Technical Information of China (English)

    HE Jing-dong; LUO Hong-lei; LI Jin; FENG Wan-ting; CHEN Long-bang

    2012-01-01

    Background Interferon-induced transmembrane protein 1 (IFITM1) has been identified as a molecular marker of the colorectal tumors; however its influences on the biological behaviors of the colorectal cancer cells are currently unknown.We aimed to study the influences of IFITM1 on the proliferation,invasion,and metastasis of the colorectal cancer SW480 cell lines.Methods We constructed IFITM1/pEGFP-C3 recombinant plasmids and transfected them into the colorectal cancer SW480 cell lines.IFITM1/pEGFP-C3 recombinant plasmids were identified by means of immunofluorescence,laser confocal scanning microscopy,and reverse transcription polymerase chain reaction.IFITM1/SW480 cells with stable over-expression of IFITM1 were confirmed by G418 screening.The influences of IFITM1 on the proliferation of the SW480 cell lines were investigated by MTT assay and tumor transplantation experiments in nude mice.Cell invasion experiments were performed to determine the invasion capacity of the IFITM1/SW480 cells.Matrix metalloproteinase 2 (MMP-2) and MMP-9 activities were detected by the gelatin zymographic analysis,and MMP-9 expression by the Western blotting analysis.Results IFITM1/pEGFP-C3 recombinant plasmids were successfully constructed in this study,and the IFITM1/SW480 cells with stable IFITM1 gene over-expression were confirmed by G418 screening.MTT results showed that the proliferation of the IFITM1/SW480 cells was significantly enhanced (P <0.01).Tumors were harvested from four weeks old mice.Tumor volumes were (1347.00±60.94) mm3,(1032.40±111.38) mm3 and (1018.78±28.83) mm3; and tumor weights were (1522.34±62.76) mg,(1137.78±97.22) mg and (1155.76±133.31) mg for mice inoculated with the IFITM1/SW480 cells,pEGFP-C3/SW480 cells and SW480 cells,respectively.Tumor volumes and weights from mice inoculated with the IFITM1/SW480 cells were significantly increased (P <0.01).In addition,the numbers of the SW480 cells and IFITM1/SW480 cells that migrated through Matrigel were

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

    Science.gov (United States)

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

    2016-06-01

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

  18. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Directory of Open Access Journals (Sweden)

    Teruyuki Hayashi

    Full Text Available Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT in combination with fluorescence lifetime imaging microscopy (FLIM. Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  19. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Science.gov (United States)

    Hayashi, Teruyuki; Fukuda, Nanaho; Uchiyama, Seiichi; Inada, Noriko

    2015-01-01

    Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  20. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl].

    Science.gov (United States)

    Shimizu, T

    1997-11-01

    Solcoseryl is composed of extracts from calf blood, and is a drug known to activate tissue respiration. In the present study, I demonstrated the cell biological effects of Solcoseryl on a human small cell lung cancer cell line, PC-6, by analyzing cell morphology, cell growth, expression of neuronal differentiation markers, and the ras proto-oncogene product(ras p21). Exposure of PC-6 cells to Solcoseryl at the concentration of 200 microliters/ml induced (1) cell morphological changes, including neurodendrite-like projections from the cell surface, and (2) complete inhibition of cell growth, that was shown by the loss of Ki-67 expression. Solcoseryl also induced the expression of neurofilament protein and acetylcholinesterase, both of which are markers of neuronal differentiation. Moreover, it upregulated the expression of the ras proto-oncogene product, ras p21. Taken together, these data suggest that Solcoseryl is composed of component(s) which can induce neuronal differentiation of the human small cell lung cancer cell line, PC-6.

  1. Heterotransplantation of human leukemic B-cell, T-cell and null-cell lines in hamsters.

    Directory of Open Access Journals (Sweden)

    Hiraki,Shunkichi

    1979-02-01

    Full Text Available Human leukemic B-cell (BALL-1, T-cell (TALL-1 and null-cell (NALL-1 lines have been established from three patients with acute lymphoblastic leukemia (ALL. To study the heterotransplantability and in vivo growth characteristics, attempts were made to transplant these ALL cell lines into newborn Syrian hamsters treated with rabbit anti-hamster thymocyte serum. Intraperitoneal implantation of 1.8-3.5 x 10(7 cells gave rise to invasive tumors in all recipients after 15 to 41 days. In addition to a common in vivo feature of mesenteric and retroperitoneal tumors, BALL-1 line was characterized by infiltration of the skin, massive ascites and bone marrow invasion. TALL-1 cells infiltrated various organs including the lymph nodes, liver, gallbladder, spleen, bone marrow, central nervous system and eyes. NALL-1 line grew slowly, producing the least tumors, although there were distant metastases in the lungs. Tumor cells were detected in the blood of 2 of 3 BALL-1-bearing hamsters and in the blood of 4 of 5 TALL-1-bearing hamsters. Thus, these three ALL cell lines were found to exhibit a characteristic biological behavior in hamsters, which might be related to the different cell lineage.

  2. Over-expression of mitochondrial antiviral signaling protein inhibits coxsackievirus B3 infection by enhancing type-I interferons production

    Directory of Open Access Journals (Sweden)

    Zhang Qing-Meng

    2012-12-01

    Full Text Available Abstract Background Recent studies have revealed that Mitochondrial Antiviral Signaling (MAVS protein plays an essential role in the inhibition of viral infection through type I interferon (IFN pathway. It has been shown that 3C (pro cysteine protease of coxsackievirus B3 (CVB3 cleaves MAVS to inhibit type I IFNs induction. Other workers also found that MAVS knock-out mice suffered CVB3 susceptibility and severe histopathological change. Accordingly,our experiments were designed to explore the protection of over-expressing MAVS against CVB3 infection and the possible mechanism. Results In this study, HeLa cells (transfected with MAVS constructs pre- or post- exposure to CVB3 were used to analyze the function of exogenous MAVS on CVB3 infection. The results revealed that though CVB3 infection induced production of type I IFNs, viral replication and cell death were not effectively inhibited. Similarly, exogenous MAVS increased type I IFNs moderately. Morever, we observed robust production of type I IFNs in CVB3 post-infected HeLa cells thereby successfully inhibiting CVB3 infection, as well formation of cytopathic effect (CPE and cell death. Finally, introduction of exogenous MAVS into CVB3 pre-infected cells also restricted viral infection efficiently by greatly up-regulating IFNs. Conclusions In summary, exogenous MAVS effectively prevents and controls CVB3 infection by modulating and promoting the production of type I IFNs. The IFNs level in MAVS over-expressing cells is still tightly regulated by CVB3 infection. Thus, the factors that up-regulate MAVS might be an alternative prescription in CVB3-related syndromes by enhancing IFNs production.

  3. Drug treatment of cancer cell lines: a way to select for cancer stem cells?

    Science.gov (United States)

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A Ivana; Mondello, Chiara

    2011-03-04

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  4. Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

    Directory of Open Access Journals (Sweden)

    Ilaria Chiodi

    2011-03-01

    Full Text Available Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  5. Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

    Energy Technology Data Exchange (ETDEWEB)

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A. Ivana; Mondello, Chiara, E-mail: mondello@igm.cnr.it [Institute of Molecular Genetics, CNR, via Abbiategrasso 207, 27100 Pavia (Italy)

    2011-03-04

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  6. Photodynamic therapy-induced programmed cell death in carcinoma cell lines

    Science.gov (United States)

    He, Xiao-Yan; Sikes, Robert A.; Thomsen, Sharon L.; Chung, L.; Jacques, Steven L.

    1993-06-01

    The mode of cell death following photodynamic therapy (PDT) was investigated from the perspective of programmed cell death (apoptosis). Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a), and rat mammary carcinoma (MTF7) were treated by PDT following sensitization with dihematoporphyrin ether (DHE). The response of these carcinoma cell lines to PDT was variable. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder pattern indicative of internucleosomal cleavage of DNA during apoptosis. MTF7 and PC3 responded to PDT by inducing apoptosis while H322a had no apoptotic response. The magnitude of the response and the PDT dosage required to induce the effect were different in PC3 and MTF7. MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal apoptosis at the LD50 but had a marked response at the LD85. Furthermore, the onset of apoptosis followed slower kinetics in PC3 (2 hr - 4 hr) than in MTF7 (cells were killed by PDT but failed to exhibit any apoptotic response. This study indicates that apoptosis may occur during PDT induced cell death, but this pathway is not universal for all cancer cell lines.

  7. Interaction between x-irradiated plateau-phase bone marrow stromal cell lines and co-cultivated factor-dependent cell lines leading to leukemogenesis in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Naparstek, E.; Anklesaria, P.; FitzGerald, T.J.; Sakakeeny, M.A.; Greenberger, J.S.

    1987-03-01

    Plateau-phase mouse clonal bone marrow stromal cell lines D2XRII and C3H cl 11 produce decreasing levels of M-CSF (CSF-1), a specific macrophage progenitor cell humoral regulator, following X-irradiation in vitro. The decrease did not go below 40% of control levels, even after irradiation doses of 50,000 rad (500 Gy). In contrast, a distinct humoral regulator stimulating growth of GM-CSF/IL-3 factor-dependent (FD) hematopoietic progenitor cell lines was detected following radiation to doses above 2000 rad. This humoral factor was not detectable in conditioned medium from irradiated cells, weakly detected using factor-dependent target cell populations in agar overlay, and was prominently detected by liquid co-cultivation of factor-dependent cells with irradiated stromal cell cultures. Subclonal lines of FD cells, derived after co-cultivation revealed karyotypic abnormalities and induced myeloblastic tumors in syngeneic mice. Five-eight weeks co-cultivation was required for induction of factor independence and malignancy and was associated with dense cell to cell contact between FD cells and stromal cells demonstrated by light and electron microscopy. Increases in hematopoietic to stromal cell surface area, total number of adherent cells per flask, total non-adherent cell colonies per flask, and cumulative non-adherent cell production were observed after irradiation. The present data may prove very relevant to an understanding of the cell to cell interactions during X-irradiation-induced leukemia.

  8. Histamine as a Radiosensitizer of Malignant Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Rivera, E. S.; Medina, V.; Cricco, G.; Mohamed, N.; Croci, M.; Martin, G.; Nunez, M.; Bergoc, R. M.

    2004-07-01

    It has been established that the treatment with Histamine (Hi) produces a significant growth inhibition of different cell lines derived from human neoplasia. In a model of Knockout mice completely depleted of endogenous Hi, it was observed a significant delay in bone marroe repopulation after whole body irradiation. These results are in agreement with the hypothesis that histamine has a role in the regulation of haematopoiesis as well as an inhibitory effect on apoptosis. The objective of this paper was to study the possible effect of Hi as protector of normal cells and radiosensitizer of malignant ones. To study the effect of Hi on small-intestine and bone marrow, thirty made mice were randomly separeted into two groups: Control irradiated (C), and irradiated receiving Histamine (HI-group). All animals received a single dose of 10 Gy on whole-body employing a ''137Cs source of 189 TB{sub q} (Dose rate: 7.7 Gy/min) calibrated with TLD 700 dosimeter. Hi-group recieved a daily se injection (0.1 mg/kg) starting 20 hs before irradiation. Mice were sacrificed 5 days after irradiation. Histopathological analysis indicated that intestinal mucosae of C group showed important injury, whist mucosae of Hi-treated mice showed mild mucosal atrophy with conservation of villous projections and absence of vascular congestive changes. In order to investigate the effect of Hi on radiosensitivity of transformed cells, MDA-MB-231 (human breast carcinoma cells) were irradiated in vitro with doses ranging from 0 to 10 Gy. Results of radiobiological parameters indicate a significant increase on radiosensitivity of malignant cells. Employing specific fluorescent dyes and flow cytometric analysis we determined that the intracellular levels of hydrogen peroxide (H{sub 2}O{sub 2}) are significant increased by Hi 10 {mu}M in control and also in irradiated MDA-MB-231 cells, while the levels of superoxide (SO{sub 2}) were not significantly modified by Hi-treatment. (Author) 9 refs.

  9. Mitotic slippage and expression of survivin are linked to differential sensitivity of human cancer cell-lines to the Kinesin-5 inhibitor monastrol.

    Directory of Open Access Journals (Sweden)

    Hila Asraf

    Full Text Available The mitotic Kinesin-5 motor proteins crosslink and slide apart antiparallel spindle microtubules, thus performing essential functions in mitotic spindle dynamics. Specific inhibition of their function by monastrol-like small molecules has been examined in clinical trials as anticancer treatment, with only partial success. Thus, strategies that improve the efficiency of monastrol-like anticancer drugs are required. In the current study, we examined the link between sensitivity to monastrol and occurrence of mitotic slippage in several human cell-lines. We found that the rank of sensitivity to monastrol, from most sensitive to least sensitive, is: AGS > HepG2 > Lovo > Du145 ≥ HT29. We show correlation between the sensitivity of a particular cell-line to monastrol and the tendency of the same cell-line to undergo mitotic slippage. We also found that in the monastrol resistant HT29 cells, prolonged monastrol treatments increase mRNA and protein levels of the chromosomal passenger protein survivin. In contrast, survivin levels are not increased by this treatment in the monastrol-sensitive AGS cells. We further show that over-expression of survivin in the monastrol-sensitive AGS cells reduces mitotic slippage and increases resistance to monastrol. Finally, we show that during short exposure to monastrol, Si RNA silencing of survivin expression reduces cell viability in both AGS and HT29 cells. Our data suggest that the efficiency of anti-cancer treatment with specific kinesin-5 inhibitors may be improved by modulation of expression levels of survivin.

  10. Rhomboid-7 over-expression results in Opa1-like processing and malfunctioning mitochondria.

    Science.gov (United States)

    Rahman, Mokhlasur; Kylsten, Per

    2011-10-22

    Rhomboid-7 (rho-7) is a mitochondrial-specific intramembranous protease. The loss-of-function mutation rho-7 results in semi-lethality, while escapers have a reduced lifespan with several neurological disorders [1]. Here we show that general, or CNS-specific expression of rho-7 can rescue the lethality of rho-7. General, or CNS-specific over-expression of rho-7 in otherwise wild-type animals caused semi-lethality, with approximately 50% of the animals escaping this lethality, developing into adults displaying a shortened life span with larval locomotory problem. On a cellular level, over-expression resulted in severe depression of ATP levels and cytochrome c oxidase subunit II mRNA levels, a lowered number of mitochondria in neurons and aggregation of mitochondria in the brain indicating mitochondrial malfunction. Over-expression of rho-7 in developing eye discs resulted in an elevated apoptotic index. In the CNS, elevated levels of rho-7 were accompanied by both isoforms of Opa1-like, a dynamin-like GTPase, a mitochondrial component involved in regulating mitochondrial dynamics and function, including apoptosis. Most, but not all, of rho-7 over-expression phenotypes were suppressed by introducing a heterozygous mutation for Opa1-like. Our results suggest that rho-7 and Opa1-like function in a common molecular pathway affecting mitochondrial function and apoptosis in Drosophila melanogaster.

  11. CXCL12/SDF-1 over-expression in human insulinomas and its biological relevance

    DEFF Research Database (Denmark)

    Ilhan, Aysegul; Nabokikh, Anastasiya; Maj, Magdalena

    2009-01-01

    This study was performed on the basis of previously obtained investigative gene array data concerning the over-expression of CXCL12/SDF-1 in human insulinomas versus human pancreatic islet preparations. The presence of CXCL12/SDF-1 was studied by RT-qPCR in human insulinomas (n=8) versus pancreatic...

  12. Evaluation of Stem Cell Markers, CD44/CD24 in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Masoud Hashemi Arabi

    2014-05-01

    Four breast cancer cell lines, MCF-7 ، T47D ، MDA-MB231 and MDA-MB468 were purchased from National cell Bank of Iran based in Iran Pasture Institute and were cultured in high glucose DMEM supplemented with 10% FCS. Cells were stained with antiCD44-PE and antiCD24-FITC antibodies and Status of CD44 and CD24 as markers of breast cancer stem cells were evaluated using flow cytometer and fluorescent microscopy.Evaluation of CD44 and CD24 as markers of breast cancer stem cells showed that MDA-MB231 with 97±1.2% CD44+/CD24-/low cells is significantly different from the others that they were mainly CD44 and CD24 positive cells(p

  13. Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity

    OpenAIRE

    D’Angelo, Rosemarie C.; Ouzounova, Maria; Davis, April; Choi, Daejin; Tchuenkam, Stevie M.; Kim, Gwangil; Luther, Tahra; Quraishi, Ahmed A.; Senbabaoglu, Yasin; Conley, Sarah J; Shawn G Clouthier; Hassan, Khaled A.; Wicha, Max S; Korkaya, Hasan

    2015-01-01

    Developmental pathways such as Notch play a pivotal role in tissue specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch-) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays we investigated the role of Notch ...

  14. In vitro evaluation of a new nitrosourea, TCNU, against human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Roed, H; Vindeløv, L L; Spang-Thomsen, M;

    1987-01-01

    The cytotoxic activity of a new nitrosourea, TCNU, was compared with that of BCNU in five human small cell lung cancer cell lines in vitro. TCNU was found to be equivalent or inferior to BCNU when compared on a microgram to microgram basis. If the potential of in vitro phase II trials for selecti...... of new drugs can be validated, it can be concluded that TCNU is not superior to other nitrosoureas for the treatment of SCCL....

  15. Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension

    DEFF Research Database (Denmark)

    Krabbe, Christina; Courtois, Elise; Jensen, Pia

    2009-01-01

    differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x(L) over-expressing subline (hVMbcl-x(L)) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10...

  16. Comparative In Vitro Immune Stimulation Analysis of Primary Human B Cells and B Cell Lines

    Science.gov (United States)

    Van Belle, Kristien; Herman, Jean; Boon, Louis; Waer, Mark

    2016-01-01

    B cell specific immunomodulatory drugs still remain an unmet medical need. Utilisation of validated simplified in vitro models would allow readily obtaining new insights in the complexity of B cell regulation. For this purpose we investigated which human B lymphocyte stimulation assays may be ideally suited to investigate new B lymphocyte immunosuppressants. Primary polyclonal human B cells underwent in vitro stimulation and their proliferation, production of immunoglobulins (Igs) and of cytokines, and expression of cell surface molecules were analysed using various stimuli. ODN2006, a toll-like receptor 9 (TLR9) agonist, was the most potent general B cell stimulus. Subsequently, we investigated on which human B cell lines ODN2006 evoked the broadest immunostimulatory effects. The Namalwa cell line proved to be the most responsive upon TLR9 stimulation and hence may serve as a relevant, homogeneous, and stable B cell model in an in vitro phenotypic assay for the discovery of new targets and inhibitors of the B cell activation processes. As for the read-out for such screening assay, it is proposed that the expression of activation and costimulatory surface markers reliably reflects B lymphocyte activation. PMID:28116319

  17. Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polym...

  18. Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidop teran Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Yan-lei Wu; Lei Jiang; Yoshifumi Hashimoto; Robert R.Granados; Guo-xun Li

    2011-01-01

    Lepidopteran heat-tolerant(ht)cell lines have been obtained with sf-9,sf-21 and several Bombyx cells.They have a distinct karyotype,membrane lipid composition,morphology and growth kinetics from the parental cell lines.In this paper,we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility.Adaptation of cell lines sf-9,BTI-TN-5131-4(High5)and BTI-TN-MG1(MG 1)to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature.The process of adaption to a higher culture temperature was accomplished over a period of 2 months.The cell lines with the temperature adaption were designated as sf9-ht33,sf9-ht35,High5-ht33,High5-ht35,MG1-ht33,MG1-ht35.These cell lines have been subcultured over 70 passages.Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines.The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines.Cell shapes did not show obvious change,however,the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption.When the cell lines were infected with Autographa californica nuclear polyhedrosis virus(AcMNPV)at 28℃,33℃,35℃ and 37℃,production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.

  19. Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines

    Directory of Open Access Journals (Sweden)

    Maria Bernard L

    2006-01-01

    Full Text Available Abstract Background Pluripotent mouse embryonic stem (ES cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321 or Stem Cell Factor (SCF. Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium. RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed.

  20. Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines

    Science.gov (United States)

    Serfozo, Peter; Schlarman, Maggie S; Pierret, Chris; Maria, Bernard L; Kirk, Mark D

    2006-01-01

    Background Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF). Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium). RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed. PMID:16436212

  1. Establishment and characterization of a cell line (OMC-9) originating from a human endometrial stromal sarcoma.

    Science.gov (United States)

    Kakuno, Yoshiteru; Yamada, Takashi; Mori, Hiroshi; Narabayashi, Isamu

    2008-05-01

    Cell lines are very useful for clinical and basic research. The establishment of uterine malignant tumor cell lines with unusual histology is especially important. We describe the establishment and characterization of a new human endometrial stromal sarcoma cell line of the uterus. The cell line OMC-9 was established from a tumor mass in the uterine body of a 55-year-old woman. Characteristics of the cell line studied include morphology, chromosome analysis, heterotransplantation, tumor markers and chemosensitivity. This cell line has grown well for 196 months and has been subcultured more than 50 times. Monolayer cultured cells are polygonal in shape, appear to be spindle-shaped or multipolar and have a tendency to pile up without contact inhibition. The