Sample records for cell line stably

  1. 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: [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)


    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.

  2. Characterization of new cell line stably expressing CHI3L1 oncogene

    Directory of Open Access Journals (Sweden)

    Chekhonin V. P.


    Full Text Available Aim. To characterize the immortalized 293 cell line after stable transfection with human oncogene (CHI3L1. Methods. 293 cells, stably transfected with pcDNA3.1_CHI3L1, and 293 cells, stably transfected with pcDNA3.1 as a negative control, were used throughout all experiments. The clones of CHI3L1-expressing 293 cells and 293 cells, transfected with pcDNA3.1, were analyzed by immunofluorescence and confocal microscopy. Cell proliferation was measured using MTT assay; analyses of ERK1/2 and AKT activation and their cellular localization were performed with anti-phospho-ERK and anti-phospho-AKT antibodies. Specific activation of MAP and PI3 kinases was measured by densitometric analysis of Western-blot signals. Results. The obtained results show quite modest ability of CHI3L1 to stimulate cell growth and reflect rather an improved cellular plating efficiency of the 293 cells stably transfected with pcDNA3.1_CHI3L1 as compared to the 293 cells transfected with an «empty» vector. ERK1/2 and AKT are activated in the 293_CHI3L1 cells. In these cells phosphorylated ERK1/2 were localized in both cell cytoplasm and nuclei while AKT only in cytoplasm. The 293_CHI3L1 cells differed from the 293 cells, transfected with an «empty» vector, in their size and ability to adhere to the culture plates. Conclusions. The overexpression of CHI3L1 is likely to have an important role in tumorigenesis via a mechanism which involves activation of PI3K and ERK1/2 pathways. The tumors which can be induced by orthotopic implantation of the transformed human cells with overexpressed human oncogene CHI3L1 into the rat brain can be used as a target for anticancer drug development.

  3. Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

    DEFF Research Database (Denmark)

    Jacobsen, Stine Engesgaard; Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie;


    receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort...... of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A...

  4. Development of Cell Lines Stably Expressing Staphylococcal Nuclease Fused to Dengue 2 Virus Capsid Protein for CTVI

    Institute of Scientific and Technical Information of China (English)

    Cheng-Feng QIN; E-De QIN


    To explore the potential application of capsid-targeted viral inactivation(CTVI)strategy in prophylactic model against dengue virus(DV)infection,here we fused a Ca2+-dependent nuclease,staphylococcal nuclease(SN),to the capsid protein of dengue 2 virus(D2C)at the carboxyl terminal,and constructed the desired expression plasmid pc/D2C-SN and control plasmids pc/D2C-SN* and pc/D2C.A mammalian cell line BHK-21 was transfected by electroporation with those plasmids and thereafter selected by 5 μg/ml blasticidin.The resistant cell clones were then expanding cultured and screened by RT-PCR and Western Blot assays.The nuclease activity of the expressed fusion protein D2C-SN was analyzed by in vitro DNA digestion assay.It was confirmed cell lines stably expressing D2C-SN and control constructs were obtained.The intracellular expressed fusion protein D2C-SN had ideal nuclease activity and no cytotoxicity on mammalian cells.Those engineered cell lines provided the experimental system for CTVI application in prophylactic model and paved the new road for combating DV infection with CTVI.

  5. Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

    Directory of Open Access Journals (Sweden)

    Shaoxing YANG


    Full Text Available Background and objective CD147, a type of transmembrane glycoprotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD147 gene was amplified by real-time polymerase chain reaction (RT-PCR, inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable, overexpressed cell line named A549-CD147. The mRNA expression of MMP-9 was examined by RT-PCR. The proliferation and invasive ability of the human lung cancer cells before and after transfection were examined by the CCK-8 and Transwell methods. Results A CD147 lentiviral expression vector (pEGFP-CD147 was successfully constructed by restrictive enzyme digestion and plasmid sequencing. RT-PCR and Western blot analyses revealed increased mRNA and protein expression of CD147 gene in cells transfected with pEGFP-CD147 compared with the control groups. Therefore, the A549-CD147 cell line was successfully established through the experiment. The mRNA expression of MMP-9 also significantly increased after the upregulation of CD147 expression. Meanwhile, CCK-8 and Transwell assays indicated that the proliferation and invasive ability significantly increased in the A549-CD147 cells. Conclusion A lentiviral CD147 expression vector and its A549 cell line (A549-CD14 were successfully constructed. CD147 overexpression upregulated the protein expression of MMP-9, and strengthened the proliferation and invasive ability of human lung adenocarcinoma cells.

  6. Activation of transgenic estrogen receptor-beta by selected phytoestrogens in a stably transduced rat serotonergic cell line. (United States)

    Amer, Dena A M; Kretzschmar, Georg; Müller, Nicole; Stanke, Nicole; Lindemann, Dirk; Vollmer, Günter


    Many flavonoids, a major group of phenolic plant-derived secondary metabolites, are known to possess estrogen-like bioactivities. However, little is known about their estrogenic properties in the central nervous system due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor beta (ERbeta). To overcome this deficit, we have created a cellular model, which is serotonergic in origin, to study properties of estrogenic substances by stably transducing RN46A-B14 cells derived from raphe nuclei region of the rat brain with a lentiviral vector encoding a human ERbeta. We clearly showed that the transgenic human ERbeta is a spontaneously expressed and a functional receptor. We have further assessed the estrogenicity of three different isoflavones and four different naringenin-type flavanones in this cell line utilizing a luciferase reporter gene assay. Genistein (GEN), Daidzein (DAI), Equol (EQ), Naringenin (NAR) and 8-prenylnaringenin (8-PN) showed strong estrogenic activity in a concentration-dependent manner as compared to 7-(O-prenyl)naringenin-4'-acetate (7-O-PN) which was only slightly estrogenic and 6-(1,1-dimethylallyl)naringenin (6-DMAN) that neither showed estrogenic nor anti-estrogenic activity in our model. All observed effects could be antagonized by the anti-estrogen fulvestrant. Moreover, co-treatment of cells with 17beta-estradiol (E2) and either GEN or DAI showed a slight additive effect as compared to EQ. On the other hand, 8-PN in addition to 7-O-PN, but not NAR and 6-DMAN, were able to slightly antagonize the responses triggered by E2. Our newly established cellular model may prove to be a useful tool in explicating basic physiological properties of ERbeta in the brain and may help unravel molecular and cellular mechanisms involved in serotonergic mood regulation by estrogen or potential plant-derived secondary metabolites.

  7. GnRH receptor activation competes at a low level with growth signaling in stably transfected human breast cell lines

    Directory of Open Access Journals (Sweden)

    Morgan Kevin


    Full Text Available Abstract Background Gonadotrophin releasing hormone (GnRH analogs lower estrogen levels in pre-menopausal breast cancer patients. GnRH receptor (GnRH-R activation also directly inhibits the growth of certain cells. The applicability of GnRH anti-proliferation to breast cancer was therefore analyzed. Methods GnRH-R expression in 298 primary breast cancer samples was measured by quantitative immunofluorescence. Levels of functional GnRH-R in breast-derived cell lines were assessed using 125I-ligand binding and stimulation of 3H-inositol phosphate production. Elevated levels of GnRH-R were stably expressed in cells by transfection. Effects of receptor activation on in vitro cell growth were investigated in comparison with IGF-I and EGF receptor inhibition, and correlated with intracellular signaling using western blotting. Results GnRH-R immunoscoring was highest in hormone receptor (triple negative and grade 3 breast tumors. However prior to transfection, functional endogenous GnRH-R were undetectable in four commonly studied breast cancer cell lines (MCF-7, ZR-75-1, T47D and MDA-MB-231. After transfection with GnRH-R, high levels of cell surface GnRH-R were detected in SVCT and MDA-MB-231 clones while low-moderate levels of GnRH-R occurred in MCF-7 clones and ZR-75-1 clones. MCF-7 sub-clones with high levels of GnRH-R were isolated following hygromycin phosphotransferase transfection. High level cell surface GnRH-R enabled induction of high levels of 3H-inositol phosphate and modest growth-inhibition in SVCT cells. In contrast, growth of MCF-7, ZR-75-1 or MDA-MB-231 clones was unaffected by GnRH-R activation. Cell growth was inhibited by IGF-I or EGF receptor inhibitors. IGF-I receptor inhibitor lowered levels of p-ERK1/2 in MCF-7 clones. Washout of IGF-I receptor inhibitor resulted in transient hyper-elevation of p-ERK1/2, but co-addition of GnRH-R agonist did not alter the dynamics of ERK1/2 re-phosphorylation. Conclusions Breast cancers


    Lindley, David J; Carl, Stephen M; Mowery, Stephanie A; Knipp, Gregory T


    There have been relatively few studies focused on the proton-dependent oligopeptide transporter (POT) superfamily member, Peptide/Histidine Transporter 1 (PHT1), with respect to its contribution to the ADME of peptides and peptide-based drugs. These studies were conducted to determine hPHT1-mediated, H(+)-dependent uptake kinetics of histidine, carnosine, Gly-Sar and valacyclovir in stably transfected hPHT1-COS-7 cells comparative to kinetics determined in an empty vector (Mock) stably transfected cell line. The results suggest that Gly-Sar appears to be a substrate for PHT1 based on efflux from the stably transfected hPHT1 COS-7 cells. Histidine and Gly-Sar concentration- and time-dependent studies suggest mixed-uptake kinetics. These studies suggest that stably transfected hPHT1-COS-7 cells exhibit different uptake kinetics than those observed in our previous studies and illustrate the requirement for experiments to delineate the physiological role of hPHT1.

  9. Establishment of Human Embryonic Stem Cell Line Stably Expressing Epstein-Barr Virus-Encoded Nuclear Antigen 1

    Institute of Scientific and Technical Information of China (English)

    Cai-Ping REN; Ming ZHAO; Wen-Jiao SHAN; Xu-Yu YANG; Zhi-Hua YIN; Xing-Jun JIANG; Hong-Bo ZHANG; Kai-Tai YAO


    Human embryonic stem (hES) cells have the capability of unlimited undifferentiated proliferation,yet maintain the potential to form perhaps any cell type in the body. Based on the high efficiency of the Epstein-Barr virus-based episomal vector in introducing exogenous genes of interest into mammalian cells,we applied this system to hES cells, expecting that this would resolve the problem of poor transfection efficiency existing in current hES cell research. Therefore, the first step was to establish EBNAl-positive hES cells. Using the Fugene 6 transfection reagent, we transfected hES cells with the EBNA1 expression vector and subsequently generated hES cell clones that stably expressed EBNA 1 under drug selection. These clones were confirmed to express EBNA1 mRNA by RT-PCR and to express EBNA1 protein by Western blotting. Furthermore, luciferase reporter gene analysis was performed on the EBNA1 clones and revealed that the expressed EBNA1 protein was functional. When the EBNAl-positive cells were injected into severe combined immunodeficient (SCID) mice, they formed teratoma tissues containing all three embryonic germ layers and EBNA1 protein was detected in these teratoma tissues by Western blotting. All the results show that we have successfully created stable EBNA1-hES cells, thus laying a good foundation for further research.

  10. Construction of a hepatocellular carcinoma cell line that stably expresses stathmin with a Ser25 phosphorylation site mutation. (United States)

    Du, J; Tao, Z H; Li, J; Liu, Y K; Gan, L


    We constructed hepatocellular carcinoma (HCC) cells that stably express stathmin with a Ser25 phosphorylation site mutation (stathmin S25A). We used the polymerase chain reaction for site-directed mutagenesis, constructed a stathmin S25A plasmid, and verified the results by restriction enzyme cleavage and sequencing technology. Using the liposome transfection method, stathmin wild-type and S25A HCCLM6 cells were established, which were identified by western blotting. The sequencing report of the stathmin S25A plasmid showed that stathmin serine at position 25 had mutated into alanine. Stable cells transfected with stathmin wild-type and S25A plasmids were constructed. Using western blotting, we confirmed that the expression level of stathmin pS25 in the stathmin S25A cells was reduced than that in the stathmin wild-type and HCCLM6 control cells (P stathmin S25A HCCLM6 cells, which offer an experimental model for further investigation of the molecular mechanism of stathmin phosphorylation in hepatocarcinogenesis.

  11. Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1. (United States)

    Nukuzuma, Souichi; Nakamichi, Kazuo; Kameoka, Masanori; Sugiura, Shigeki; Nukuzuma, Chiyoko; Miyoshi, Isao; Takegami, Tsutomu


    The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.

  12. Propagation of Hepatitis B Virus in a Rat Hepatoma Cell Line Stably Transfected with Human Annexin-V

    Directory of Open Access Journals (Sweden)

    Seyed Mohammad Jazayeri


    Full Text Available Background and Aims: Hepatitis B virus (HBV displays a distinct hepatotropism and a narrow host range in vivo. However, very little is known about the interaction of HBV with its host cells, mainly because of difficulties in the development of suitable tissue culture system. We present here confirmatory evidence of a putative role of annexin-V in HBV infection. Methods: HBV from both human sera and from culture supernatants from HepG2 2.15 cells were used to infect FTO9.1 cells (a rat hepatoma cell line transfected with a construct containing human annexin-V. Cells and culture supernatants were assayed at various times post-infection by immunofluorescent microscopy (HBcAg staining in nucleus, and by HBV cccDNA-specific PCR. Supernatants from these initially infected cells were then used to infect fresh FTO9.1 cells with a similar outcome to primary infection. Results: Core and surface gene PCRs were positive on days 2, 5 and following transfer experiments. cccDNA-specific PCR confirmed internalisation of the virus into the nucleus. HBcAg fluorescence showed nuclear staining on days 2, 5 and following transfer experiments. Addition of recombinant annexin-V and DMSO to the cell culture medium resulted in a greater efficiency of infection. Later washes were negative for HBV-DNA, ruling out contamination of the cells by external HBV particles. Conclusions: This cell line does appear to be useful in the study of the early stages of HBV infection, but requires further evaluation.

  13. Expression of particulate-form of Japanese encephalitis virus envelope protein in a stably transfected Drosophila cell line

    Directory of Open Access Journals (Sweden)

    Zhang Li


    Full Text Available Abstract Background Japanese encephalitis virus (JEV, a member of the family Flaviviridae, is an important mosquito-borne human pathogen. Its envelope glycoprotein (E is the major determinant of the pathogenicity and host immune responses. In the present study, we explored the feasibility of producing recombinant JEV E protein in the virus-free Drosophila expression system. Results The coding sequence for the signal sequence of premembrane and E protein was cloned into the Drosophila expression vector pAc5.1/V5-His. A Drosophila cell line S2 was cotransfected with this construct as well as a plasmid providing hygromycin B resistance. A cell line expressing the JEV E protein was selected by immunofluoresence, confocal microscopy, and western blot analysis using three different monoclonal antibodies directed against JEV E protein. This cell line was stable in the yield of JEV E protein during two months in vitro maintenance in the presence of hygromycin B. The results showed that the recombinant E protein had an expected molecular weight of about 50 kilodalton, was immunoreactive with all three monoclonal antibodies, and found in both the cytoplasm and culture supernatant. Sucrose gradient ultracentrifugation analysis revealed that the secreted E protein product was in a particulate form. It migrated to the sucrose fraction with a density of 1.13 g/ml. Balb/c mice immunised with the sucrose fraction containing the E protein particles developed specific antibodies. These data show that functioning JEV E protein was expressed in the stable S2 cell line. Conclusion The Drosophila expression system is a more convenient, cheaper and safer approach to the production of vaccine candidates and diagnostic reagents for JEV.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. (United States)

    Legler, J; van den Brink, C E; Brouwer, A; Murk, A J; van der Saag, P T; Vethaak, A D; van der Burg, B


    Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, non-estradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 microM. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.

  16. The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha(1A1)-adrenoceptors

    DEFF Research Database (Denmark)

    Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork


    challenged with phenylephrine (EC(50)=1.61x10(-8) M). From Schild analysis, prazosin, sertindole, risperidone, and haloperidol caused a concentration-dependent, rightward shift of the cumulative concentration-response curves for phenylephrine in cells expressing human recombinant alpha(1A1)-adrenoceptors...... human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole...... for human alpha(1A1)-adrenoceptors compared to haloperidol. These findings are consistent with the observation that risperidone and sertindole have a higher incidence of orthostatic hypotension than haloperidol....

  17. Development of a human breast-cancer derived cell line stably expressing a bioluminescence resonance energy transfer (BRET-based phosphatidyl inositol-3 phosphate (PIP3 biosensor.

    Directory of Open Access Journals (Sweden)

    Mei-Shiue Kuo

    Full Text Available Stimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP2 to generate PIP3, which recruit Akt to the plasma membrane through its pleckstrin homology (PH domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP3 production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP3 production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP3 and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer.

  18. Manganese superoxide dismutase: effect of the ala16val polymorphism on protein, activity, and mRNA levels in human breast cancer cell lines and stably transfected mouse embryonic fibroblasts. (United States)

    McAtee, Britt L; Yager, James D


    The manganese superoxide dismutase (MnSOD) ala16val polymorphism has been associated with various diseases including breast cancer. In the present study, we investigated levels of MnSOD protein, enzymatic activity, and mRNA with respect to MnSOD genotype in several human breast carcinoma cell lines and in mouse embryonic fibroblasts (MEF), developed from the MnSOD knockout mouse, stably expressing human MnSOD-ala and MnSOD-val. In human breast cell lines, the MnSOD-ala allele was associated with increased levels of MnSOD protein and MnSOD protein per unit mRNA. In the MEF transformants, MnSOD activity correlated fairly well with MnSOD protein levels. MnSOD mRNA expression was significantly lower in MnSOD-ala versus MnSOD-val lines. MnSOD protein and activity levels were not related to MnSOD genotype in the transformed MEF, although, as observed in the human breast cell lines, the MEF human MnSOD-ala lines produced significantly more human MnSOD protein per unit mRNA than the human MnSOD-val lines. This suggests that there is more efficient production of MnSOD-ala protein compared to MnSOD-val protein. Examination of several indicators of reactive oxygen species levels, including superoxide and hydrogen peroxide, in wild-type MEF and in MEF expressing similar elevated amounts of MnSOD-ala or val activity did not show differences related to the levels of MnSOD protein expression. In conclusion, in both human breast carcinoma cell lines and MEF cell lines stably transfected with human MnSOD, the MnSOD-ala allele was associated with increased production of MnSOD protein per unit mRNA indicating a possible imbalance in MnSOD protein production from the MnSOD-val mRNA.

  19. Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing

    Institute of Scientific and Technical Information of China (English)

    Jian Zhu-Ge; Ying-Nian Yu; Yu-Li Qian; Xin Li


    AIM: The human cytochrome P-450 2C18(CYP2C18) hasbeen characterized. However, the protein has not beenpurified from liver and very little is known regarding thespecific substrate of CYP2C18. In order to study its enzymaticactivity for drug metabolism, the CYP2C18cDNA was clonedand a stable CHL cell line expressing recombinant CYP 2C18was established.METHODS: The human CYP2C18cDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned intopGEM-T vector. The cDNA segment was identified by DNAsequencing and subcloned into a mammalian expressionvector pREP9. A transgenic cell line was established bytransfecting the recombinant plasmid of pREPg-CYP2C18toChinese hamster lung (CHL) cell. The enzyme activity ofCYP2C18 catalyzing oxidation of tolbutamide tohydroxytolbutamide in postmitochondrial supernant(Sg)fraction of the cell was determined by high performanceliquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from thecloned cDNA segment was identical to that of reported byRomkes et al(GenBank accession number: M61856,J05326).The S9 fraction of the established cell line metabolizestolbutamide to hydroxytolbutamide. Tolbutamide hydroxylaseactivity was found to be 0.509±0.052 μmol.min-1.g-1 S9protein or 8.82±0.90 mol.min-1.mol-1 CYP, but wasundetectable in parental CHL cell. In addition, we haveidentified a CYP2C18cDNA clone with exon 5 missing.CONCLUSION: The cDNA of human CYP2C18 wassuccessfully cloned and a cell line, CHL-CYP2C18, efficientlyexpressing the protein of CYP2C18, was established. Aspliced variant of CYP2C18 with exon 5 missing was identifiedin the cloning process.

  20. A Recombinant Human Pluripotent Stem Cell Line Stably Expressing Halide-Sensitive YFP-I152L for GABAAR and GlyR-Targeted High-Throughput Drug Screening and Toxicity Testing (United States)

    Kuenzel, Katharina; Friedrich, Oliver; Gilbert, Daniel F.


    GABAARs and GlyRs are considered attractive drug targets for therapeutic intervention and are also increasingly recognized in the context of in vitro neurotoxicity (NT) and developmental neurotoxicity (DNT) testing. However, systematic human-specific GABAAR and GlyR-targeted drug screening and toxicity testing is hampered due to lack of appropriate in vitro models that express native GABAARs and GlyRs. We have established a human pluripotent stem cell line (NT2) stably expressing YFP-I152L, a halide-sensitive variant of yellow fluorescent protein (YFP), allowing for fluorescence-based functional analysis of chloride channels. Upon stimulation with retinoic acid, NT2 cells undergo neuronal differentiation and allow pharmacological and toxicological evaluation of native GABAARs and GlyRs at different stages of brain maturation. We applied the cell line in concentration-response experiments with the neurotransmitters GABA and glycine as well as with the drugs strychnine, picrotoxin, fipronil, lindane, bicuculline, and zinc and demonstrate that the established in vitro model is applicable to GABAAR and GlyR-targeted pharmacological and toxicological profiling. We quantified the proportion of GABAAR and GlyR-sensitive cells, respectively, and identified percentages of approximately 20% each within the overall populations, rendering the cells a suitable model for systematic in vitro GABAAR and GlyR-targeted screening in the context of drug development and NT/DNT testing. PMID:27445687

  1. Construction and Identification of a Vero Cell Line Stably Expressing Canine SLAM Gene%稳定表达犬信号淋巴细胞激活因子基因细胞株的建立与鉴定

    Institute of Scientific and Technical Information of China (English)

    苏建青; 褚秀玲; 马秀亮; 江成; 张吉清


    [目的]构建稳定表达犬瘟热病毒细胞受体——犬信号淋巴细胞激活因子(SLAM)的非洲绿猴肾细胞株(Vero).[方法]采用RT-PCR方法从犬外周血淋巴细胞中扩增出SLAM基因,将其克隆到哺乳动物真核表达载体pcDNA3.1(+)中,构建重组质粒pcDNA3.1/SLAM.采用脂质体将peDNA3.1/SLAM转染到Vero细胞中,利用G418加压筛选和纯化培养获得稳定表达SLAM的重组Vero细胞株.应用RT-PCR和间接免疫荧光试验检测SLAM的表达.[结果]重组蛋白SLAM在Vero细胞中获得表达,并且在不同代次的阳性细胞株中均能稳定表达目的蛋白.[结论]该研究建立了稳定表达犬SLAM的细胞株Vero/SLAM,为犬瘟热病毒的分离和生物学特性研究提供了平台.%[Objective] The aim was to construction a Vero cell line stably expressing SLAM. [ Method]SLAM gene was amplified by RT-PCR from canine peripheral blood lymphocytes and inserted into the eukaryotic expression vector pcDNA3.1 { + ) to construct a recombinant plasmid pcDNA3.1( + )/SlAM. The pcI)NA3.1( + )/SLAM was then transfected into the Vero cells by Lipofectamine 2000. The Vero cell stably expressing the SLAM was screened and purified under the drug selection of G4I8. The expression of SLAM was confirmed by KT-PCR and indirect immunofluorescence assay. [ Result ] The recombinant SLAM was correctly and effectively expressed in Vero cells. [ Conclusion ] The study establishes SLAM-expressing cell line, which provides a platform for studying the isolation and biological functions of canine distemper virus.

  2. Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule (United States)

    Chen, Weiye; Li, Cuicui; Xie, Meimei; Bu, Zhigao


    From 2013 to 2015, peste des petits ruminants (PPR) broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA) was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM) cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP) (rPPRV-GFP), an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT). Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field. PMID:27768770

  3. 人血小板生成素基因的克隆及CHO稳定细胞系的建立%Cloning of Human Thrombopoietin Gene and Establishment of CHO Cell Lines Stably Expressing TPO Gene

    Institute of Scientific and Technical Information of China (English)

    马倩倩; 宋玲玲; 王红梅; 方永志; 王立群; 何洪彬


    以人肝cDNA为模板克隆了人血小板生成素(Human Thrombopoietin,hTPO)基因,利用基因重组技术构建了带有新霉素抗性基因(neo)筛选标记的pcDNA3.1(+)-hTPO真核表达载体,将重组质粒瞬时转染293T细胞,用鼠抗人TPO单抗Western blot检测TPO蛋白的瞬时表达;再将重组质粒转染中国仓鼠卵巢细胞(Chinese Hamster Ovary,CHO),应用400 μg/mL的G418筛选克隆,经PCR及Western blot验证,获得了3株hTPO蛋白表达水平不同的CHO细胞系,为获得大量蛋白并进行活性功能试验及临床应用奠定基础.%Human thrombopoietin (Human Thrombopoietin, hTPO) gene was cloned by PCR using the human liver cDNA as template, then fragment of hTPO gene was cloned into pcDNA3. 1 ( + ) eukaryotic expression vector which has the neomycin resistance gene (neo) selection marker, named as pcDNA3. 1 ( + )-hTPO. First, the recombinant plasmid was transiently transfected into 293T cells. The transient expression of TPO in eukaryotic cells was determined by Western blot using mouse anti-human TPO monoclonal antibody; then Chinese hamster ovary cells (Chinese hamster ovary, CHO) were transfected with pcDNA3. 1 ( + ) -hTPO recombinant plasmid, using G418 of 400 jug/ml to select CHO stable cell lines, and verified by PCR and Western blot. The above results showed that three CHO cell lines stably expressing TPO gene were established. The cell lines may get a large number of proteins applied to actual medical and activity functional experiments and even laid a certain foundation for the next stage.

  4. 稳定表达人CCR5基因CHO细胞系的建立及鉴定%Establishment and characterization of CHO cell line stably expressing human CCR5 gene

    Institute of Scientific and Technical Information of China (English)

    程林; 吴喜林; 袁钟平; 吴稚伟


    CCR5 is one of the most important co-receptors required for HIV-I infection and a potential target for anti viral agents. In this study,the eukaryotic expression plasmid pcDNA3.1 CCR5 carrying human CCR5 gene was stably transfected into CHO-K1 cells. After 2 weeks selection by G418, cell clones were selected from limited dilution in 96-well plates,and 22 clones were obtained. All the clones were analyzed for cell surface CCR5 expression using flow cytometry, and clone 10 was identified as a high expression clone. The CCR5 gene transcription of the clone 10 was further analyzed using RT PCR and gel electrophoresis,and the target band was visible in the expected location. Cellular ELISA indicated that the surface CCR5 expression of clone 10 was 13. 6 fold higher than the control cells. Our results indicated that the CHO cell line stably expressing human CCR5 can be a useful tool for study viral co receptor,specific antibody screening and anti-viral agents.%CC型趋化因子受体5(CCR5)是HIV-1感染机体所需的最重要的辅助受体和潜在的抗病毒药物靶点之一.将含有人CCR5基因的真核表达质粒pcDNA3.1CCR5稳定转染CHOK1细胞,G418筛选2周后,在96孔板内通过有限稀释法培养细胞单克隆,最后得到22个细胞克隆,用流式细胞术检测细胞表面CCR5蛋白,发现克隆10能够高表达人CCR5基因.使用RT—PCR鉴定克隆10CCR5基因转录情况,结果在预期的位置检测出目的条带.采用细胞ELISA的方法进一步鉴定克隆10细胞表面CCR5的表达,结果该克隆的405nm光密度值是对照组的13.6倍.结果表明,本研究建立的稳定转染人CCR5的CHO细胞系能够高效表达CCR5基因,为研究HIV—1共受体、筛选病毒中和抗体、以及抗病毒药物奠定了基础.

  5. Establishment and characterization of human engineered cells stably expressing large extracellular matrix proteins. (United States)

    Kwon, Daekee; Kang, Gwang-Sik; Han, Dong Keun; Park, Kwideok; Kim, Jae-Hwan; Lee, Soo-Hong


    Commercially available extracellular matrix (ECM) hydrogel-coated culture plates have been used to study the relationship between the ECM microenvironment and stem cell behavior. However, it is unclear whether ECM-coated dishes mimic the natural ECM microenvironment because the architecture of the ECM is constructed of randomly distributed fibers. The purpose of this study was the production and confirmation of human engineered cell lines stably expressing large ECM proteins such as collagen I/II and fibronectin. First, large (over 10 kb) ECM vectors encoding human collagen I/II and fibronectin were constructed and the circular vectors were linearized. Second, the linear ECM vectors were introduced into immortalized human embryonic kidney cells using various transfection methods. The polyethylenimine and liposome methods showed higher efficiencies than electroporation for transfection of these large vectors. Third, human ECM engineered cells were established by stable integration of the vector into the genomic DNA and resulted in stable overexpression of mRNA and proteins. In summary, human engineered cell lines stably expressing large ECM proteins such as human collagen I/II and fibronectin were successfully prepared, and secretion of the ECM components into the surrounding environment was confirmed by immunocytochemistry. Thus, human ECM engineered cells naturally secreting ECM components could be valuable for studying the relationship between the native ECM microenvironment and stem cell behavior.

  6. Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat. (United States)

    Nukuzuma, Souichi; Kameoka, Masanori; Sugiura, Shigeki; Nakamichi, Kazuo; Nukuzuma, Chiyoko; Miyoshi, Isao; Takegami, Tsutomu


    Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter. In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.

  7. Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

    Institute of Scientific and Technical Information of China (English)

    朱长虹; 田红


    In order to assess the impact of mRNA degradation on steady state levels of follicle-stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half-life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time-dependent changes in FSHR mRNA content were determined by nuclease protection-solution hybridization assay (NPA) or by qualitative reverse transcription-competitive polymerase chain reaction (RT-PCR) in cultured hFSHR-YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR-Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT-PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of [3 H]uridine into total RNA, by 90 % within 1 h in hFSHR-Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time-dependent decrease in FSHR mRNA content in hFSHR-Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half-life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT-PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

  8. Stem cells expanded from the human embryonic hindbrain stably retain regional specification and high neurogenic potency. (United States)

    Tailor, Jignesh; Kittappa, Raja; Leto, Ketty; Gates, Monte; Borel, Melodie; Paulsen, Ole; Spitzer, Sonia; Karadottir, Ragnhildur Thora; Rossi, Ferdinando; Falk, Anna; Smith, Austin


    Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

  9. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells (United States)

    Mandegar, Mohammad A.; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y.L.; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P.; Volpi, Emanuela V.; Thrasher, Adrian J.; James, William; Monaco, Zoia L.


    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  10. 流感病毒NS1蛋白稳定表达的A549细胞系建立%Establishment of A549 Cell Line Stably Expressing NS1 Protein of Influenza Virus

    Institute of Scientific and Technical Information of China (English)

    李志辉; 曾琳姣; 王慧煜; 梅琳; 刘永飞; 韩雪清


    NS1 of influenza A virus is a key multifunctional protein that plays various roles in regulating viral replication mechanisms, disease pathogenesis. In order to establish stable A549 cell line expressing NS1 protein of influenza A Virus, NSl cDNA was obtained by RT-PCR using 2009 A(H1N1) influenza virus total RNA as template. The fragment was cloned in the pMD19-T vector, then the fragment was obtained by BamHI and NdeI digestion, and ligated with pCMV-HA. Linearized pCMV-HA-NS1 and neo were transfect-ed into A549 cells. The stable expressing NSl protein cell line was screened by G418. DNA, RNA, protein levels of NS1 were detected in A549 cells by PCR, RT-PCR and Western blot, the location of the NSl protein in cells was observed by immunofluorescence. The result indicated that NS1 protein was stable expressed in A549 cell line, suggesting that NSl stable expression A549 cell line was successfully constructed, and the NS1 protein is located in nucleus. This stable cell line can be used for further study of biological functions of NS1.%A型流感病毒的NSl(Nonstructurol 1 protein,NSl)蛋白是病毒复制、毒力等的重要调节蛋白.运用RT-PCR方法扩增A/Beijing/501/2009 (H1N1)流感病毒NS1基因,克隆至真核表达载体pCMV-HA,用Lipofectamine 2000将线性化pCMV-HA-NS1与neo基因共同转染A549细胞,通过G418筛选获得阳性重组细胞,并采用PCR、RT-PCR、Western blot技术检测重组细胞中NS1蛋白的表达,通过免疫荧光技术观察NS1蛋白在细胞中的定位.PCR、RT-PCR检测显示NS1基因成功整合进入细胞基因组,并转录为mRNA;Western blot检测显示重组细胞系稳定表达NS1蛋白,免疫荧光显示NS1蛋白定位于细胞核内.表明通过G418筛选,成功构建稳定表达NS1蛋白的重组A549-HA-NS1细胞系,且NS1蛋白定位于细胞核内,为进一步研究NS1蛋白的生物学功能奠定基础.

  11. Establishment of cell lines stably co-expressing Japanese encephalitis virus prM and E protein with a mutant in prM furin cleavage site%流行性乙型脑炎prM蛋白剪切位点突变的prM-E蛋白表达细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    李业南; 陈振师; 步志高; 华荣虹


    In order to establish the BHK-21 cell lines stably co-expressing prM-E fusion protein of Japanese encephalitis virus (JEV) with a mutant furin site to disable the pre-peptide cleavage of prM, BHK-21 cells was transfected with the recombinant plasmid pCAG-JEV-prM(R89A)E, which was constructed by introducing a point mutation at the cleavage site of prM gene by PCR and cloned into eukaryotic vector pCAG-neo with E gene. Cells stably co-expressing prM-E fusion protein were selected in the present of G418 and identified by indirect immunofluorescence assay and western blot, In addition, the cells displaying high-level protein expression were purified through limiting dilution cloning. Eventfully, the results showed that the cell line was able to express the intact prM of prM-E fusion protein. The established cell line would provide a basis for further study the effect of the cleavage event on mature mechanism of JEV and the development of JEV subunit vaccine.%为建立稳定共表达乙型脑炎病-毒(JEV)完整M前体蛋白(prM)和E蛋白的BHK-21细胞系,本研究在prM蛋白furin蛋白酶剪切位点编码基因中引入突变,将突变后的prM基因克隆于质粒中构建重组表达质粒pCAG-JEV-prM(R89A)E.将重组质粒转染BHK-21细胞,转染48 h后细胞用含G418的选择性培养基选择培养,进一步经细胞克隆纯化制备表达剪切位点突变的JEV prM-E蛋白的稳定细胞系.经IFA和western blot鉴定表明,该细胞系能表达JEV prM与E蛋白,所表达的prM蛋白未发生剪切;细胞经多次传代后仍能够稳定地共表达prM与E蛋白.该细胞系的建立为研究prM蛋白剪切对JEV粒子形成的影响以及亚单位疫苗的制备奠定了基础.

  12. Human dendritic cells derived from embryonic stem cells stably modified with CD1d efficiently stimulate antitumor invariant natural killer T cell response. (United States)

    Zeng, Jieming; Wang, Shu


    Invariant natural killer T (iNKT) cells are a unique lymphocyte subpopulation that mediates antitumor activities upon activation. A current strategy to harness iNKT cells for cancer treatment is endogenous iNKT cell activation using patient-derived dendritic cells (DCs). However, the limited number and functional defects of patient DCs are still the major challenges for this therapeutic approach. In this study, we investigated whether human embryonic stem cells (hESCs) with an ectopically expressed CD1d gene could be exploited to address this issue. Using a lentivector carrying an optimized expression cassette, we generated stably modified hESC lines that consistently overexpressed CD1d. These modified hESC lines were able to differentiate into DCs as efficiently as the parental line. Most importantly, more than 50% of such derived DCs were CD1d+. These CD1d-overexpressing DCs were more efficient in inducing iNKT cell response than those without modification, and their ability was comparable to that of DCs generated from monocytes of healthy donors. The iNKT cells expanded by the CD1d-overexpressing DCs were functional, as demonstrated by their ability to lyse iNKT cell-sensitive glioma cells. Therefore, hESCs stably modified with the CD1d gene may serve as a convenient, unlimited, and competent DC source for iNKT cell-based cancer immunotherapy.

  13. 稳定表达甜味受体蛋白T1R2/T1R3的HEK293细胞系的建立%Establishment of HEK293 Cell Line Stably Expressing Sweet Preceptor Protein T1R2/T1R3

    Institute of Scientific and Technical Information of China (English)

    钱玲玲; 秦玉梅; 邓少平


    以小鼠舌组织为对象,提取总mRNA,并以此为模板,使用自行设计的引物通过RT-PCR扩增Gα15、T1R2和T1R3目的片段.构建重组质粒pEGFP-C1-Gα 15、pDsRed1-N1-T1R2、pcDNATM6.2/N-YFP-DEST-T1R3.以脂质体介导的方法转染HEK293细胞,经抗性筛选后,通过极限稀释法获得稳定表达T1R2/T1R3的HEK293细胞系,最后通过RT-PCR,荧光显微镜及Western blot方法在基因及蛋白质水平上对建立的稳定表达细胞系进行鉴定.基因及蛋白质水平上的结果均表明,目的基因Gα15、T1R2/T1R3成功导入HEK293细胞中,并且稳定表达.该细胞系的建立为细胞水平上甜味机理的体外研究(如甜味识别热动力学等)提供了稳定的细胞来源.%It is to establish HEK293 cell line which could stably express sweet taste receptor protein T1R2/T1R3. Firstly,extract total mRNA from mouse tongue tissue,then amplify Gal5,TlR2 and T1R3 target gene fragment by RT-PCR with self-designed primers and above total mRNA template. Then,establish the recombinant plasmid pEGFP -C1 -Gα15,pDsRedl -N1 -T1R2, pcDNA TM6.2/N-YFP-DEST-TlR3 and introduce them into the HEK293 cells by liposome. After resistance screening,the HEK293 cell line with the ability of stable expressing T1R2/T1R3 was obtained in a limit dilution method. Finally,the stable cell line with sweet receptor protein T1R2/ T1R3 was identified by RT-PCR,fluorescence microscopy and Western blot. The results in gene and protein level show that Gαl5、T1R2/T1R3 are successfully introduced into HEK293 cell line and are stably expressed. The establishment of HEK293 cell line provides a stable cell source for the study of sweetness mechanism in vitro (such as sweet recognition thermodynamics,etc.) at cell level.

  14. Endogenous gustatory responses and gene expression profile of stably proliferating human taste cells isolated from fungiform papillae. (United States)

    Hochheimer, Andreas; Krohn, Michael; Rudert, Kerstin; Riedel, Katja; Becker, Sven; Thirion, Christian; Zinke, Holger


    Investigating molecular mechanisms underlying human taste sensation requires functionally dedicated and at the same time proliferating human taste cells. Here, we isolated viable human fungiform taste papillae cells from biopsy samples, adenovirally transduced proliferation promoting genes, and obtained stably proliferating cell lines. Analysis of gene expression of 1 human taste cell line termed HTC-8 revealed that these cells express 13 TAS2R bitter taste receptor genes, CD36, OXTR encoding oxytocin receptor, as well as genes implicated with signal transduction and cell fate control. Bitter tastants triggered functionally distinct signaling pathways in HTC-8 cells. Salicin elicited phospholipase C-dependent calcium signaling and no cell depolarization. In contrast, stimulation with saccharin, aristolochic acid, or phenylthiocarbamide triggered cell depolarization and phospholipase C-independent calcium influx. Simultaneous stimulation with salicin and saccharin revealed that saccharin can enhance the phospholipase C-dependent response to salicin indicating crosstalk of signaling pathways. Our results show that HTC-8 cells are programmed to bitter taste reception but are also responsive to fatty acids, oxytocin, and somatosensory stimuli, whereas HTC-8 cells are insensitive to compounds representing other basic taste qualities.

  15. Thyroid cell lines in research on goitrogenesis. (United States)

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


    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.

  16. 14-3-3σ干扰逆转录病毒载体的构建及其稳定转染HaCat细胞系的建立%Construction of RNAi Recombinant Retroviral Vector of 14-3-3P and Its Stably Transfected HaCat Cell Lines

    Institute of Scientific and Technical Information of China (English)

    周美娟; 丁振华


    目的:构建14-3-3σ干扰逆转录病毒载体,建立稳定转染的HaCat细胞系.方法:人工合成14-3-3σ基因干扰序列并定向插入到pSuper-retro-neo-EGFP质粒,并在STBL3菌内进行质粒扩增,刷选阳性克隆,酶切测序鉴定,转染293FT细胞进行病毒包装、扩增、纯化、获取逆转录病毒载体,将逆转录病毒栽体感染HaCat细胞后Western免疫印迹法、Real-time PCR法检测14-3-3σ的表达情况.结果:连接重组后经酶切和测序筛选出pSuper-retro-neo-EGFP-si14-3-3σ;干扰质粒稳定转染的HaCat细胞系在倒置荧光显微镜下呈绿色荧光,Western免疫印迹法和Real-time PCR法表明14-3-3σ表达明显抑制.结论:成功构建了14-3-3σ干扰的逆转录病毒载体,并构建了其稳定转染的HaCat细胞系.%Objective: To construct the RNAi retroviral vector of 14-3-3σ and establish the stable transfected HaCat cell lines.Methods: Hairpin siRNA of 14-3-3σ was synthesized and inserted into pSuper-retro-neo-EGFP plasmid.PSuper-retro-neo-EGFP-si14-3-3σ was transformed into competent STBL3 cells.Then the positive clones were confirmed by sequencing and transfected into the packaging 293FT cells to amplificate and depurate virus.HaCat cells were infected by the recombinant retroviral vector and the expression of 14-3-3σ was detected by Western blot and real time PCR.Results: The recombinant retroviral plasmid PSuper-retro-neo-EGFP-si 14-3-3σ was successfully constructed and green fluorescence of the stable transfected HaCat cell lines were observed under inverted fluorescence microscope.The expression of 14-3-3 was down-regulated by the RNAi-14-3-3σ.Conclusion: The RNAi retroviral vector targeting 14-3-3σ was successfully constructed and stably transfected HaCat cell lines were established.

  17. [Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone]. (United States)

    Zeng, Qing-Ping; Feng, Li-Ling; Yang, Rui-Yi; Chen, Zhu-Hua


    To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.

  18. Establishment and identification of human osteosarcoma cell line stably expressing wild-type p53 gene%稳定表达野生型p53基因人骨肉瘤细胞株的建立与鉴定

    Institute of Scientific and Technical Information of China (English)

    雷晓晶; 韩鹏飞; 吕智


    ,the stably transfected osteosarcoma cell line was manually screened. Results A recombinant plasmid pIRES-EGFP-p53 containing wild-type p53 cDNA fragment was successfully constructed. The results of PCR,enzyme electrophoresis and other methods to identify the size and position of plasmid,were consistent with the experimental design. After transfection,the human osteosarcoma cell line was named U-2-p53 OS. The transfected osteosarcoma cells showed weak apoptosis. Conclusion The establishment of human osteosarcoma U-2-p53 OS cell lines by gene transfer technology lay a foundation for the further study of wild-type p53 gene in osteosarcoma suicide gene therapy.

  19. CD147慢病毒表达载体的构建及稳定转染A549细胞系的建立%Construction of a CD147 Lentiviral Expression Vector and Establishment of Its Stably Transfected A549 Cell Line

    Institute of Scientific and Technical Information of China (English)

    杨绍兴; 汤传昊; 王思涵; 宋三泰; 刘晓晴


    背景与目的 CD147是一类位于肿瘤细胞膜表面的跨膜糖蛋白,可促进肿瘤的浸润和转移.本研究拟构建CD147慢病毒表达载体,建立稳定过表达CD147的人肺腺癌A549细胞系,观察过表达CD147后对MMP-9及细胞增殖、侵袭能力的影响.方法 RT-PCR扩增CD147基因全长序列,将序列插入pEGFP载体,构建pEGFP-CD147慢病毒表达载体,随后转入293FT细胞中进行慢病毒包装,用获得的慢病毒毒液感染人肺腺癌细胞系A549,建立稳定过表达CD147的A549细胞系.Real-time PCR检测MMP-9的变化情况,CCK-8及Transwell法检测人肺腺癌细胞增殖、侵袭能力的变化.结果 经限制性内切酶鉴定及测序分析,成功构建了pEGFP-CD 147慢病毒表达载体质粒.Real-time PCR和Western blot检测显示,与对照组相比,转染pEGFP-CD147慢病毒表达载体组的细胞,CD147的表达在mRNA和蛋白两个水平均增高,成功建立了A549-CD147细胞系.上调CD147的表达后,MMP-9的mRNA表达水平明显升高.同时,A549-CD147细胞增殖和侵袭能力明显增加(P<0.05).结论 成功构建CD147慢病毒表达载体和A549-CD147细胞系,过表达CD 147可上调MMP-9的表达,增强人肺腺癌细胞的增殖和侵袭能力.%Background and objective CD 147, a type of transmembrane glycopiotein embedded on the surface of tumor cells, can promote tumor invasion and metastasis. This aim of this study is to construct a CD147 lentiviral expression vector, establish its stably transfected A549 cell line, and observe the effect of CD 147 on MMP-9 proliferation as well as on the invasive ability of human lung adenocarcinoma cells. Methods Full-length CD 147 gene was amplified by real-time polymerase chain reaction (RT-PCR), inserted into a pEGFP vector to construct pEGFP-CD147 and pEGFP vectors, and then transfected into 293FT cells to precede the lentivirus equipment package. Subsequently, we collected the lentivirus venom to infect the A549 cells and establish a stable

  20. NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I

    Energy Technology Data Exchange (ETDEWEB)

    Lacoste, J.; Cohen, L.; Hiscott, J. (Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec (Canada))


    The effect of constitutive Tax expression on the interaction of NF-{kappa} B with its recognition sequence and on NF-{kappa} B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-{kappa} B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-{kappa} B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters.

  1. L-FABP T94A decreased fatty acid uptake and altered hepatic triglyceride and cholesterol accumulation in Chang liver cells stably transfected with L-FABP. (United States)

    Gao, Na; Qu, Xia; Yan, Jin; Huang, Qi; Yuan, Hao-Yong; Ouyang, Dong-Sheng


    Liver fatty acid-binding protein (L-FABP, FABP1) is a highly conserved key factor in lipid metabolism. This study was undertaken to verify whether the T94A mutation in the L-FABP gene affects fatty acid uptake and intracellular esterification into specific lipid pools. Candidate SNPs were recreated using site-directed mutagenesis and tested for physical function in stably transfected Chang liver cell lines. We found that the T94A mutant of L-FABP lowered FFA uptake but had no effect on FFA efflux. L-FABP T94A-expressing cells showed decreased triglyceride content and increased cholesterol accumulation compared to the wild-type control for cells incubated with an FFA mixture (oleate: palmitate, 2:1 ratio). In conclusion, our study provided additional indications of the functional relevance of the L-FABP T94A SNP in hepatic fatty acid and lipid metabolism in humans.

  2. HTLV-1 Tax mediated downregulation of miRNAs associated with chromatin remodeling factors in T cells with stably integrated viral promoter.

    Directory of Open Access Journals (Sweden)

    Saifur Rahman

    Full Text Available RNA interference (RNAi is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1 transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR using a CD4(+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type.

  3. Identification of a HEK-293 cell line containing stably-transfected H3 R gene and screening for novel non-imidazole histamine H3 receptor antagonists%稳定转染H3受体基因的HEK293细胞株的鉴定及新型非咪唑类H3受体拮抗剂的筛选

    Institute of Scientific and Technical Information of China (English)

    何萍; 谭丽; 胡薇薇; 戴海斌; 胡永洲; 陈忠


    目的:鉴定转染大鼠组胺H3受体基因的HEK293细胞株受体表达情况,筛选具有H3受体拮抗活性的新型非咪唑类化合物.方法:利用RT-PCR和免疫印迹的方法检测转染细胞株组胺H3受体的表达,以阳性刺激物forskolin诱导细胞内cAMP含量升高为指标,初步筛选具有拮抗作用的化合物.结果:细胞鉴定结果显示,稳定转染H3受体基因的HEK293细胞株高表达大鼠H3受体.H3受体选择性激动剂(R)-α-甲基组胺可浓度依赖性抑制阳性刺激物forskolin(10 μmol/L)诱导的cAMP含量升高(P<0.05),当(R)-α-甲基组胺浓度达到30 nmol/L时,抑制率接近平台值(P>0.05).而H3受体拮抗剂噻普酰胺和新合成的非咪唑类化合物XHA23及XHA25则能够浓度依赖性阻断(R)-α-甲基组胺对forskolin诱导的cAMP含量升高的抑制作用(P<0.05),IC50分别为3.62 μmol/L、0.49 μmol/L、0.14 μmol/L.结论:高表达H3受体的HEK293细胞株可用于有H3受体拮抗活性化合物的筛选,而非咪唑类化合物XHA23和XHA25具有一定的H3受体拮抗剂样活性.%Objective:To identify a HEK293 cell line containing stably-transfected H3R gene,and to screen the novel non-imidazole compounds with H3 R antagonist activity.Methods:The expression of rat H3receptor in cell line was detected by RT-PCR and Western blot.An elevation of intercellular cAMP concentration induced by forskolin was measured as the index for screening compounds with H3 R antagonist activity.Results:The H3 R-transfected HEK-293 cells stably expressed high level of rat H3 receptor mRNA and protein.Forskolin significantly increased intercellular cAMP concentration in the H3 R-transfected HEK-293 cells.H3 R agonist (R)-α-methyhistamine inhibited the forskolin-induced production of intercellular cAMP.H3 R antagonist thioperamide and newly synthesized non-imidazole compounds XHA23 and XHA25 blocked (R)-c-methyhistamine reversal of forskolin-induced cAMP formation in a concentration

  4. Regulated expression of the rat recombinant P2X(3) receptor in stably transfected CHO-K1 tTA cells. (United States)

    Lachnit, W G; Oglesby, I B; Gever, J R; Gever, M; Huang, C; Li, X C; Jin, H; McGivern, J G; Ford, A P


    ATP yielding a pK(B) of 5.6. PPADS produced non-parallel, dextral shifts of E/[A] curves to alpha beta MeATP which were insurmountable. These results show for the first time, expression of a functional, homomeric recombinant rat P2X(3) receptor which is under regulated expression in a stably transfected mammalian cell line.

  5. Construction of HEK293 cells stably expressing wild-type organic anion transporting polypeptide 1B1 (OATP1B1*1a) and variant OATP1B1*1b and OATP1B1*15. (United States)

    Chen, M; Qu, B X; Chen, X L; Hu, H H; Jiang, H D; Yu, L S; Zhou, Q; Zeng, S


    A transgenic cell line stably expressing the human organic anion transporting polypeptide (OATP1B1) was established. Human Embryonic Kidney 293 (HEK293) cell line stably expressing OATP1B1*1a sequence was amplified through PCR with the extracted total RNA as templates from human liver, then subcloned into the plasmid pMD19-T and verified by sequencing. OATP1B1*1b/OATP1B1*15 mutant sequences were obtained by site-directed mutation PCR with pMD19-T/ OATP1B1*1a as templates. The plasmids pcDNA3.1(+)/OATP1B1*1a, *1b and *15 were constructed and transfected into HEK293 cell line using Lipofectamine 2000 transfection reagent. Several stable transfected clones were obtained after selection with G418. Using rosuvastatin as a probe substrate of OATP1B1, the intracellular rosuvastatin accumulation in HEK293 and HEK-OATP1B1*1a, *1b and *15 monoclone cells were validated by a ultra-performance liquid chromatography-tandem mass spectrometry. OATP1B1 mRNA and protein expression were detected by RT-PCR and Western blot, respectively. The results from RT-PCR, rosuvastatin uptake and Western blot assay indicated that human OATP1B1 was highly expressed in transfected cells compared with controls. The HEK-293 cell lines stably expressing human OATP1B1-wild and variant (HEK-OATP1B1, *1b and *15) are potential models to study drug transport in vitro.

  6. Generating a transgenic mouse line stably expressing human MHC surface antigen from a HAC carrying multiple genomic BACs. (United States)

    Hasegawa, Yoshinori; Ishikura, Tomoyuki; Hasegawa, Takanori; Watanabe, Takashi; Suzuki, Junpei; Nakayama, Manabu; Okamura, Yoshiaki; Okazaki, Tuneko; Koseki, Haruhiko; Ohara, Osamu; Ikeno, Masashi; Masumoto, Hiroshi


    The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.

  7. Inhibitory synapse formation in a co-culture model incorporating GABAergic medium spiny neurons and HEK293 cells stably expressing GABAA receptors. (United States)

    Brown, Laura E; Fuchs, Celine; Nicholson, Martin W; Stephenson, F Anne; Thomson, Alex M; Jovanovic, Jasmina N


    Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed

  8. Storage of cell lines. (United States)

    Parker, Katharine A


    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.

  9. 大鼠结缔组织生长因子基因miRNA表达质粒的构建及其稳定转染大鼠肝星状细胞系的建立%Construction of miRNA expression vector for rat connective tissue growth factor and establishment of stably transfected rat hepatic stellate cell line

    Institute of Scientific and Technical Information of China (English)

    阳宏; 李孝生; 向颖; 邢旎旎; 沈艳


    目的 构建大鼠结缔组织生长因子(Connective tissue growth factor,CTGF)基因miRNA表达质粒,并建立稳定转染大鼠肝星状细胞(Hepatic stellate cell,HSC)系.方法 根据大鼠CTGF基因mRNA序列,设计并合成3对寡聚单链DNA X191-1、X191-2和X191-3及1对阴性对照序列DNA X191-4,将4对寡聚单链DNA退火成双链后,分别与载体pcDNA6.2-GW/EmGFP-miR连接,构建CTGF基因miRNA重组表达质粒,分别转染HSC-T6细胞,荧光显微镜观察细胞的转染效率,RT-PCR检测转染细胞中CTGF基因mRNA的转录水平;取干扰效率最高的重组质粒及阴性对照质粒,分别转染HSC-T6细胞,经杀稻瘟菌素持续加压筛选.结果 经测序鉴定,重组表达质粒构建正确,插入片段的碱基序列与设计相符;细胞的瞬时转染效率约为50%;3组干扰质粒转染的HSC-T6细胞中,CTGF基因mRNA的转录水平明显低于空白对照组(P<0.01),其中X191-2质粒对CTGF基因转录的干扰效率最高;获得了稳定转染的HSC-T6细胞.结论 成功构建了CTGF基因miRNA表达质粒,并获得了稳定转染的肝星状细胞系,为进一步研究肝纤维化的形成机制及其治疗奠定了基础.

  10. Establishment of Stably Transfected Cells Constitutively Expressing the Full-Length and Truncated Antigenic Proteins of Two Genetically Distinct Mink Astroviruses

    DEFF Research Database (Denmark)

    Bidokhti, Mehdi R. M.; Ullman, Karin; Jensen, Trine Hammer


    Astroviruses are becoming a growing concern in veterinary and public health. To date there are no registered vaccines against astrovirus-induced disease, mostly due to the difficulty to cultivate astroviruses to high titer for vaccine development using conventional techniques. As means to circumv......Astroviruses are becoming a growing concern in veterinary and public health. To date there are no registered vaccines against astrovirus-induced disease, mostly due to the difficulty to cultivate astroviruses to high titer for vaccine development using conventional techniques. As means...... to circumvent this drawback, we have developed stably transfected mink fetal cells and BHK21 cells constitutively expressing the full-length and truncated capsid proteins of two distinct genotypes of mink astrovirus. Protein expression in these stably transfected cells was demonstrated by strong signals...... shedding was observed in mink kits born from immunized females. The gene integration and protein expression were sustained through cell passage, showing that the used approach is robust and reliable for expression of functional capsid proteins for vaccine and diagnostic applications....

  11. Differences in Gene Regulation by Dual Ligands of Nuclear Receptors Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) in HepG2 Cells Stably Expressing CAR/PXR. (United States)

    Kanno, Yuichiro; Tanuma, Nobuaki; Yazawa, Saki; Zhao, Shuai; Inaba, Miki; Nakamura, Satoshi; Nemoto, Kiyomitsu; Inouye, Yoshio


    The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate various genes involved in xenobiotics and drug metabolism. In many cases, CAR/PXR share ligands termed dual ligands of CAR/PXR. It is difficult to investigate the effect of CAR/PXR dual ligands in cell lines because CAR and PXR expression is scarcely detected in cultured cell lines. Here, we established a tetracycline-inducible human CAR and stably human PXR-overexpressing HepG2 cell line (HepTR/hCAR/hPXR) to examine CAR/PXR dual ligands. In the present study, we investigated the regulation of CYP2B6, CYP2C9, CYP3A4, and UDP-glucuronosyl transferase, which are target genes of CAR/PXR, by dual ligands of CAR/PXR in two transfectants. Activation of CAR and PXR in cells treated with a high dose of CITCO [6-(4-chlorophenyl)-imidazo(2,1-b)thiazole-5-carbaldehyde] or cotreated with rifampicin and tetracycline resulted in synergistic enhancement of CYP3A4, but not CYP2B6, CYP2C9, or UGT1A1, mRNA expression in HepTR/hCAR/hPXR cells. In contrast, this synergistic effect was not observed in HepTR/hCAR cells. These observations were also demonstrated in human primary hepatocytes. Taken together, our results suggest that dual ligands of CAR/PXR show distinct gene regulation patterns by cross-talk between CAR and PXR. Furthermore, the two newly established cell lines are useful tools to investigate dual ligands of CAR/PXR.

  12. 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


    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

  13. SY5Y cells stably- expressing CLN3 have better proliferating ability than wild type cells%稳定过表达CLN3的SY5Y细胞增殖功能加强

    Institute of Scientific and Technical Information of China (English)

    刘静; 杨萱; 迟洪滨; 吴丹; 吴白燕


    Objective To establish a human neuroblastoma (SH-SY5Y)cell line,which can stably-express human ceroid-lipofuscinosis,neuronal 3 ( CLN3 ) and 1.02 kb deletion within CLN3 ( CLN3Δex7/8 ),and to detect the proliferation of CLN3 and CLN3Δex7/8 in SH-SY5Y.Methods After screening culture by Zeocin,the stable SH-SY5Y cells expressing CLN3 and CLN3Δex7/8 were established.The transcription and expression of CLN3 and CLN3Δex7/8 were identified by RT-PCR,Real Time PCR and Western blotting.To compare the proliferation of SH-SY5Y/C and SH-SY5Y/Ct using Cell Counting Kit-8 (CCK-8).Results The cell line SH-SY5Y/C expressing CLN3 stably and cell line SH-SY5 Y/Ct expressing C LN3Δex7/8 stably have been established successfully.The mRNA overexpress of CLN3 and CLN3Δex7/8 were 43 times and 124 times more respectively than the control cell line transfected vector using method of Real Time PCR.The overexpression,at protein level,of CLN3 and CLN3Δex7/8 were found to be 1.5 times and 20 times more respectively than the control cell line transfected with vector usingWestern blot.The cell growth rate of overexpressing the full length of CLN3 was significantly higher than empty vector control ( P =0.044 527 ).The cell grouth rate of overexpressing CLN3Δex7/8 was not significantly different from empty vector control ( P =0.345 329 ).Conclusion The full length of CLN3 protein results in an increase in cell growth rate of SH-SYSY cell and the CLN3Δex7/8 truncated protein has no effect on the growth rate of SH-SY5 Y cell.These results indicate that the exon of 7 and 8 maybe play an important role in regulating proliferation of SH-SYSY cell.%目的 建立稳定表达CLN3(ceroid-lipofuscinosis,neuronal 3)和CLN3 1.02 kb缺失突变体基因(CLN3Δex7/8)的稳定细胞系,研究全长CLN3在人神经母细胞瘤(SH-SY5Y)细胞中的促增殖作用及7、8号外显子缺失突变体对细胞增殖的影响.方法 利用药物Zeocin筛选构建稳定过表达CLN3全长基因和CLN 3Δex7/8

  14. Generation of induced pluripotent stem cells (iPSCs) stably expressing CRISPR-based synergistic activation mediator (SAM)

    DEFF Research Database (Denmark)

    Xiong, Kai; Zhou, Yan; Hyttel, Poul


    Human fibroblasts were engineered to express the CRISPR-based synergistic activation mediator (SAM) complex: dCas9-VP64 and MS2-P65-HSF1. Two induced pluripotent stem cells (iPSCs) clones expressing SAM were established by transducing these fibroblasts with lentivirus expressing OCT4, SOX2, KLF4...... a novel, useful tool to investigate genetic regulation of stem cell proliferation and differentiation through CRISPR-mediated activation of endogenous genes....

  15. Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor

    Directory of Open Access Journals (Sweden)

    Ebrahim Shahbazi


    Full Text Available Direct conversion of somatic cells into neural stem cells (NSCs by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.

  16. Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells. (United States)

    Moguilevsky, N; Varsalona, F; Guillaume, J P; Noyer, M; Gillard, M; Daliers, J; Henichart, J P; Bollen, A


    A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

  17. Rank of Stably Dissipative Graphs

    CERN Document Server

    Duarte, Pedro


    For the class of stably dissipative Lotka-Volterra systems we prove that the rank of its defining matrix, which is the dimension of the associated invariant foliation, is completely determined by the system's graph.

  18. Human delta opioid receptor: functional studies on stably transfected Chinese hamster ovary cells after acute and chronic treatment with the selective nonpeptidic agonist SNC-80. (United States)

    Malatynska, E; Wang, Y; Knapp, R J; Waite, S; Calderon, S; Rice, K; Hruby, V J; Yamamura, H I; Roeske, W R


    The SNC-80 series of nonpeptidic agonists for the delta-opioid receptor are being developed as potential analgesic drugs. It is important to understand their acute and chronic effects at human delta-opioid receptors. Thus, we measured the ability of SNC-80 and [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin to inhibit forskolin-stimulated adenylyl cyclase activity in recombinant Chinese hamster ovary cells stably expressing the cloned human delta-opioid receptor. The calculated EC50 values for [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin and SNC-80 were 0.6 +/- 0.1 nM and 6.3 +/- 0.1 nM, respectively. Pretreatment of these cells with SNC-80 (100 nM) for 24 hr produced 1) a time-dependent reduction of delta receptor density, as measured by radioligand binding studies with [3H]naltrindole; 2) a shift in the EC50 value of SNC-80 from 7.7 +/- 4.2 nM to 44.1 +/- 12 nM, as measured by the cyclic AMP assay; 3) a reduction in the maximum inhibition of adenylyl cyclase activity from 86% to 48%; 4) a marked increase in the forskolin stimulation of basal cyclic AMP accumulation by nearly 100% (from 442 pmol/mg of protein to 824 pmol/mg of protein); and 5) a 5-fold increase in forskolin-stimulated cyclic AMP accumulation after addition of naltrindole. These studies showed that SNC-80 produced desensitization and down-regulation of human delta-opioid receptors in recombinant Chinese hamster ovary cells after chronic treatment and that this effect was associated with an increase in adenylyl cyclase activity.


    NARCIS (Netherlands)



    A cDNA clone encoding the rat metabotropic glutamate receptor mGluR3 was stably transfected into human embryonic kidney 293 cells. Receptor-expressing cell lines were characterized by centrifugation binding assays using [H-3]glutamate as radioligand. The rank order of affinity was L-glutamate>(1S,3R

  20. 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.

  1. Multiple Defects of Cell Cycle Checkpoints in U937-ASPI3K, an U937 Cell Mutant Stably Expressing Anti-Sense ATM Gene cDNA

    Institute of Scientific and Technical Information of China (English)


    (Ataxia-telangiectasia mutated gene (ATM) functions in control of cell cycle checkpoints in responding to DNA damage and protects cells from undergoing apoptosis. Knock-out within tumor cells of endogenous ATM will achieve therapeutic benefits and nable a better understanding of the decisive mechanisms of cell death or survival in response to DNA damaging agents. ) In present paper, we sought to characterize the cell cycle checkpoint profiles in U937-ASPI3K, a U937 cell mutant that was previously established with endogenous ATM knock-out phenotype. Synchronized U937-ASPI3K was exposed to 137Cs irradiation, G1, S, G2/M cell cycle checkpoint profiles were evaluated by determining cell cycle kinetics, p53/p21 protein, cyclin dependent kinase 2 (CDK2) and p34CDC2 kinase activity in response to irradiation. U937-ASPI3K exhibited multiple defects in cell cycle checkpoints as defined by failing to arrest cells upon irradiation. The accumulation of cellular p53/p21 protein and inhibition of CDK kinase was also abolished in U937-ASPI3K. It was concluded that the stable expression of anti-sense PI3K cDNA fragment completely abolished multiple cell cycle checkpoints in U937-ASPI3K, and hence U937-ASPI3K with an AT-like phenotype could serves as a valuable model system for investigating the signal transduction pathway in responding to DNA damaging-based cancer therapy.


    Institute of Scientific and Technical Information of China (English)

    Lin Qi; Rajesh Agarwal; Rana Singh; Gail S. Harrisona; L.Michael Glodea


    Since the epidermal growth factor receptor (EGFR) is a key regulator in cell signaling pathways of cancer cell. To investigate the mechanism between cancer cells survival and its EGFR expression, drug selection of cancer cells target therapy, we generated a cell line, 9L-EGFR, which stably expressed human EGFR; the parental rat glioma cell line, 9L, does not contain endogenous EGFR message or protein. Our results show that 9L-EGFR cells had high levels of EGFR on their cell surface by using RT-PCR, Western analysis and Flow cytometry analysis. The EGFR transfected into 9L cells was capable of being activated by EGF, in which either phosphorylated (p-EGFR) or total (EGFR) was showed by Western blot. This investigation may contribute to the further studies of cancer cells bearing EGFR.

  3. Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Harjeet Singh

    Full Text Available Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28 that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC in the presence of interleukin (IL-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT. We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

  4. Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shengtao; LIU Wenli; HE Peigen; GONG Feili; YANG Dongliang


    In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

  5. Transport of the placental estriol precursor 16α-hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) by stably transfected OAT4-, SOAT-, and NTCP-HEK293 cells. (United States)

    Schweigmann, H; Sánchez-Guijo, A; Ugele, B; Hartmann, K; Hartmann, M F; Bergmann, M; Pfarrer, C; Döring, B; Wudy, S A; Petzinger, E; Geyer, J; Grosser, G


    16α-Hydroxy-dehydroepiandrosterone sulfate (16α-OH-DHEAS) mainly originates from the fetus and serves as precursor for placental estriol biosynthesis. For conversion of 16α-OH-DHEAS to estriol several intracellular enzymes are required. However, prior to enzymatic conversion, 16α-OH-DHEAS must enter the cells by carrier mediated transport. To identify these carriers, uptake of 16α-OH-DHEAS by the candidate carriers organic anion transporter OAT4, sodium-dependent organic anion transporter SOAT, Na(+)-taurocholate cotransporting polypeptide NTCP, and organic anion transporting polypeptide OATP2B1 was measured in stably transfected HEK293 cells by LC-MS-MS. Furthermore, the study aimed to localize SOAT in the human placenta. Stably transfected OAT4-HEK293 cells revealed a partly sodium-dependent transport for 16α-OH-DHEAS with an apparent Km of 23.1 ± 5.1 μM and Vmax of 485.0 ± 39.1 pmol/mg protein/min, while stably transfected SOAT- and NTCP-HEK293 cells showed uptake only under sodium conditions with Km of 319.0 ± 59.5 μM and Vmax of 1465.8 ± 118.8 pmol/mg protein/min for SOAT and Km of 51.4 ± 9.9 μM and Vmax of 1423.3 ± 109.6 pmol/mg protein/min for NTCP. In contrast, stably transfected OATP2B1-HEK293 cells did not transport 16α-OH-DHEAS at all. Immunohistochemical studies and in situ hybridization of formalin fixed and paraffin embedded sections of human late term placenta showed expression of SOAT in syncytiotrophoblasts, predominantly at the apical membrane as well as in the vessel endothelium. In conclusion, OAT4, SOAT, and NTCP were identified as carriers for the estriol precursor 16α-OH-DHEAS. At least SOAT and OAT4 seem to play a functional role for the placental estriol synthesis as both are expressed in the syncytiotrophoblast of human placenta.

  6. Development of an androgen reporter gene assay (AR-LUX) utilizing a human cell line with an endogenously regulated androgen receptor

    NARCIS (Netherlands)

    Blankvoort, B.M.G.; Groene, E.M. de; Meeteren-Kreikamp, A.P. van; Witkamp, R.F.; Rodenburg, R.J.T.; Aarts, J.M.M.J.G.


    The aim of the work described in this report is to develop and characterize a cell-based androgen reporter assay. For this purpose, the androgen receptor (AR) expressing human breast cancer cell line T47D was stably transfected with a luciferase gene under transcriptional control of the PB-ARE-2 and

  7. Kinetics of desensitization and recovery from desensitization for human a4β2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells

    Institute of Scientific and Technical Information of China (English)

    Kewei D YU; Qiang LIU; Jie WU; Ronald J LUKAS


    Aim: Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human a4p2-nicotinic acetylcholine receptors (nAChR) heterologously expressed in the SH-EP1 human epithelial cell line. Methods: Whole-cell patch clamp recordings were performed to evaluate a4p2-nAChR currents.Results: Application of 0.1 nmol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of ~70 and ~700 ms, for the onset of a4(32-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of a4p2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs'1' from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human a4p2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings'21 using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse.Conclusion: These findings indicate the existence of a4p2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.

  8. Molluscan cells in culture: primary cell cultures and cell lines. (United States)

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


    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.

  9. Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity

    NARCIS (Netherlands)

    Boerboom, A.M.J.F.; Vermeulen, M.; Woude, van der H.; Bremer, B.I.; Lee, Y.Y.; Kampman, E.; Bladeren, van P.J.; Rietjens, I.M.C.M.; Aarts, J.M.M.J.G.


    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

  10. Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity.

    NARCIS (Netherlands)

    Boerboom, A.M.A.; Vermeulen, M.; Woude, H. van der; Bremer, B.I.; Lee-Hilz, Y.Y.; Kampman, E.; Bladeren, P.J. van; Rietjens, I.M.C.M.; Aarts, J.


    The electrophile-responsive element (EpRE) is a transcriptional enhancer involved in cancer-chemoprotective gene expression modulation by certain food components. Two stably transfected luciferase reporter cell lines were developed, EpRE(hNQO1)-LUX and EpRE(mGST-Ya)-LUX, based on EpRE sequences from

  11. Construction of eukaryotic vector pEGFP-N1-IL-17 and screening of stably transfected pEGFP-N1-IL-17-U87MG cell%IL-17真核表达载体的构建及其在胶质瘤细胞株U87MG中的表达与筛选

    Institute of Scientific and Technical Information of China (English)

    胡锦辉; 邱潮林; 华玲; 谢秋华; 何小雪; 沈振华; 胡珺


    IL-17 has been demonstrated to promote tumor growth via certain pathways. In current studies IL-17 has been found to be highly expressed in glioma. This research aims to construct the eukaryotic vector pEGFP-Nl-IL-17 and makes it to be stably expressed in glioma cell line U87MG in order to provide the basis for studying its function in glioma progress. PBMC was collected from an idiopathic thrombocytopenic purpura (ITP) patient and the RNA was extracted. IL-17 cDNA was synthesized and cloned into PMD 19-T and pEGFP-N1 plasmid by SaI I and BamH enzymatic digestion. The clones were identified by enzymatic digestion and sequencing. The correct clone was amplified and plasmid DNA was then transfected into glioma cell line U87MG. pEGFP-N1-IL-17-U87MG cells that stably expressed IL-17 were obtained by limited dilution by G418 added. Correct clone was identified by fluorescence, qRTPCR and ELISA. The results showed that IL-17 cDNA、PMDR 19-T-IL-17, and pEGFP-N1-IL-17 were successfully obtained. And pEGFP-Nl-IL-17-U87MG cells that stably express IL-17 were identified through fluorescence, qRT-PCR and ELISA. MCP-1 mRNA was down regulated after U87MG cell was transfected by IL-17. Therefore the eukaryotic expression vector pEGFP-Nl-IL-17 has been successfully constructed and stably transfected into U87MG cell which could be used for study on IL-17's function in glioma tumorigenesis.%IL-17可以通过多种途径促进肿瘤发展,前期工作发现胶质瘤组织中IL-17高表达,本研究拟构建IL-17真核表达载体pEGFP-N1-IL-17,并且稳定转染胶质瘤细胞株U87MG,为研究IL-17在胶质瘤中作用提供基础.取血小板减少性紫癜自愿患者外周血2ml,Ficoll分离PBMC,提取RNA后经含BamH Ⅰ及Sal Ⅰ酶切位点引物逆转录成cDNA,连接T载体,酶切后与经相同酶切的载体pEGFP-N1连接,卡那霉素筛选,重组载体经Xfect试剂转染胶质瘤细胞U87MG,以G418筛选,单克隆阳性株扩大培养,经荧光、Real Time PCR及ELISA

  12. Development of improved vaccine cell lines against rotavirus (United States)

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


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

  13. Immunosenescent CD57+CD4+ T-cells Accumulate and Contribute to Interferon-γ Responses in HIV Patients Responding Stably to ART

    Directory of Open Access Journals (Sweden)

    Sonia Fernandez


    Full Text Available HIV-infected individuals responding to antiretroviral therapy (ART after severe CD4+ T-cell depletion may retain low responses to recall antigens [eg: cytomegalovirus (CMV] and altered expression of T-cell co-stimulatory molecules consistent with immunosenescence. We investigated the capacity of phenotypically senescent cells to generate cytokines in HIV patients receiving long-term ART (n = 18 and in healthy controls (n = 10. Memory T-cells were assessed by interferon (IFN-γ ELISpot assay and flow cytometrically via IFN-γ or IL-2. Proportions of CD57brightCD28null CD4+ T-cells correlated with IFN-γ responses to CMV (p = 0.009 and anti-CD3 (p = 0.002 in HIV patients only. Proportions of CD57brightCD28null CD8+ T-cells and CD8+ T-cell IFN-γ responses to CMV peptides correlated in controls but not HIV patients. IL-2 was predominantly produced by CD28+T-cells from all donors, whereas IFN-γ was mostly produced by CD57+ T-cells. The findings provide evidence of an accumulation of immunosenescent T-cells able to make IFN-γ. This may influence the pathogenesis of secondary viral infections in HIV patients receiving ART.

  14. 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


    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...

  15. Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection

    Directory of Open Access Journals (Sweden)

    Mjaaland Siri


    Full Text Available Abstract Background Cell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV, an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines. Findings Here we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxa's cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells. Conclusions The results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.

  16. 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


    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.

  17. Retrovirus-Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yuan; ZHAI Suo-qiang; SONG Wei; GUO Wei; ZHENG Gui-liang; HU Yin-yan


    Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possibl utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through morphology observation, immunnocytochemical staining and RT-PCR analysis. The Hathl gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1 (a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differenti-ate into hair cell-like cells when forced to express Hathl. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.


    Institute of Scientific and Technical Information of China (English)

    肖成祖; 黄子才; 刘凤云; 郭志霞; 高丽华


    Genetically-enginecred CHO cell lines,rβ-13and CLF-8B2,were cultivated with the MC-1 microcarrier cul-ture system.The cell density could be enhanced by increasing the concentration of microcarrier.At a microcarrier concentration of 10 mg/ml.the cell density could reach 4 to 5×106 cells/ml.It was shown that these cell itnes would spontaneously release from the microcarrier to attach to and proliferate on fresh microcatriera.We were thus able to scale up cultivation using a simple methcd, adding fresh mlcrocarriers and medium directiy in-to the culture system to about 2,4 or 8 times the original volume.Using a 2 L bioreactor for several weeks at medium perfusion rates of 0.5 to 3working volumes.Prourokinase was stably secreted.

  19. Difference in Membrane Repair Capacity Between Cancer Cell Lines and a Normal Cell Line. (United States)

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


    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.

  20. Classification of Stably Dissipative 3D Lotka-Volterra Systems and Their Necessary and Sufficient Condition for Being Stably Dissipative

    Institute of Scientific and Technical Information of China (English)

    LIU Hai-ying


    By introducing the concepts of stably dissipative matrix and graph, some criteria conditions for stably dissipative matrix were given. On this basis, the method of graph theory was used to classify all stably dissipative 3D Lotka-Volterra systems and five classes of maximal stably dissipative graphs were obtained for these systems. Finally, the necessary and sufficient condition of being stably dissipative for every class was studied, under which the matrix associated with the graph is stably dissipative.

  1. Stably stratified magnetized stars in general relativity

    CERN Document Server

    Yoshida, Shijun; Shibata, Masaru


    We construct magnetized stars composed of a fluid stably stratified by entropy gradients in the framework of general relativity, assuming ideal magnetohydrodynamics and employing a barotropic equation of state. We first revisit basic equations for describing stably-stratified stationary axisymmetric stars containing both poloidal and toroidal magnetic fields. As sample models, the magnetized stars considered by Ioka and Sasaki (2004), inside which the magnetic fields are confined, are modified to the ones stably stratified. The magnetized stars newly constructed in this study are believed to be more stable than the existing relativistic models because they have both poloidal and toroidal magnetic fields with comparable strength, and magnetic buoyancy instabilities near the surface of the star, which can be stabilized by the stratification, are suppressed.

  2. Suppression of bcl-2 Gene by RNA Interference Increases Chemosensitivity to Cisplatin in Nasopharyngeal Carcinoma Cell Line CNE1

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hua YIN; Cai-Ping REN; Feng LI; Xu-Yu YANG; Hui LI; Ming ZHAO; Kai-Tai YAO


    To explore the effect of suppressing BCL-2 expression using RNA interference (RNAi) technique in nasopharyngeal carcinoma cell line CNE1. CNE1 cell lines stably expressing shRNAs targeted bcl-2 and GL3 gene were established and gene expression inhibition was assessed by Western blotting analysis. The effect of suppressing bcl-2 by RNAi on cell growth was studied, the apoptosis induction and the sensitization of CNE 1 cells to cisplatin were quantified by MTT assay and flow cytometry. The results showed that: stable transfection of CNE 1 cells with vectors expressing shRNAs against bcl-2 decreased the expression of BCL-2 protein; suppression of BCL-2 expression did not affect cell proliferation but could increase the chemosensitivity to cisplatin in CNE1 cells. This will help physicians to make some clinical trials of gene therapy on nasopharyngeal carcinoma by RNAi.

  3. Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines. (United States)

    Zou, Chunlin; Chou, Bin-Kuan; Dowey, Sarah N; Tsang, Kitman; Huang, Xiaosong; Liu, Cyndi F; Smith, Cory; Yen, Jonathan; Mali, Prashant; Zhang, Yu Alex; Cheng, Linzhao; Ye, Zhaohui


    Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

  4. False leukemia-lymphoma cell lines: an update on over 500 cell lines. (United States)

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


    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.

  5. Virus Discovery Using Tick Cell Lines (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam


    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

  6. [Establishment and identification of the near-infrared fluorescence labeled exosomes in breast cancer cell lines]. (United States)

    Li, Taiming; Lan, Wenjun; Huang, Can; Zhang, Chun; Liu, Xiaomei


    Exosomes, a population of extracellular membrane vesicles of 30-100 nm in diameter, play important roles in cell biological functions, intercellular signal transduction and especially in cancer diagnosis and therapy. To better apply exosomes in mechanistic study of breast cancer signal transduction, we constructed recombinant eukaryotic expression vector expressing the near-infrared fluorescence protein and CD63 fusion protein through cloning iRFP682 gene and exosomal marker protein CD63 gene into plasmid containing the ITR of AAV. The constructed plasmids were co-transfected with helper plasmid in AAV-293 cell lines and were packaged into rAAV. After titer measurement, the recombinant plasmids were transfected into breast cancer cell lines. The cell lines that stably expressing near-infrared fluorescence protein were selected by fluorescence. Through isolation, purification and identification, we finally obtained a new biomarker: iRFP682 labeled exosomes secreted by breast cancer cell lines, which could be used in further studies of the distribution and signal transduction of exosomes in breast cancer microenvironment.

  7. High-throughput viability assay using an autonomously bioluminescent cell line with a bacterial Lux reporter. (United States)

    Class, Bradley; Thorne, Natasha; Aguisanda, Francis; Southall, Noel; McKew, John C; Zheng, Wei


    Cell viability assays are extensively used to determine cell health, evaluate growth conditions, and assess compound cytotoxicity. Most existing assays are endpoint assays, in which data are collected at one time point after termination of the experiment. The time point at which toxicity of a compound is evident, however, depends on the mechanism of that compound. An ideal cell viability assay allows the determination of compound toxicity kinetically without having to terminate the assay prematurely. We optimized and validated a reagent-addition-free cell viability assay using an autoluminescent HEK293 cell line that stably expresses bacterial luciferase and all substrates necessary for bioluminescence. This cell viability assay can be used for real-time, long-term measurement of compound cytotoxicity in live cells with a signal-to-basal ratio of 20- to 200-fold and Z-factors of ~0.6 after 24-, 48- 72-, or 96-h incubation with compound. We also found that the potencies of nine cytotoxic compounds correlated well with those measured by four other commonly used cell viability assays. The results demonstrated that this kinetic cell viability assay using the HEK293(lux) autoluminescent cell line is useful for high-throughput evaluation of compound cytotoxicity.

  8. Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines. (United States)

    Wu, Yong; Zhang, Tian-Ying; Fang, Lin-Lin; Chen, Zi-Xuan; Song, Liu-Wei; Cao, Jia-Li; Yang, Lin; Yuan, Quan; Xia, Ning-Shao


    The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A-D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study.

  9. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines. (United States)

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


    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 ( 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 (, that allows to link authentication data to actual cell lines.

  10. Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation. (United States)

    Hou, Jingmei; Niu, Minghui; Liu, Linhong; Zhu, Zijue; Wang, Xiaobo; Sun, Min; Yuan, Qingqing; Yang, Shi; Zeng, Wenxian; Liu, Yang; Li, Zheng; He, Zuping


    Spermatogonial stem cells (SSCs) have significant applications in both reproductive and regenerative medicine. However, primary human SSCs are very rare, and a human SSC line has not yet been available. In this study, we have for the first time reported a stable human SSC line by stably expressing human SV40 large T antigen. RT-PCR, immunocytochemistry, and Western blots revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks, including VASA, DAZL, MAGEA4, GFRA1, RET, UCHL1, GPR125, PLZF and THY1, suggesting that these cells are human SSCs phenotypically. Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years, and high levels of PCNA, UCHL1 and SV40 were maintained for long-term culture. Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine.

  11. Development of avian sarcoma and leukosis virus-based vector-packaging cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Stoker, A.W.; Bissell, M.J. (Univ. of California, Berkeley (USA))


    The authors have constructed an avian leukosis virus derivative with a 5{prime} deletion extending from within the tRNA primer binding site to a SacI site in the leader region. The aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1{Psi}{sup {minus}}, to develop vector-packaging cell lines. They show that RAV-1{Psi}{sup {minus}} can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, they have demonstrated that QT6-derived lines expressing RAV-1{Psi}{sup {minus}} can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-{Psi}{sup {minus}}-expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. They show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.

  12. Chemical genetic inhibition of Mps1 in stable human cell lines reveals novel aspects of Mps1 function in mitosis.

    Directory of Open Access Journals (Sweden)

    Tale Sliedrecht

    Full Text Available BACKGROUND: Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by the mitotic checkpoint that allows chromosome segregation only when all chromosomes are stably attached. Proper functioning of the attachment and checkpoint processes is thus important to prevent chromosomal instability. Both processes rely on the mitotic kinase Mps1. PRINCIPAL FINDING: We present here two cell lines in which endogenous Mps1 has been stably replaced with a mutant kinase (Mps1-as that is specifically inhibited by bulky PP1 analogs. Mps1 inhibition in these cell lines is highly penetrant and reversible. Timed inhibition during bipolar spindle assembly shows that Mps1 is critical for attachment error-correction and confirms its role in Aurora B regulation. We furthermore show that Mps1 has multiple controls over mitotic checkpoint activity. Mps1 inhibition precludes Mad1 localization to unattached kinetochores but also accelerates mitosis. This acceleration correlates with absence of detectable mitotic checkpoint complex after Mps1 inhibition. Finally, we show that short-term inhibition of Mps1 catalytic activity is sufficient to kill cells. CONCLUSIONS/SIGNIFICANCE: Mps1 is involved in the regulation of multiple key processes that ensure correct chromosome segregation and is a promising target for inhibition in anti-cancer strategies. We report here two cell lines that allow specific and highly penetrant inhibition of Mps1 in a reproducible manner through the use of chemical genetics. Using these cell lines we confirm previously suggested roles for Mps1 activity in mitosis, present evidence for novel functions and examine cell viability after short and prolonged Mps1 inhibition. These cell lines present the best cellular model system to date for investigations into Mps1 biology and the effects of penetrance and duration of Mps1 inhibition on cell viability.

  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


    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.


    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. Low expression of SLOOP associated with paclitaxel resistance in ovarian cancer cell line

    Institute of Scientific and Technical Information of China (English)

    GAO Jian-hua; HE Zhi-juan; WANG Qi; LI Xin; LI Yi-xuan; LIU Min; ZHENG Jian-hua; TANG Hua


    Background Recent studies indicate that Sl 00P expression may be a biomarker that can predict the success of cancer chemotherapy. Whether it is relevant to chemOtherapeutics in ovarian cancer is unknown.In this study,we investigated the association of S1OOP expression with paclitaxel sensitivity in ovarian cancer cell lines.Methods We measured S1 OOP expression and paclitaxel resistance profiles in parent SKOV3 and OVCAH3 cell lines.Then,the two cell lines were transiently transfected with SlOOP siRNA.We also constructed an OVCAR3 cell clone that stably overexpressed SIOOP The effect of S100P expression level on the survival of cells exposed to paclitaxel was measured using the MTT assav.S1OOP expression was evaluated by semi-quantitative RT.PCR and Western blotting.Significance of differences was calculated using independent samples t-test and one way analysis of variance(ANOVA).Results Lower S1 00P expression was associated with a survival advantage in OVCAR3 cells exposed to paclitaxel;the survival advantage in SKOV3 cells was smaller Pcells that had been transfected with S1 00P siRNA before being exposed to paciitaxel(P<0.05).Consistent with this,the OVCAR3 cell clone that was transfected to overexpress S1 00P was more sensitive to paclitaxelf P<0.05).Conclusions Low S1 00P expression contributes to drug resistance to paclitaxel in ovarian cancer cell lines.S100P expression thus might be a marker that can predict the effectiveness of paclitaxel based chemotherapy.Such a marker could be helpful in improving individual medication regimens for ovarian cancer patients.

  16. 稳定表达CRFR1的HEK293细胞株建立与功能鉴定%Development and evaluation of HEK293 cells stably expressing CRFR1

    Institute of Scientific and Technical Information of China (English)

    葛治娟; 于金梅; 马晓芸; 刘晓燕; 郑建全


    目的:建立稳定表达CRFR1的HEK293细胞株,并鉴定cAMP评价体系构建是否成功。方法采用Lipofectamine 2000将CRFR1质粒转染至HEK293细胞中,经G418筛选单克隆阳性细胞,采用Western blot技术、RT-PCR法、免疫荧光法证实稳定表达CRFR1细胞株构建成功。并用CRF刺激稳定表达 CRFR1的 HEK293细胞株,绘制 CRF 刺激HEK293-CRFR1细胞释放cAMP的量效曲线。结果 West-ern blot、RT-PCR、免疫荧光结果表明, CRFR1受体在HEK293细胞系中成功表达。 cAMP释放量效实验显示,CRF刺激 HEK293-CRFR1细胞释放 cAMP 的 EC50为(5.64±0.05)×10-10 mol · L-1。结论成功建立了稳定表达CRFR1的HEK293细胞株,并且cAMP含量评价体系构建成功,为研究CRFR1的生物学功能及筛选CRFR1受体靶向药物奠定了基础。%Aim ToconstructHEK293cellsstablyex-pressing corticotropin releasing factor receptor 1 ( CRFR1 ) , and evaluate its function by the cAMP as-say.Methods CulturedHEK293cellsweretransfect-ed with CRFR1-expressing vector by Lipofectamine 2000 and were selected by using G418 . CRFR1 ex-pression was detected by Western blot, RT-PCR and immunofluorescence.Results Westernblot,RT-PCR and immunofluorescence data revealed that the HEK293 cells expressed CRFR1 protein stably. The dose-responsive relationship experiment revealed that CRF induced a CRFR1-mediated cAMP production in HEK293 cells with EC50 =(5. 64 ± 0. 05) × 10 -10 mol ·L-1.Conclusion HEK293celllinesstablyex-pressing CRFR1 were constructed successfully, which would provide a cellular model to facilitate the research on the biological function of CRFR1 and CRFR1-targe-ted drug screening.

  17. Genetic engineering of cell lines using lentiviral vectors to achieve antibody secretion following encapsulated implantation. (United States)

    Lathuilière, Aurélien; Bohrmann, Bernd; Kopetzki, Erhard; Schweitzer, Christoph; Jacobsen, Helmut; Moniatte, Marc; Aebischer, Patrick; Schneider, Bernard L


    The controlled delivery of antibodies by immunoisolated bioimplants containing genetically engineered cells is an attractive and safe approach for chronic treatments. To reach therapeutic antibody levels there is a need to generate renewable cell lines, which can long-term survive in macroencapsulation devices while maintaining high antibody specific productivity. Here we have developed a dual lentiviral vector strategy for the genetic engineering of cell lines compatible with macroencapsulation, using separate vectors encoding IgG light and heavy chains. We show that IgG expression level can be maximized as a function of vector dose and transgene ratio. This approach allows for the generation of stable populations of IgG-expressing C2C12 mouse myoblasts, and for the subsequent isolation of clones stably secreting high IgG levels. Moreover, we demonstrate that cell transduction using this lentiviral system leads to the production of a functional glycosylated antibody by myogenic cells. Subsequent implantation of antibody-secreting cells in a high-capacity macroencapsulation device enables continuous delivery of recombinant antibodies in the mouse subcutaneous tissue, leading to substantial levels of therapeutic IgG detectable in the plasma.

  18. Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis) (United States)

    Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko


    Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

  19. Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

    Energy Technology Data Exchange (ETDEWEB)

    Yokoo, Masako [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Fujita, Ryosuke [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Innate Immunity Laboratory, Graduate School of Life Science and Creative Research Institution, Hokkaido University, Sapporo 001-0021 (Japan); Nakajima, Yumiko [Functional Genomics Group, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213 (Japan); Yoshimizu, Mamoru; Kasai, Hisae [Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611 (Japan); Asano, Shin-ichiro [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Bando, Hisanori, E-mail: [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan)


    Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.

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

    DEFF Research Database (Denmark)

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


    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...

  1. Hepatitis B virus X protein modulates the apoptosis of hepatoma cell line induced by TRAIL

    Institute of Scientific and Technical Information of China (English)

    LIANG Xiaohong; SUN Wensheng; GAO Lifen; MA Chunhong; HAN Lihui; CHEN Youhai


    The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study preliminary molecular mechanisms for its effects. In order to set up a model in vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test,caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL receptors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 μg/L TRAIL was 41.4%±7.2%, significantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly confirms the effects of HBx on TRAIL- induced apoptosis from two different points and it is not related with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.

  2. Toward an ideal animal model to trace donor cell fates after stem cell therapy: production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene. (United States)

    Hsiao, F S H; Lian, W S; Lin, S P; Lin, C J; Lin, Y S; Cheng, E C H; Liu, C W; Cheng, C C; Cheng, P H; Ding, S T; Lee, K H; Kuo, T F; Cheng, C F; Cheng, W T K; Wu, S C


    The discovery of postnatal mesenchymal stem cells (MSC) with their general multipotentiality has fueled much interest in the development of cell-based therapies. Proper identification of transplanted MSC is crucial for evaluating donor cell distribution, differentiation, and migration. Lack of an efficient marker of transplanted MSC has precluded our understanding of MSC-related regenerative studies, especially in large animal models such as pigs. In the present study, we produced transgenic pigs harboring an enhanced green fluorescent protein (EGFP) gene. The pigs provide a reliable and reproducible source for obtaining stable EGFP-labeled MSC, which is very useful for donor cell tracking after transplantation. The undifferentiated EGFP-tagged MSC expressed a greater quantity of EGFP while maintaining MSC multipotentiality. These cells exhibited homogeneous surface epitopes and possessed classic trilineage differentiation potential into osteogenic, adipogenic, and chondrogenic lineages, with robust EGFP expression maintained in all differentiated progeny. Injection of donor MSC can dramatically increase the thickness of infarcted myocardium and improve cardiac function in mice. Moreover, the MSC, with their strong EGFP expression, can be easily distinguished from the background autofluorescence in myocardial infarcts. We demonstrated an efficient, effective, and easy way to identify MSC after long-term culture and transplantation. With the transgenic model, we were able to obtain stem or progenitor cells in earlier passages compared with the transfection of traceable markers into established MSC. Because the integration site of the transgene was the same for all cells, we lessened the potential for positional effects and the heterogeneity of the stem cells. The EGFP-transgenic pigs may serve as useful biomedical and agricultural models of somatic stem cell biology.

  3. Inhibiting effect of antisense hTRT on telomerase activity of human liver cancer cell line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    牟娇; 李晓冬; 杨庆; 贾凤岐; 卫立辛; 郭亚军; 吴孟超


    Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity; the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion: Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.


    Institute of Scientific and Technical Information of China (English)

    肖成祖; 黄子才; 张正光; 叶建新; 高丽华; 郭智霞; 程度胜; 周鹤山; 孔惟惟


    CD-1,a genetically-engineered CHO cell line,was cultivated with a Biosilon microcarrier culture system.We successfully cultivated CD-1 cells to a very high density(over 1 ×107 cells/ml).Prourokinase was stably secreted at about 180IU/106 cells/24h.Experiments showed that CD-1 cells growing on Biosilon microcarriers were able to spontaneously release from the microcarriers,then reattach and proliferate on fresh microcarries.This makes it very easy to scale up production.The microcarries could be reused several times without affecting adhesion ,proliferation and prourokinase secretion.With CM-PECC membrane radial flow chromatography and MPG chromatography,the prouroknase in conditioned medium could be purfied to a specific activity of 1×105IU/mg of protein.The purification factor was about 600 fold,and approxiamately 90% of the biological activity was recovered.

  5. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

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


    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...

  6. Induced pluripotent stem cell lines derived from human somatic cells. (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


    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.

  7. Splice variants DNMT3B4 and DNMT3B7 overexpression inhibit cell proliferation in 293A cell line. (United States)

    Shao, Guo; Zhang, Ran; Zhang, Shu; Jiang, Shuyuan; Liu, You; Zhang, Wei; Zhang, Yanbo; Li, Jinping; Gong, Kerui; Gong, Keri; Hu, Xin-Rong; Jiang, Shi-Wen


    DNA methyltransferase 3B (DNMT3B) is critical in abnormal DNA methylation patterns in cancer cells. Nearly 40 alternatively spliced variants of DNMT3B have been reported. DNMT3B4 and DNMT3B7 are two kinds of splice variants of DNMT3B lacking the conserved methyltransferase motif. In this study, the effect of inactivation of DNMT3B variants, DNMT3B4 and DNMT3B7, on cell proliferation was assessed. pCMV-DNMT3B4 and pCMV-DNMT3B7 recombinant plasmids were developed and stably transfected into 293A cells. 293A cells transfected with plasmid pCMV-DNMT3B4 or pCMV-2B were then treated with G418 to the stable cell lines. After that, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method was used for testing the proliferation level, and flow cytometry was used to test cell cycle distribution of the cell line. The expression of p21 was detected by real-time PCR and Western blot. The methylation status of p21 promoter was detected by methylation-specific PCR (MS-PCR). It was found that DNMT3B4 and DNMT3B7 overexpression could inhibit cell proliferation and increase the expression of p21. Cell cycle analysis demonstrated that inactivation of DNMT3B variants overexpression inhibited cell cycle progression. Inactivation of DNMT3B variants overexpression facilitated p21 expression to delay 293A cell proliferation. These findings indicate that inactivation of DNMT3B variants might play an important role in cell proliferation correlating with the change of p21.

  8. Slashing the timelines: Opting to generate high-titer clonal lines faster via viability-based single cell sorting. (United States)

    Misaghi, Shahram; Shaw, David; Louie, Salina; Nava, Adrian; Simmons, Laura; Snedecor, Brad; Poon, Chungkee; Paw, Jonathan S; Gilmour-Appling, Laurie; Cupp, James E


    Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines.

  9. 77 FR 5489 - Identification of Human Cell Lines Project (United States)


    ... Genetics Group Web site at . Once the total number... methods for human cell line authentication the identity of a cell line need no longer be in doubt. NIST...

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

    Institute of Scientific and Technical Information of China (English)


    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.

  11. 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.


    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

  12. Characterization of the estrogen receptor transfected MCF10A breast cell line 139B6. (United States)

    Pilat, M J; Christman, J K; Brooks, S C


    There has been increasing evidence which suggests that abnormal expression of the estrogen receptor (ER) protein in nonmalignant breast tissue may be important in the carcinogenic process. To examine the effects of ER expression in immortalized nonmalignant mammary epithelial cells, an expression vector containing human ER cDNA was transfected into the ER negative human breast cells, MCF10A. Characterization of a clone stably expressing ER, 139B6, provided evidence for the regulated synthesis of a functional ER capable of binding estradiol-17 beta (E2) and undergoing processing. Expression of the ER gene did not enable E2 to stimulate endogenous genes [progesterone receptor (PgR), pS2, cathepsin D and TGF alpha] which normally respond to estrogens in breast cancer cells. The ER in 139B6 cells was, however, capable of inducing expression of an ERE-regulated reporter gene, indicating its ability to interact with transcriptional machinery. Furthermore, cultures in log growth displayed a slight increase in doubling time in the presence of E2. These results indicate that ER expression alone is not sufficient to induce a transformed phenotype. Thus, the 139B6 cell line should provide a new model for determining what additional changes lead to increased growth potential in response to E2 and for exploring how E2 itself may help bring about changes leading to progression of preneoplastic breast epithelial cells.

  13. Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP). (United States)

    Ducray, A; Bloquel, M; Hess, K; Hammond, G L; Gérard, H; Gérard, A


    The ultimate goal of this study was to compare the fate of rat testicular germ cells cocultured with mouse Sertoli cells that either do or do not produce rat androgen-binding protein (ABP). As a first step, we stably transfected a rat ABP expression construct into an immortalized mouse Sertoli cell line (TM4), which does not produce ABP when growing on plastic without hormones. The transfection of the pRc/CMV- rat ABP cDNA expression vector containing a neomycin resistance gene was made by either the liposome method (Dotap) or by polyethyleneimine transfection (PEI) into TM4 cell cultures. Neomycin-resistant clones were selected by adding Geneticin to the culture medium for 3 weeks. Analysis of over 25 clones revealed the presence of recombinant rat ABP when cell extracts and culture media were probed with a rabbit polyclonal antibody raised against rat testicular ABP, indicating the translation and secretion of a protein similar to rat testicular ABP. Transfected TM4 cells maintain the secretion of rat ABP for more than 40 days, with immunopositive rat ABP localized within cytoplasmic granules in the Golgi region and along cytoplasmic processes in TM4 transfected with either vector. Electron microscopic study revealed a higher development of cytoplasmic organelles involved in protein secretion.

  14. Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines. (United States)

    Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C


    Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival.

  15. Glial cell line-derived neurotrophic factor induced the differentiation of amniotic fluid-derived stem cells into vascular endothelial-like cells in vitro. (United States)

    Zhang, Ruyu; Lu, Ying; Li, Ju; Wang, Jia; Liu, Caixia; Gao, Fang; Sun, Dong


    Amniotic fluid-derived stem cells (AFSCs) are a novel source of stem cells that are isolated and cultured from second trimester amniocentesis. Glial cell line-derived neurotrophic factor (GDNF) acts as a tissue morphogen and regulates stem cell proliferation and differentiation. This study investigated the effect of an adenovirus-mediated GDNF gene, which was engineered into AFSCs, on the cells' biological properties and whether GDNF in combination with AFSCs can be directionally differentiated into vascular endothelial-like cells in vitro. AFSCs were isolated and cultured using the plastic adherence method in vitro and identified by the transcription factor Oct-4, which is the primary marker of pluripotent stem cells. AFSCs were efficiently transfected by a GFP-labeled plasmid system of an adenovirus vector carrying the GDNF gene (Ad-GDNF-GFP). Transfected AFSCs stably expressed GDNF. Transfected AFSCs were cultured in endothelial growth medium-2 containing vascular endothelial growth factor. After 1 week, AFSCs were positive for von Willebrand factor (vWF) and CD31, which are markers of endothelial cells, and the recombinant GDNF group was significantly higher than undifferentiated controls and the GFP only group. These results demonstrated that AFSCs differentiated into vascular endothelial-like cells in vitro, and recombinant GDNF promoted differentiation. The differentiation-induced AFSCs may be used as seed cells to provide a new manner of cell and gene therapies for transplantation into the vascular injury site to promote angiogenesis.

  16. A stably expressed llama single-domain intrabody targeting Rev displays broad-spectrum anti-HIV activity. (United States)

    Boons, Eline; Li, Guangdi; Vanstreels, Els; Vercruysse, Thomas; Pannecouque, Christophe; Vandamme, Anne-Mieke; Daelemans, Dirk


    The HIV Rev protein mediates the transport of partially and unspliced HIV mRNA from the nucleus to the cytoplasm. Rev multimerizes on a secondary stem-loop structure present in the viral intron-containing mRNA species and recruits the cellular karyopherin CRM1 to export viral mRNAs from the nucleus to the cytoplasm. Previously we have identified a single-domain intrabody (Nb(190)), derived from a llama heavy-chain antibody, which efficiently inhibits Rev multimerization and suppresses the production of infectious virus. We recently mapped the epitope of this nanobody and demonstrated that Rev residues K20 and Y23 are crucial for interaction while residues V16, H53 and L60 are important to a lesser extent. Here, we generated cell lines stably expressing Nb(190) and assessed the capacity of these cell lines to suppress the replication of different HIV-1 subtypes. These cells stably expressing the single-domain antibody are protected from virus-induced cytopathogenic effect even in the context of high multiplicity of infection. In addition, the replication of different subtypes of group M and one strain of group O is significantly suppressed in these cell lines. Next, we analysed the natural variations of Rev amino acids in sequence samples from HIV-1 infected patients worldwide and assessed the effect of Nb(190) on the most prevalent polymorphisms occurring at the key epitope positions (K20 and Y23) in Rev. We found that Nb(190) was able to suppress the function of these Rev variants except for the K20N mutant, which was present in only 0.7% of HIV-1 sequence populations (n = 4632). Cells stably expressing the single-domain intrabody Nb(190) are protected against virus-induced cytopathogenic effect and display a selective survival advantage upon infection. In addition, Nb(190) suppresses the replication of a wide range of different HIV-1 subtypes. Large-scale sequence analysis reveals that the Nb(190) epitope positions in Rev are well conserved across major HIV-1

  17. Tumstatin transfected into human glioma cell line U251 represses tumor growth by inhibiting angiogenesis

    Institute of Scientific and Technical Information of China (English)

    YE Hong-xing; YAO Yu; JIANG Xin-jun; YUAN Xian-rui


    Background Angiogenesis is a prerequisite for tumor growth and plays an important role in rapidly growing tumors,such as malignant gliomas.A variety of factors controlling the angiogenic balance have been described,and among these,the endogenous inhibitor of angiogenesis,tumstatin,has drawn considerable attention.The current study investigated whether expression of tumstatin by glioma cells could alter this balance and prevent tumor formation.Methods We engineered stable transfectants from human glioma cell line U251 to constitutively secrete a human tumstatin protein with c-myc and polyhistidine tags.Production and secretion of the tumstatin-c-myc-His fusion protein by tumstatin-transfected cells were confirmed by Western blotting analysis.In the present study,we identify the anti-angiogenic capacity of tumstatin using several in vitro and in vivo assays.Student's t-test and one-way analysis of variance (ANOVA) test were used to determine the statistical significance in this study.Results The tumstatin transfectants and control transfectants (stably transfected with a control plasmid) had similar in vitro growth rates compared to their parental cell lines.However,the conditioned medium from the tumstatin transfected tumor cells significantly inhibits proliferation and causes apoptosis of endothelial cells.It also inhibits tube formation of endothelial cells on Matrigel.Examination of armpit tumors arising from cells overexpressing tumstatin repress the growth of tumor,accompanying the decreased density of CD31 positive vessels in tumors ((5.62±1.32)/HP),compared to the control-transfectants group ((23.84+1.71)/HP) and wild type U251 glioma cells group ((29.33+4.45)/HP).Conclusion Anti-angiogenic gene therapy using human tumstatin gene may be an effective strategy for the treatment of glioma.

  18. Establishment and evaluation of a murine ανβ3-integrin-expressing cell line with increased susceptibility to Foot-and-mouth disease virus. (United States)

    Zhang, Wei; Lian, Kaiqi; Yang, Fan; Yang, Yang; Zhu, Zhijian; Zhu, Zixiang; Cao, Weijun; Mao, Ruoqing; Jin, Ye; He, Jijun; Guo, Jianhong; Liu, Xiangtao; Zheng, Haixue


    Integrin ανβ3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of ανβ3 integrin, a stable CHO-677 cell line expressing the murine ανβ3 heterodimer (designated as "CHO-677-mανβ3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits αν and β3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-mανβ3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-mανβ3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable ανβ3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of ανβ3 integrin, and as a cell model for FMDV research.


    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁


    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


    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



    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.


    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. Effect of TSLC1 Gene on Proliferation, Invasion and Apoptosis of Human Hepatocellular Carcinoma Cell Line HepG2

    Institute of Scientific and Technical Information of China (English)

    QIN Li; ZHU Wentao; XU Tao; HAO Youhua; ZHANG Zhengmao; TIAN Yongjun; YANG Dongliang


    The recombinant plasmid pCI-TSLC1 carrying TSLC1 gene was stably transfected into human hepatocellular carcinoma cell line HepG2. Cell proliferation was analyzed by MTT assay. The ability of migration was determined by transwell and FACSort flow cytometry was used to detect the cell cycle distribution and apoptosis. Western blotting revealed that H4 expressed higher amounts of TSLC1 protein than H15 and H0 did. The growth of TSLC1-transfected cells was significantly sup- pressed in vitro, and the ability of migration was reduced as well. The re-expression of TSLC1 could induce cell apoptosis. It was concluded that TSLC1 strongly inhibited the growth and ability of mi- gration of HepG2 cell line in vitro and also induced apoptosis, suggesting that TSLC1 could reduce the tumorigenicity of human hepatocellular carcinoma cell line HepG2 in vitro, which provided a ba-sis for further exploring the roles of TSLC1 in hepatocellular cellular carcinoma.

  4. Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene. (United States)

    Johnson, B E; Battey, J; Linnoila, I; Becker, K L; Makuch, R W; Snider, R H; Carney, D N; Minna, J D


    Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c-myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines. Images PMID:3016030

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

    Institute of Scientific and Technical Information of China (English)


    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.

  6. Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals

    Institute of Scientific and Technical Information of China (English)

    Zhi-Ren Zhang; Hong Yan; Shun-Qing Xu; Xi Sun; Yong-Jun Xu; Xiao-Kun Cai; Zhi-Wei Liu; Xiang-Lin Tan; Yi-Kai Zhou; Jun-Yue Zhang


    AIM: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs.METHODS: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs)and MMTV promoter segments into the pGL3-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG2, both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course,responsive period, sensitivity, structure-inducibility and doseeffect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG2 cells stably transfected by the recombinant vector (HepG2-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG2 cells (HepG2-wt).RESULTS: The inducible luciferase expression of HepG2-Luc cells wa s noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG2-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG2-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4′-PCB in HepG2-Luc cells was much less than that induced by TCDD suggests a structureinducibility relationship existing among DLCs. Within the concentrations from 3.5× 10-12 to 5× 10-9 mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG2-luc and HepG2-wt cells. The correlation between TCDD doses from 1.1×10-13 to 1×10-8 mol/L and luciferase activities was also found to be significant in HepG2-luc cells (r=0.997, P<0.001), but not in their HepG2-wt counterparts. For the comparison of the


    NARCIS (Netherlands)



    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


    Directory of Open Access Journals (Sweden)

    Mohan Durga


    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.

  9. Derivation and Utilization of Functional CD8(+) Dendritic Cell Lines. (United States)

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


    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.

  10. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

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


    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...

  11. P53 but not cyclin E acts in a negative regulatory loop to control HER-2 expression in MCF-7 breast carcinoma cell line.

    Directory of Open Access Journals (Sweden)

    Hamed Montazeri


    Full Text Available Cyclin E, HER-2 and p53, are considered as major prognostic markers in breast cancer. As they are related in patho-clinical level, we aimed to check if they have any direct interaction on expression of each other. To study the effect of cyclin E on HER-2 expression, cell lines stably overexpressing cyclin E or its low molecular weight (LMW isoforms were generated. To understand the results of p53 silencing either alone or in combination with cyclin E overexpression, we created three different p53 stably knocked down cell lines. Protein expression was analyzed by western blot, HER-2 expression in the established cell lines were determined using SYBR green real time PCR and data analyzed by REST software. Results indicate that HER-2 expression is only downregulated following p53 silencing and none of cyclin E isoforms can alter its expression. The presence of cyclin E isoforms in p53 silenced clones also does not altered HER-2 expression. Given the fact that p53 degradation is increased by HER-2 overexpression, these data can draw a regulatory loop in which a non-mutated functional p53 and HER-2 can bidirectionally regulate the expression of these two genes. This study improves our understandings of these pathways and these proteins can be introduced either as a marker or as a target in cancer treatment.

  12. Establishment of a functional cell line expressing both subunits of H1a and H2c of human hepatocyte surface molecule ASGPR. (United States)

    Hu, Bin; Yang, Yan; Liu, Jia; Ma, Zhiyong; Huang, Hongping; Liu, Shenpei; Yu, Yuan; Hao, Youhua; Wang, Baoju; Lu, Mengji; Yang, Dongliang


    To better understand the effect of a new split variant of human asialoglycoprotein receptor (ASGPR H1b) on ASGPR ligands' binding ability, we established a functional cell line which expresses ASGPR. The full lengths of ASGPRH1a and H2c fragments from human liver were amplified by reverse transcript PCR (RT-PCR) and inserted into eukaryotic expression vector pIRES2EGFP, pCDNA3.1 (Zeo+) respectively. The recombinants were co-transfected into HeLa cells. After selection by using Neocin and Zeocin, a stably transfected cell line was established, which was designated 4-1-6. The transcription and expression of ASGPRH1a and H2c in 4-1-6 were confirmed by RT-PCR, Western blotting and immunofluorescence. The endocytosis function of the artificial "ASGPR" on the surface of 4-1-6 was tested by FACS. It was found that the cell line 4-1-6 could bind ASGPR natural ligand molecular asialo-orosomucoid (ASOR). After the eukaryotic plasmid H1b/pCDNA3.1 (neo) was transfected into cell line 4-1-6, H1b did not down-regulate the ligand binding ability of ASGPR. The eukaryotic expression plasmid H1b/pcDNA3.1 (neo) and H2c/pcDNA3.1 (neo) were co-transfected transiently into Hela cell. Neither single H1b nor H1b and H2c could bind ASOR. In conclusion, a functional cell line of human asialoglycoprotein receptor (ASGPR) which expresses both H1a and H2c stably was established. The new split variant H1b has no effect on ASGPR binding to ASOR. ASGPRH1b alone can't bind to ASOR, it yet can't form functional complex with ASGPRH2c.

  13. Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines (United States)

    Chandhok, Gursimran; Horvath, Judit; Aggarwal, Annu; Bhatt, Mohit; Zibert, Andree; Schmidt, Hartmut HJ


    AIM: To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture. METHODS: The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined. RESULTS: Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability. CONCLUSION: The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines. PMID:27122662

  14. Authentication of the R06E Fruit Bat Cell Line

    Directory of Open Access Journals (Sweden)

    Ingo Jordan


    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.

  15. 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


    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.

  16. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning


    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

  17. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa


    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.

  18. 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)


    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.

  19. Beryllium-stimulated apoptosis in macrophage cell lines. (United States)

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


    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.

  20. 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.


    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 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.

  1. 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


    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.

  2. Motoneuron differentiation of immortalized human spinal cord cell lines. (United States)

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


    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.

  3. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska


    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.

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


    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.

  5. 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


    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.

  6. 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


    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.

  7. Deriving cell lines from zebrafish embryos and tumors. (United States)

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


    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.

  8. Recombinant protein production from stable mammalian cell lines and pools. (United States)

    Hacker, David L; Balasubramanian, Sowmya


    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.

  9. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,


    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.

  11. JKT-1 is not a human seminoma cell line. (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


    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.

  12. Establishment of a Novel Permissive Cell Line for the Propagation of Hepatitis C Virus by Expression of MicroRNA miR122 (United States)

    Kambara, Hiroto; Fukuhara, Takasuke; Shiokawa, Mai; Ono, Chikako; Ohara, Yuri; Kamitani, Wataru


    The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of “cured” cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics. PMID:22114337

  13. 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.

  14. Establishment of a Tumor-bearing Mouse Model Stably Expressing Human Tumor Antigens Survivin and MUC1 VNTRs

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li-xing; DU Jian-shi; WANG Yu-qian; LIU Chen-lu; XIA Qiu; ZHANG Xi-zhen; CONG Xian-ling; ZHANG Hai-hong


    The eukaryotic vectors VR1012 expressing survivin or 33 tandem repeats of human mucin 1(MUC1)(VNTRs),namely,VR1012-S and VR1012-VNTR(VNTR=variable number of tandem repeat),were constructed by cloning survivin and VNTR genes into VR1012,respectively.The eukaryotic vector pEGFP expressing survivin and MUC1 VNTRs fusion gene pEGFP-MS was also constructed.Mouse melanoma cell line(B16)stably expressing survivin and MUC1 VNTRs(MS+B16)was established by Lipofectamine-mediated transfection of pEGFP-MS into B16 cells.EGFP expression in MS+B16 cells was observed using a fluorescent microscope and survivin and MUC1 VNTRs(MS)expression was confirmed by means of Western blot analysis.A syngenic graft tumor model was generated by subcutaneous injection of MS+B16 cells into C57/BL6 mice and tumor size increased rapidly with time in a cell number dependent manner.After the third immunization,mice were challenged subcutaneously with 5×105 MS+B16 cells.Compared with that of the negative control immunized with phosphate-buffered saline(PBS),a significant reduction of tumor growth was observed in groups immunized with survivin plasmid DNA and MUC1 VNTRs plasmid DNA.Thus,the suppression of subcutaneous tumor was antigen-specific.This model is useful for the development of tumor vaccines targeting survivin and MUCI VNTRs.

  15. Development of a cell line from Echinococcus granulosus germinal layer. (United States)

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


    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.

  16. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line. (United States)

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


    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.

  19. Apoptotic effect of noscapine in breast cancer cell lines. (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M


    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

  20. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

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


    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

  1. Investigation of the selenium metabolism in cancer cell lines. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Julian Biau


    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.

  3. 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

  4. Profile of differentially expressed genes mediated by the type III epidermal growth factor receptor mutation expressed in a small-cell lung cancer cell line

    DEFF Research Database (Denmark)

    Pedersen, M.W.; Andersen, Thomas Thykjær; Ørntoft, Torben Falck;


    understanding of how the EGFRvIII contributes to the malignant phenotype is of major importance for future therapy. The GeneChip Hu6800Set developed by Affymetrix was used to identify changes in gene expression caused by the expression of EGFRvIII. The cell line selected for the study was an EGF receptor......Previous studies have shown a correlation between expression of the EGF receptor type III mutation (EGFRvIII) and a more malignant phenotype of various cancers including: non-small-cell lung cancer, glioblastoma multiforme, prostate cancer and breast cancer. Thus, a detailed molecular genetic...... negative small-cell-lung cancer cell line, GLC3, stably transfected with the EGFRvIII gene in a Tet-On system. By comparison of mRNA levels in EGFRvIII-GLC3 with those of Tet-On-GLC3, it was found that the levels of mRNAs encoding several transcription factors (ATF-3, JunD, and c-Myb), cell adhesion...

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

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin


    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

  6. 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


    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

  7. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren


    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...

  8. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud


    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.

  9. RSK4过表达真核表达载体对人乳腺癌移植瘤体内侵袭和转移的影响%Effects of breast cancer cells stably overexpressing RSK4 on growth of transplanted human breast cancer in severe combined immunodeficiency mice

    Institute of Scientific and Technical Information of China (English)

    杨华伟; 刘剑仑; 廖德仲; 孙源; 陈祖舜; 张晓丽


    Objective To construct a breast cancer cell line MD-MB-231 stably overexpressing RSK4 gene and study its in vivo effects on tumor tumorigenesis.Methods The MD-MB-231 cells were transfected with pcDNA 3.1/Neo and pcDNA3.1/Neo-RSK4 by lipofectamin transfection respectively.The stable expression of RSK4 (MR11 and MR12) and control vector (MN10 and MN11) were inoculated into severe combined immunodeficiency (SCID) mice subcutis to establish a model of human breast cancer in SCID mice.The xenograft tumor growth,invasion and metastasis were observed after 6-10 weeks.Results The stable cell lines MR11,MR12 and MN10,MN11 were screened successfully.We constructed the human breast cancer transplanted model and dissected SCID mice.After 6 weeks,SCID mice subcutis of the MN10/MN11 group yielded 10/10 metastatic tumors versus 6/10 and 7/10 in the MR11/MR12 group respectively.MR11 and MR12 showed much smaller tumor sizes and significantly reduced tumor volume and weight versus MN10 and MN11 (P <0.001 ).In the control group,visceral metastasis developed in 80%(8/10) of mice while in metastasis developed in 40% (4/10) of mice injected with RSK4-overexpressing MDA-MB-231 cells.Histological examination of hematoxylin and eosin-stained paraffin sections of lungs revealed numerous metastases in mice injected with vector control cells whereas RSK4-overexpressing cells showed markedly decreased metastatic lesions.Conclusion Transplanted human breast cancer in SCID mice closely correlates with the disease course of clinical tumor patients.Overexpression of RSK4 can inhibit tumor growth of transplanted human breast cancer in SCID mice.%目的 构建稳定过表达RSK4基因的MD-MB-231乳腺癌细胞株,观察过表达RSK4基因对乳腺癌体内成瘤的影响.方法 将pcDNA3.1/Neo和pcDNA3.1/Neo-RSK4分别转染进人乳腺癌细胞MD-MB-231,筛选出稳定的细胞株,命名为空载体组(MN10,MN11)和转染组(MR11,MR12)并种植至免疫缺陷小鼠(SCID)皮

  10. Establishment of a new bovine leukosis virus producing cell line. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Slavc Irene


    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.

  12. Metronidazole decreases viability of DLD-1 colorectal cancer cell line. (United States)

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


    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.

  13. CRIF1 enhances p53 activity via the chromatin remodeler SNF5 in the HCT116 colon cancer cell lines. (United States)

    Yan, Hai-Xia; Zhang, Yan-Jun; Zhang, Yuan; Ren, Xue; Shen, Yu-Fei; Cheng, Mo-Bin; Zhang, Ye


    CR6-interacting factor 1 (CRIF1) is ubiquitously expressed in human tissues. CRIF1 was first identified as a Gadd45γ (also known as CR6)-interacting protein, and it was also identified in a human colon cancer cell line stably transformed with p53. These results suggested that CRIF1 functions in the nucleus with p53 and Gadd45 family proteins in the suppression of cell growth and tumor development. Here, we found that CRIF1 could be recruited to a specific region in the promoter of the p53 gene, eliciting an increase in the mRNA and protein levels of p53 as well as p53 functional target genes. These functions required CRIF1 to interact with SNF5. CRIF1 was further recruited to the upstream promoter region of the p53 gene to suppress cell cycle progression in HCT116 cells. To our knowledge, this is the first evidence indicating that SNF5 is indispensable for CRIF1-enhanced p53 activity and its function in the suppression of cell cycle arrest in human cancer cells.

  14. Inhibitory Effects of Anti-sense PTTG on Malignant Phenotype of Human Ovarian Carcinoma Cell Line SK-OV-3

    Institute of Scientific and Technical Information of China (English)

    陈刚; 李静; 李辅军; 李箫; 周剑锋; 卢运萍; 马丁


    To construct eukaryotic expression vector expressing full length anti-sense pituitary tumor transforming gene (PTTG) mRNA and observe its blocking effect on the potential invasion of human ovarian carcinoma cell line SK-OV-3. PCR primers containing designed enzyme cut sites were used for cloning full-length PTTG gene fragment, and the resulting PCR product was inserted into the eukaryotic vector pcDNA3. 1 in the antisense direction. The recombinant vector was then transfected into SK-OV-3 by Lipofectamine. The positive cell clone was screened by G418, PTTG and bFGF at protein level expression were detected by Western blot. The biological behavior change of transfection positive cells was observed by colony formation in soft agar assay. Our results showed that SK-OV-3 clones stably expressing full-length recombinant pcDNA3. 1-PTTGas were obtained. The expressions of PTTG and bFGF protein in transfected cells were decreased by 61.5 % and 52.3%, respectively as compared with non-transfected ones. The number of colony formation was reduced significantly in transfected cells as compared with empty vector transfected and non-transfected cells. It is concluded that the recombinant vector pcDNA3. 1-PTTGas is a novel tool and provides an alternative anti-sense gene therapy targeted at PTTG in human carcinoma.

  15. 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.


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  17. Efficacy of ribavirin against malignant glioma cell lines (United States)



    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

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

    Directory of Open Access Journals (Sweden)

    Ritter Peter R


    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.

  19. Exometabolom analysis of breast cancer cell lines: Metabolic signature. (United States)

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


    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.

  20. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren


    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...

  1. Characterisation of thyroid medullary carcinoma TT cell line. (United States)

    Zabel, M; Grzeszkowiak, J


    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.

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

    Directory of Open Access Journals (Sweden)

    I. Hansenne


    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.

  3. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

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


    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...

  4. Generation of H9 T-cells stably expressing a membrane-bound form of the cytoplasmic tail of the Env-glycoprotein: lack of transcomplementation of defective HIV-1 virions encoding C-terminally truncated Env

    Directory of Open Access Journals (Sweden)

    Bosch Valerie


    Full Text Available Abstract H9-T-cells do not support the replication of mutant HIV-1 encoding Env protein lacking its long cytoplasmic C-terminal domain (Env-CT. Here we describe the generation of a H9-T-cell population constitutively expressing the HIV-1 Env-CT protein domain anchored in the cellular membrane by it homologous membrane-spanning domain (TMD. We confirmed that the Env-TMD-CT protein was associated with cellular membranes, that its expression did not have any obvious cytotoxic effects on the cells and that it did not affect wild-type HIV-1 replication. However, as measured in both a single-round assay as well as in spreading infections, replication competence of mutant pNL-Tr712, lacking the Env-CT, was not restored in this H9 T-cell population. This means that the Env-CT per se cannot transcomplement the replication block of HIV-1 virions encoding C-terminally truncated Env proteins and suggests that the Env-CT likely exerts its function only in the context of the complete Env protein.

  5. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes. (United States)

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


    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.

  6. 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


    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.

  7. 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.


    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.

  8. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska


    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.

  9. Guidelines for the use of cell lines in biomedical research. (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


    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.

  10. Inhibitory effects of hesperetin on Kv1.5 potassium channels stably expressed in HEK 293 cells and ultra-rapid delayed rectifier K(+) current in human atrial myocytes. (United States)

    Wang, Huan; Wang, Hong-Fei; Wang, Chen; Chen, Yu-Fang; Ma, Rong; Xiang, Ji-Zhou; Du, Xin-Ling; Tang, Qiang


    In the present study, the inhibitory effects of hesperetin (HSP) on human cardiac Kv1.5 channels expressed in HEK 293 cells and the ultra-rapid delayed rectifier K(+) current (Ikur) in human atrial myocytes were examined by using the whole-cell configuration of the patch-clamp techniques. We found that hesperetin rapidly and reversibly suppressed human Kv1.5 current in a concentration dependent manner with a half-maximal inhibition (IC50) of 23.15 μΜ with a Hill coefficient of 0.89. The current was maximally diminished about 71.36% at a concentration of 300μM hesperetin. Hesperetin significantly positive shifted the steady-state activation curve of Kv1.5, while negative shifted the steady-state inactivation curve. Hesperetin also accelerated the inactivation and markedly slowed the recovery from the inactivation of Kv1.5 currents. Block of Kv1.5 currents by hesperetin was in a frequency dependent manner. However, inclusion of 30μM hesperetin in pipette solution produced no effect on Kv1.5 channel current, while the current were remarkable and reversibly inhibited by extracellular application of 30μM hesperetin. We also found that hesperetin potently and reversibly inhibited the ultra-repaid delayed K(+) current (Ikur) in human atrial myocytes, which is in consistent with the effects of hesperetin on Kv1.5 currents in HEK 293 cells. In conclusion, hesperetin is a potent inhibitor of Ikur (which is encoded by Kv1.5), with blockade probably due to blocking of both open state and inactivated state channels from outside of the cell.

  11. 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


    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.

  12. 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.

  13. 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.

  14. Monoclonal antibodies against the human leukemia cell line K 562. (United States)

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


    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.

  15. Comparative Metabolic Flux Profiling of Melanoma Cell Lines (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.


    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

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


    Krampe, Britta; Al-Rubeai, Mohamed


    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...

  17. Skin Biopsy and Patient-Specific Stem Cell Lines (United States)

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


    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

  18. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations. (United States)

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


    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.

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


    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.

  20. 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))


    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.

  1. Analysis of LINE-1 expression in human pluripotent cells. (United States)

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


    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.

  2. 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


    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.

  3. Bcl-2 and N-Myc Coexpression Increases IGF-IR and Features of Malignant Growth in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Rama Jasty


    Full Text Available The bcl-2 and c-myc oncogenes cooperate to transform multiple cell types. In the pediatric malignancy NB2, Bcl2 is highly expressed. In tumors with a poor prognosis, N-Myc, a protein homologous to c-Myc, is overexpressed as a result of gene amplification. The present study was designed to determine whether Bcl-2 cooperates with N-Myc to bestow a tumorigenic phenotype to neuroblastoma (NB cells. NB cell lines that at baseline express neither Bcl-2 nor N-Myc were stably transfected to express these gene products. In this model, we found Bcl-2 rescues N-Myc-expressing cells from apoptosis induced by serum withdrawal. Coexpression of Bcl-2 and N-Myc supports growth in low serum conditions and anchorage-independent growth in soft agar. Similarly, in vivo tumorigenic and angiogenic activity was dependent on coexpression. Our data further suggests that the mechanism underlying these changes involves the receptor for insulin growth factor type I (IGF-IR.

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

    Directory of Open Access Journals (Sweden)

    Emanuela Mari


    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.

  5. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo


    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC] 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.

  6. Oral bioavailability of glyphosate: studies using two intestinal cell lines. (United States)

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


    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.

  7. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line. (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


    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.

  8. Heterologous expression of human cytochrome P450 2E1 in HepG2 cell line

    Institute of Scientific and Technical Information of China (English)

    Jian Zhuge; Ye Luo; Ying-Nian Yu


    AIM: Human cytochrome P-450 2E1 (CYP2E1) takes part in the biotransformation of ethanol, acetone, many smallmolecule substrates and volatile anesthetics. CYP2E1 is involved in chemical activation of many carcinogens,procarcinogens, and toxicants. To assess the metabolic and toxicological characteristics of CYP2E1, we cloned CYP2E1 cDNA and established a HepG2 cell line stably expressing recombinant CYP 2E1.METHODS: Human CYP2E1 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned into pGEM-T vector. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP2E1 to HepG2 cells. The expression of CYP2E1 mRNA was validated by RT-PCR. The enzyme activity of CYP2E1 catalyzing oxidation of 4-nitrophenol in postmitochondrial supernate (S9) fraction of the cells was determined by spectrophotometry. The metabolic activation of HepG2-CYP2E1 cells was assayed by N-nitrosodiethylamine (NDEA)cytotoxicity and micronucleus test.RESULTS: The cloned CYP2E1 cDNA segment was identical to that reported by Umeno et al(GenBank access No.J02843). HepG2-CYP2E1 cells expressed CYP2E1 mRNA and had 4-nitrophenol hydroxylase activity (0.162± S9 protein), which were undetectable in parent HepG2 cells. HepG2-CYP2E1 cells increased the cytotoxicity and micronucleus rate of NDEA in comparison with those of HepG2 cells.CONCLUSION: The cDNA of human CYP2E1 can be successfully cloned, and a cell line, HepG2-CYP2E1, which can efficiently express mRNA and has CYP2E1 activity, is established. The cell line is useful for testing the cytotoxicity,mutagenicity and metabolism of xenobiotics, which may possibly be activated or metabolized by CYP2E1.

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

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M


    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.

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

    Directory of Open Access Journals (Sweden)

    Valentina Pozzi


    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.

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


    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.

  12. 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.


    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).

  13. LINEing germ and embryonic stem cells' silencing of retrotransposons. (United States)

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena


    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.

  14. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies. (United States)

    Krampe, Britta; Al-Rubeai, Mohamed


    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.

  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


    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. U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3

    Directory of Open Access Journals (Sweden)

    Chan Wai-Yee


    stably expressing U94 implicate up-regulation of FN 1 and downregulation of ANGPTL 4 in anti tumor activity of U94. Further studies are necessary to determine functional roles of differentially expressed genes in U94 recombinant PC3 cell line, and hopefully provide leads to potential therapeutic targets in prostate cancer.

  17. 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


    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.

  18. Optimized protocol for derivation of human embryonic stem cell lines. (United States)

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


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yslas, Edith I., E-mail: [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)


    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.

  20. Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

    Directory of Open Access Journals (Sweden)

    Lin-Lin Liu

    Full Text Available Reverse transcription-quantitative polymerase chain reaction (RT-qPCR is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct, and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2 expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

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


    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


    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


    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. Characteristics of stably expressed human dopamine D1a and D1b receptors: atypical behavior of the dopamine D1b receptor

    DEFF Research Database (Denmark)

    Pedersen, U B; Norby, B; Jensen, Anders A.


    Human dopamine D1a and D1b receptors were stably expressed in Baby Hamster Kidney (BHK) or Chinese Hamster Ovary (CHO) cells. [3H]SCH23390 saturation experiments indicated the presence of only a single binding site in the D1a expressing cell line with a Kd of 0.5 nM. In D1b expressing cell lines...... for these receptors. Besides SCH 23390, only NNC 112, fluphenazine and bulbocapnine were able to discriminate between the two states of the D1b receptor. In case of the D1a receptor, the Ki values obtained in binding experiments were very similar to Ki values obtained from inhibition of dopamine stimulated adenylyl...... cyclase. In the D1b expressing cell line, the Ki values obtained from inhibition of the dopamine stimulated adenylyl cyclase indicated a significantly better correlation with the state of the D1b receptor showing high affinity for antagonists. In agreement with observations from binding experiments...

  6. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project (United States)


    ...; 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...

  7. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul


    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.

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

    Directory of Open Access Journals (Sweden)

    Chen Lei


    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.

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

    NARCIS (Netherlands)

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


    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

  10. 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)


    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...

  11. Cytotoxicity and genotoxicity of phenazine in two human cell lines. (United States)

    McGuigan, Claire F; Li, Xing-Fang


    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.

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


    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.

  13. Relation of cell proliferation to expression of peripheral benzodiazepine receptors in human breast cancer cell lines. (United States)

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


    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.

  14. 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


    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.

  15. Centrosomal dysregulation in human metastatic melanoma cell lines. (United States)

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


    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.

  16. 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)


    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.)

  17. 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


    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.

  18. Plasmids and packaging cell lines for use in phage display (United States)

    Bradbury, Andrew M.


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

  19. Effect of environmental estrogens on IL-1beta promoter activity in a macrophage cell line. (United States)

    Ruh, M F; Bi, Y; Cox, L; Berk, D; Howlett, A C; Bellone, C J


    Environmental estrogens or estrogen disrupters have recently received a great deal of attention because of their potential health impact on reproductive tissues. Few, if any, studies have been made on the impact of these compounds on the immune system. We sought to determine the activities of various environmental estrogens on the modulation of the interleukin-1beta (IL-1beta) gene in a model monocytic cell line, hER + IL-1beta-CAT+. This cell line stably transfected with the human estrogen receptor, and an IL-1beta promoter construct fused to the CAT reporter gene allows us to monitor the effect of estrogenic compounds on IL-1beta promoter activity. 17beta-estradiol (E2) markedly enhanced lipopolysaccharide- (LPS) induced IL-1beta promoter-driven CAT activity in a dose-dependent manner. The mycotoxins alpha-zearalenol and zearalenone both exhibited full agonist activity, but at lower potencies, with EC50 values of 1.8 and 54 nM, respectively, compared with E2 at 0.5 nM. In addition, genistein was a very low-potency agonist, having an EC50 of 1.5 microM. Similar to the E2 response, the slope factors for alpha-zearalenol, zearalenone, and genistein were close to 3.0, suggesting positive cooperativity in the estrogenic response. The activity of the mycotoxins appeared to be mediated through the estrogen receptor, since both the antiestrogens H1285 and ICI 182,780 effectively inhibited their agonist activity in a dose-dependent manner. Representative environmental estrogenic compounds both from plant and industrial sources were also tested. Unlike the mycoestrogens, none of the compounds, with the exception of genistein, synergized with LPS to enhance IL-1beta promoter activity. When tested for antiestrogenic activity, the industrial compound 4-octylphenol was able to antagonize the response to E2; however, the response was three orders of magnitude less potent than H 1285. Naringenin, a plant flavonoid, showed little or no ability to antagonize the response to E2

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

    Directory of Open Access Journals (Sweden)

    Yipeng Wang


    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.

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

    Directory of Open Access Journals (Sweden)

    Michael Iacovos P


    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

  2. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells. (United States)

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


    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.

  3. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    N. Rahimi-Fard


    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.

  5. 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


    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...

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

  7. Cytolytic replication of echoviruses in colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Gullberg Maria


    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.

  8. 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


    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...

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

    Institute of Scientific and Technical Information of China (English)


    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...

  10. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines. (United States)

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


    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

  11. 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


    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.

  12. Computation of mixing in large stably stratified enclosures (United States)

    Zhao, Haihua

    This dissertation presents a set of new numerical models for the mixing and heat transfer problems in large stably stratified enclosures. Basing on these models, a new computer code, BMIX++ (Berkeley mechanistic MIXing code in C++), was developed by Christensen (2001) and the author. Traditional lumped control volume methods and zone models cannot model the detailed information about the distributions of temperature, density, and pressure in enclosures and therefore can have significant errors. 2-D and 3-D CFD methods require very fine grid resolution to resolve thin substructures such as jets, wall boundaries, yet such fine grid resolution is difficult or impossible to provide due to computational expense. Peterson's scaling (1994) showed that stratified mixing processes in large stably stratified enclosures can be described using one-dimensional differential equations, with the vertical transport by free and wall jets modeled using standard integral techniques. This allows very large reductions in computational effort compared to three-dimensional numerical modeling of turbulent mixing in large enclosures. The BMIX++ code was developed to implement the above ideas. The code uses a Lagrangian approach to solve 1-D transient governing equations for the ambient fluid and uses analytical models or 1-D integral models to compute substructures. 1-D transient conduction model for the solid boundaries, pressure computation and opening models are also included to make the code more versatile. The BMIX++ code was implemented in C++ and the Object-Oriented-Programming (OOP) technique was intensively used. The BMIX++ code was successfully applied to different types of mixing problems such as stratification in a water tank due to a heater inside, water tank exchange flow experiment simulation, early stage building fire analysis, stratification produced by multiple plumes, and simulations for the UCB large enclosure experiments. Most of these simulations gave satisfying

  13. 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


    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.

  14. 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


    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.

  15. 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


    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.

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

    Directory of Open Access Journals (Sweden)



    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.

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

    Directory of Open Access Journals (Sweden)

    Edyta Biskup


    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.

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


    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.

  19. 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


    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.

  20. Efficient genetic method for establishing Drosophila cell lines unlocks the potential to create lines of specific genotypes. (United States)

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


    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.

  1. 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


    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.

  2. Apoptosis induced by propolis in human hepatocellular carcinoma cell line. (United States)

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


    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.

  3. Insulin sensitivity and inhibition by forskolin, dipyridamole and pentobarbital of glucose transport in three L6 muscle cell lines

    Institute of Scientific and Technical Information of China (English)


    L6 skeletal muscle myoblasts stably overexpressing glucose transporter GLUT1 or GLUT4 with exofacial myc-epitope tags were characterized for their response to insulin. In clonally selected cultures, 2-deoxyglucose uptake into L6-GLUT1myc myoblasts and myotubes was linear within the time of study. In L6-GLUT1myc and L6-GLUT4myc myoblasts, 100 nmol/L insulin treatment increased the GLUT1 content of the plasma membrane by 1.58±0.01 fold and the GLUT4 content 1.96±0.11 fold, as well as the 2-deoxyglucose uptake 1.53±0.09 and 1.86±0.17 fold respectively, all by a wortmannin-inhibitable manner. The phosphorylation of Akt in these two cell lines was increased by insulin. L6-GLUT1myc myoblasts showed a dose-dependent stimulation of glucose uptake by insulin, with unaltered sensitivity and maximal responsiveness compared with wild type cells. By contrast, the improved insulin responsiveness and sensitivity of glucose uptake were observed in L6-GLUT4myc myoblasts. Earlier studies indicated that forskolin might affect insulin-stimulated GLUT4 translocation. A 65% decrease of insulin-stimulated 2-deoxyglucose uptake in GLUT4myc cells was not due to an effect on GLUT4 mobilization to the plasma membrane, but instead on direct inhibition of GLUT4. Forskolin and dipyridamole are more potent inhibitors of GLUT4 than GLUT1. Alternatively, pentobarbital inhibits GLUT1 more than GLUT4. The use of these inhibitors confirmed that the overexpressed GLUT1 or GLUT4 are the major functional glucose transporters in unstimulated and insulin-stimulated L6 myoblasts. Therefore, L6-GLUT1myc and L6-GLUT4myc cells provide a platform to screen compounds that may have differential effects on GLUT isoform activity or may influence GLUT isoform mobilization to the cell surface of muscle cells.

  4. 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


    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.

  5. Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

    Directory of Open Access Journals (Sweden)

    Gourzones Claire


    Full Text Available Abstract Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2. To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2. Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to

  6. Pseudoislet of hybrid cellular spheroids from commercial cell lines. (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


    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.

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

    Directory of Open Access Journals (Sweden)

    Mozhgan Sedigh-Ardekani


    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.

  8. Off-line test of the KISS gas cell

    Energy Technology Data Exchange (ETDEWEB)

    Hirayama, Yoshikazu, E-mail: [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)


    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.

  9. 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;


    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...

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

    DEFF Research Database (Denmark)

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


    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...

  11. Impairment of cell cycle progression by aflatoxin B1 in human cell lines. (United States)

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


    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.

  12. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics. (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M


    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.

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

  14. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research. (United States)

    Kniss, Douglas A; Summerfield, Taryn L


    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.

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

    Institute of Scientific and Technical Information of China (English)


    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.

  16. 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


    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.

  17. Chromosome abnormalities in Japanese Burkitt lymphoma cell lines.

    Directory of Open Access Journals (Sweden)



    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.

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

    Institute of Scientific and Technical Information of China (English)


    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).

  19. LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines. (United States)

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


    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.

  20. 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: [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: [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Korea Centers for Disease Control and Prevention (Korea, Republic of)


    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.

  1. Effective and Steady Differentiation of a Clonal Derivative of P19CL6 Embryonal Carcinoma Cell Line into Beating Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Itsuki Mueller


    Full Text Available The P19CL6 cell line is a useful model to study cardiac differentiation in vitro. However, large variations were noticed in the differentiation rates among previous reports as well as our individual experiments. To overcome the unstable differentiation, we established P19CL6-A1, a new clonal derivative of P19CL6 that could differentiate into cardiomyocytes more efficiently and stably than the parent using the double stimulation with 5-Aza and DMSO based on the previous report. We also introduced a new software, Visorhythm, that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms. Using P19CL6-A1-derived cardiomyocytes and the software, we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents. These indicate that a combination of P19CL6-A1 and Visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery, drug screening, and toxicity testing.

  2. Internalization of cystatin C in human cell lines. (United States)

    Ekström, Ulf; Wallin, Hanna; Lorenzo, Julia; Holmqvist, Bo; Abrahamson, Magnus; Avilés, Francesc X


    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.

  3. 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


    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....

  4. Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection (United States)

    Chen, Zhilei; Simeon, Rudo; Chockalingam, Karuppiah; Rice, Charles M.


    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

  5. Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines. (United States)

    Chinchar, V G; Floyd, A D; Chinchar, G D; Taylor, M W


    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.

  6. 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.

  7. Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. (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


    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.

  8. Fluorouracil selectively enriches stem-like cells in the lung adenocarcinoma cell line SPC. (United States)

    Shi, Mu-mu; Xiong, Yan-lei; Jia, Xin-shan; Li, Xin; Zhang, Li; Li, Xiao-lei; Wang, En-Hua


    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.

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

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta


    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.

  10. 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


    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.

  11. Effects of small interfering RNAs targeting fascin on human esophageal squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Garcia Jose


    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.

  12. Study on the Iodine 125 Uptake of H460 Lung Cancer Cell Line by Co-transfection with the Human Sodium/Iodide Symporter and the Human Thyroperoxidase

    Directory of Open Access Journals (Sweden)

    Wei LI


    Full Text Available Background and objective Lung cancer harms people’s health or even lives severely. Especially, the therapy of non-small cell lung cancer (NSCLC has not been obviously improved for many years. The aim of this study is to transfer the human sodium/iodide symporter (hNIS and the human thyroperoxidase (hTPO genes into H460 lung cancer cell line, and to study the uptake ability of iodide after co-transfected hTPO and hNIS gene in cell lines. Methods Through cloning, recombination, packaging and amplifying, the recombinant adenosine virus (AdTPO was constructed. Then the protein expression of AdTPO was tested by Western blot. After transfected hNIS gene into human lung cancer cell line H460 through liposome, stably expressing hNIS gene cell lines (hNIS-H460 selected by G418 antibiotics was determined as hNIS-H460 group. Using AdTPO, hTPO gene was transducted into hNIS-H460, as AdTPO-hNIS-H460 group. H460 cell without hNIS gene was applied as control group (H460. Then, we investigated the 125I uptake assay of the above cells. Results We were successful in co-transfecting hNIS and hTPO gene into human lung cell lines H460, and were obtained hNIS and hTPO gene lung cancer cell lines (hNIS-H460 and AdTPO-hNIS-H460. In AdTPO-hNIS-H460, hNIS-H460 and H460, the uptake ability of 125I was (59 637.67±1 281.13, (48 622.17±2 242.28 and (1 440.17±372.86 counts•min-1. The uptake ability of 125I was 41 fold higher in AdTPO-hNIS-H460 than in blank control H460 (P<0.01, and 34 fold higher in hNIS-460 than in blank control H460 (P<0.01, and 1.2 fold higher in AdTPO-hNIS-H460 than in hNIS-H460 (P<0.01. Conclusion The uptake ability of 125I could increase by co-transfected hNIS and hTPO genes into human lung cancer cell lines H460.

  13. Stably propagating trains of attosecond electron bunches generated along the target back (United States)

    Pan, K. Q.; Zheng, C. Y.; Cao, L. H.; Liu, Z. J.; He, X. T.


    With the help of particle-in-cell simulations, we show a stably propagating train of attosecond ( 10 - 18 s) electron bunches which are generated along the target back surface via laser-solid interactions. The electron bunches are generated by the oscillating electric fields of the surface plasma wave. Because of the combinational effects of the electrostatic field and the static magnetic field on the target back surface, the electron bunches are stably propagating along the target back surface, which means they are totally separated from the laser pulse. The averaged energy of these electron bunches is over 20 MeV , the maximum averaged density is about 6 n c (where n c ≈ 1.1 × 10 21 cm - 3 is the critical density of the incident laser), and the averaged duration is less than 200 as. Such electron bunches are easily applied to the generation of attosecond x-rays via Compton backscattering. The energy conversion efficiency from the laser to the attosecond electron bunches is about 1.5%.

  14. Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines. (United States)

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


    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.

  15. Specific binding of benzodiazepines to human breast cancer cell lines. (United States)

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


    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.

  16. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah


    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.

  17. File list: Pol.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306580,SRX306578,SRX306576,SRX306575 ...

  18. File list: His.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 Histone Blood Lymphoblastoid cell line...5,SRX306570,SRX106080,SRX306566,SRX306568 ...

  19. File list: ALL.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 All antigens Blood Lymphoblastoid cell ...

  20. File list: Oth.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell ...

  1. File list: Oth.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell ...

  2. File list: His.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 Histone Blood Lymphoblastoid cell line...2,SRX306570,SRX356718,SRX356754,SRX026069 ...

  3. File list: DNS.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 DNase-seq Blood Lymphoblastoid cell line...091606,SRX469953,SRX091596 ...

  4. File list: Unc.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... ...

  5. File list: Pol.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306580,SRX306575,SRX306578,SRX306576 ...

  6. File list: Unc.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... ...

  7. File list: His.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 Histone Blood Lymphoblastoid cell line...7,SRX356718,SRX356754,SRX026054,SRX026069 ...

  8. File list: Unc.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... ...

  9. File list: His.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 Histone Blood Lymphoblastoid cell line...8,SRX356735,SRX356754,SRX306535,SRX026069 ...

  10. File list: DNS.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 DNase-seq Blood Lymphoblastoid cell line...8,SRX091598,SRX091626,SRX469951,SRX469953,SRX469955 ...

  11. File list: Pol.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306575,SRX306580,SRX306576,SRX306578 ...

  12. File list: DNS.Bld.20.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.20.AllAg.Lymphoblastoid_cell_line hg19 DNase-seq Blood Lymphoblastoid cell line...0,SRX091618,SRX091595,SRX091598,SRX469953 ...

  13. File list: Unc.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 Unclassified Blood Lymphoblastoid cell line... ...

  14. File list: Pol.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 RNA polymerase Blood Lymphoblastoid cell line... SRX306575,SRX306580,SRX306576,SRX306578 ...

  15. File list: DNS.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 DNase-seq Blood Lymphoblastoid cell line...8,SRX091623,SRX091605,SRX469953,SRX469951,SRX469955 ...

  16. File list: ALL.Bld.10.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.10.AllAg.Lymphoblastoid_cell_line hg19 All antigens Blood Lymphoblastoid cell line...:// ...

  17. File list: ALL.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 All antigens Blood Lymphoblastoid cell line...:// ...

  18. File list: Oth.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell ...

  19. File list: ALL.Bld.50.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.50.AllAg.Lymphoblastoid_cell_line hg19 All antigens Blood Lymphoblastoid cell line...6,SRX306568,SRX469953,SRX144527,SRX144520,SRX091596 ...

  20. File list: Oth.Bld.05.AllAg.Lymphoblastoid_cell_line [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.05.AllAg.Lymphoblastoid_cell_line hg19 TFs and others Blood Lymphoblastoid cell ...

  1. 稳定表达重组小鼠FAPα蛋白细胞株的构建%Establishment of a Cell Line Stably Expressing Recombinant Mouse FAPa

    Institute of Scientific and Technical Information of China (English)

    张欢; 方瑞; 黄思超; 杨倩之; 杜军; 蔡绍晖


    采用RT-PCR方法从BALB/c胚鼠成纤维细胞克隆FAPα基因,将其连接至表达栽体pTd-FL-N,转化Stbl3感受态.筛选重组质粒,经酶切、PCR检测及测序鉴定,证实表达质粒构建正确.将表达载体转染HEK293细胞,经G418筛选单克隆阳性细胞,采用Western blot技术证实稳定表达FAPα细胞株构建成功.通过流式细胞术确定FAPa蛋白可定位表达于细胞膜上.

  2. The Establishment of HEK293 Cell Line Stably Expressing GJB2 Mutation Common in Chinese%稳定表达中国人常见GJB2基因突变细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    张延平; 张晓强; 刘雅莉; 黄玮; 孙玉蕊


    目的 将中国人常见GJB2基因突变c.235deIC真核表达载体稳定转染入HEK293细胞,建立稳定表达人c.235deIC的HEK293细胞系.方法 将pCx26-EGFP和pCx26-235deIC-EGFP转染HEK293细胞,24 h后加G418筛选两周,使用FCM分选GFP阳性细胞,继续培养.蛋白免疫印迹检测GFP表达,激光共聚焦显微镜检测蛋白表达.结果 蛋白免疫印迹显示稳定转染人pCx26-EGFP和pCx26-235deIC-EGFP的HEK293细胞都能够检测到GFP的蛋白条带,显微镜下可见稳定表达基因的HEK293细胞普遍表达GFP.结论 本研究成功构建了稳定表达中国人常见GJB2基因突变的HEK293细胞系,为今后的研究工作打下基础.

  3. 稳定高效表达正反义Alu-Sx细胞系的建立%Establishment of HEK293 cell line expressing sense and antisense Alu-Sx stably and efficiently

    Institute of Scientific and Technical Information of China (English)

    彭亮; 吴刚; 蒋继志; 靳风烁; 丁瑞


    目的建立稳定高效表达正反义Alu-Sx的细胞系.方法根据Alu亚家族Sx序列合成两对两端带有限制性酶切位点的引物,提取HEK293细胞的总DNA后PCR扩增,产物连接到真核表达载体pcDNA3.1/myc-His A中构建成重组体.经酶切及测序鉴定后,阳离子脂质体转染法转染HEK293细胞后G418筛选稳定表达的细胞克隆,Northern杂交检测并挑选出表达最强的亚克隆.结果成功构建Alu亚家族Sx的正反义真核表达载体并获得高效稳定表达的HEK293细胞亚克隆.结论高效稳定表达正反义Alu Sx的HEK293细胞亚克隆可用于下一步研究.

  4. M-CSF核内稳定表达细胞系的构建和鉴定%Construction of nuclear localization vector pCMV/M-CSF and establishment of its stably expressing cell line

    Institute of Scientific and Technical Information of China (English)

    涂剑; 滕淑静; 张晓红; 赵雪琴; 唐圣松


    目的:构建真核细胞pCMV/nuc/M-CSF载体,建立M-CSF核内稳定表达细胞系,为进一步研究M-CSF的核内作用奠定基础.方法:采用PCR扩增人M-CSF活性片段,将M-CSF片段插入真核表达载体pCMV/myc/nuc,强制性把M-CSF引入细胞核内,通过PCR、测序鉴定筛选阳性重组体pCMV/M-CSF,脂质体介导转染HeLa细胞,经G418筛选后,用RT-PCR、间接免疫荧光鉴定其在HeLa细胞中的表达及定位分布.结果:琼脂糖电泳结果显示插入片段为1 400 bp左右,与预期M-CSF分子大小相当;DNA测序分析表明插入质粒的M-CSF无读码框移位,并与来源序列一致.RT-PCR和间接免疫荧光检测表明,转染pCMV/M-CSF的HeLa细胞能稳定表达M-CSF mRNA与M-CSF蛋白,且表达的M-CSF定位于HeLa细胞的细胞核.结论:成功构建了真核细胞pCMV/nuc/M-CSF载体,建立了M-CSF核内稳定表达细胞系.

  5. Oxidative stress induces hypomethylation of LINE-1 and hypermethylation of the RUNX3 promoter in a bladder cancer cell line. (United States)

    Wongpaiboonwattana, Wikrom; Tosukhowong, Piyaratana; Dissayabutra, Thasinas; Mutirangura, Apiwat; Boonla, Chanchai


    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.

  6. 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


    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

  7. Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Zhang Ping


    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

  8. A vertically integrated dynamic RAM-cell: Buried bit line memory cell with floating transfer layer

    NARCIS (Netherlands)

    Mouthaan, Ton; Vertregt, Maarten


    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

  9. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines. (United States)

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


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

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

    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: [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States)


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

  11. Characterisation and Manipulation of Docetaxel Resistant Prostate Cancer Cell Lines

    LENUS (Irish Health Repository)

    O'Neill, Amanda J


    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.

  12. Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

    DEFF Research Database (Denmark)

    Norén, K; Hansen, Gert Helge; Clausen, H;


    In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...

  13. Isolation and Enrichment of Mouse Female Germ Line Stem Cells

    Directory of Open Access Journals (Sweden)

    Somayeh Khosravi-Farsani


    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.

  14. 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


    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.

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

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


    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.

  16. Origin of the U87MG glioma cell line: Good news and bad news. (United States)

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


    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.

  17. Growth inhibitory activity of Ankaferd hemostat on primary melanoma cells and cell lines (United States)

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


    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

  18. Neuroblastoma cell lines contain pluripotent tumor initiating cells that are susceptible to a targeted oncolytic virus.

    Directory of Open Access Journals (Sweden)

    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.

  19. 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;


    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...

  20. Cell Motility and Invasiveness of Neurofibromin-Deficient Neural Crest Cells and Malignant Triton Tumor Lines (United States)


    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

  1. Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line.

    Directory of Open Access Journals (Sweden)

    Keiichi Ohshima

    Full Text Available BACKGROUND: Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene down-regulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well. METHODOLOGY/PRINCIPAL FINDINGS: Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity. CONCLUSIONS/SIGNIFICANCE: The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosomes from AZ-P7a cells may reflect their oncogenic characteristics including tumorigenesis and metastasis. Since let-7 miRNAs generally play a tumor-suppressive role as targeting oncogenes such as RAS and HMGA2, our results suggest that AZ-P7a cells

  2. Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

    Directory of Open Access Journals (Sweden)

    Weyhe Dirk


    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.

  3. In vitro Acute Cytotoxicity of Abamectin to the Gill Cell Line of Flounder Paralichthy olivaceus

    Institute of Scientific and Technical Information of China (English)


    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.

  4. Tooth regeneration from newly established cell lines from a molar tooth germ epithelium. (United States)

    Komine, Akihiko; Suenaga, Momoko; Nakao, Kazuhisa; Tsuji, Takashi; Tomooka, Yasuhiro


    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.

  5. Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

    Directory of Open Access Journals (Sweden)

    Tobias Hannes


    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.

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

    Directory of Open Access Journals (Sweden)

    Behra Martine


    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.

  7. Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection. (United States)

    Throm, Robert E; Ouma, Annastasia A; Zhou, Sheng; Chandrasekaran, Anantharaman; Lockey, Timothy; Greene, Michael; De Ravin, Suk See; Moayeri, Morvarid; Malech, Harry L; Sorrentino, Brian P; Gray, John T


    Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 10(7) transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common gamma chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 x 10(7) TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1).

  8. 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


    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.

  9. In vitro platinum drug chemosensitivity of human cervical squamous cell carcinoma cell lines with intrinsic and acquired resistance to cisplatin.


    Mellish, K. J.; Kelland, L R; Harrap, K. R.


    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...

  10. Stably Doped Conducting Polymer Nanoshells by Surface Initiated Polymerization. (United States)

    Li, Junwei; Yoon, Soon Joon; Hsieh, Bao-Yu; Tai, Wanyi; O'Donnell, Matthew; Gao, Xiaohu


    Despite broad applications ranging from electronics to biomedical sensing and imaging, a long-standing problem of conducting polymers is the poor resistance to dedoping, which directly affects their signature electrical and optical properties. This problem is particularly significant for biomedical uses because of fast leaching of dopant ions in physiological environments. Here, we describe a new approach to engineer multimodal core-shell nanoparticles with a stably doped conductive polymer shell in biological environments. It was achieved by making a densely packed polymer brush rather than changing its molecular structure. Polyaniline (PANI) was used as a model compound due to its concentrated near-infrared (NIR) absorption. It was grafted onto a magnetic nanoparticle via a polydopamine intermediate layer. Remarkably, at pH 7 its conductivity is ca. 2000× higher than conventional PANI nanoshells. Similarly, its NIR absorption is enhanced by 2 orders of magnitude, ideal for photothermal imaging and therapy. Another surprising finding is its nonfouling property, even outperforming polyethylene glycol. This platform technology is also expected to open exciting opportunities in engineering stable conductive materials for electronics, imaging, and sensing.

  11. Turbulence comes in bursts in stably stratified flows

    CERN Document Server

    Rorai, C; Pouquet, A


    There is a clear distinction between simple laminar and complex turbulent fluids. But in some cases, as for the nocturnal planetary boundary layer, a stable and well-ordered flow can develop intense and sporadic bursts of turbulent activity which disappear slowly in time. This phenomenon is ill-understood and poorly modeled; and yet, it is central to our understanding of weather and climate dynamics. We present here a simple model which shows that in stably stratified turbulence, the stronger bursts can occur when the flow is expected to be more stable. The bursts are generated by a rapid non-linear amplification of energy stored in waves, and are associated with energetic interchanges between vertical velocity and temperature (or density) fluctuations. Direct numerical simulations on grids of 2048^3 points confirm this somewhat paradoxical result of measurably stronger events for more stable flows, displayed not only in the temperature and vertical velocity derivatives, but also in the amplitude of the field...

  12. Mixing efficiency of turbulent patches in stably stratified flows (United States)

    Garanaik, Amrapalli; Venayagamoorthy, Subhas Karan


    A key quantity that is essential for estimating the turbulent diapycnal (irreversible) mixing in stably stratified flow is the mixing efficiency Rf*, which is a measure of the amount of turbulent kinetic energy that is irreversibly converted into background potential energy. In particular, there is an ongoing debate in the oceanographic mixing community regarding the utility of the buoyancy Reynolds number (Reb) , particularly with regard to how mixing efficiency and diapycnal diffusivity vary with Reb . Specifically, is there a universal relationship between the intensity of turbulence and the strength of the stratification that supports an unambiguous description of mixing efficiency based on Reb ? The focus of the present study is to investigate the variability of Rf* by considering oceanic turbulence data obtained from microstructure profiles in conjunction with data from laboratory experiments and DNS. Field data analysis has done by identifying turbulent patches using Thorpe sorting method for potential density. The analysis clearly shows that high mixing efficiencies can persist at high buoyancy Reynolds numbers. This is contradiction to previous studies which predict that mixing efficiency should decrease universally for Reb greater than O (100) . Funded by NSF and ONR.

  13. Expression of tumor antigens on primary ovarian cancer cells compared to established ovarian cancer cell lines (United States)

    Kloudová, Kamila; Hromádková, Hana; Partlová, Simona; Brtnický, Tomáš; Rob, Lukáš; Bartůňková, Jiřina; Hensler, Michal; Halaška, Michael J.; Špíšek, Radek; Fialová, Anna


    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

  14. 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.


    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.

  15. 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


    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.

  16. Lipid analysis of eight human breast cancer cell lines with ToF-SIMS. (United States)

    Robinson, Michael A; Graham, Daniel J; Morrish, Fionnuala; Hockenbery, David; Gamble, Lara J


    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.

  17. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines. (United States)

    Varamini, P; Soltani, M; Ghaderi, A


    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.

  18. 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:


    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.

  19. Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

    Directory of Open Access Journals (Sweden)

    Létourneau Isabelle J


    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

  20. Effect of Docosahexaenoic Acid on Cell Cycle Pathways in Breast Cell Lines With Different Transformation Degree. (United States)

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


    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.

  1. Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2

    Institute of Scientific and Technical Information of China (English)

    Xiao-Na Zhou; Guang-Ming Li; Ying-Chen Xu; Tuan-Jie Zhao; Ji-Xiang Wu


    Background:Decoy receptor 3 (DcR3) binds to Fas ligand (FasL) and inhibits FasL-induced apoptosis.The receptor is overexpressed in hepatocellular carcinoma (HCC),and it is associated with the growth and metastatic spread of tumors.DcR3 holds promises as a new target for the treatment of HCC,but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3.The present work,therefore,examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2.Methods:HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3.After the knockdown of DcR3 was confirmed,cell proliferation,clone formation,ability of migrating across transwell membrane,and wound healing were assessed in vitro.Matrix metalloproteinase-9 (MMP 9) and vascular epithelial growth factor (VEGF)-C and D expressions of the DcR3 knockdown were also studied.Comparisons between multiple groups were done using one-way analysis of variance (ANOVA),while pairwise comparisons were performed using Student's t test.P < 0.05 was regarded statistically significant.Results:DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2.Stable knockdown of DcR3 slowed down the growth of HepG2 (P < 0.05) and reduced the number of clones formed by 50% compared to those without DcR3 knockdown (P < 0.05).The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control (P < 0.05) and suppressed the closure of scratch wound (P < 0.05).In addition,the messenger RNA levels of MMP 9,VEGF-C,and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control (P < 0.05).Conclusions:Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2.Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC.

  2. 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.

  3. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines. (United States)

    Hayashi, Teruyuki; Fukuda, Nanaho; Uchiyama, Seiichi; Inada, Noriko


    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.

  4. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl]. (United States)

    Shimizu, T


    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.

  5. Heterotransplantation of human leukemic B-cell, T-cell and null-cell lines in hamsters.

    Directory of Open Access Journals (Sweden)



    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.

  6. Establishment of a Tumor-bearing Mouse Model Stably Expressing EGFP Labeled Human MUC1 VNTRs

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shu-zi; ZHANG Hai-hong; ZHANG Wa; SHI He-liang; YU Xiang-hui; KONG Wei; LI Wei


    Two eukaryotic vectors expressing 9 tandem repeats of human MUCI(VNTR),VRI012-VNTR,and pEGFP-VNTR,were constructed by cloning VNTR gene into VR1012 and pEGFP,respectively,VNTR stably expressing murine Lewis lung carcinoma(LLC) cell Iine(VNTR+ LLC) was established by Lipofectamine-mediated transfection of pEGFP-VNTR into LLC cells,The EGFP expression was observed under a fluorescent microscope and VNTR expression in VNTR+ LLC cells was confirmed by means of Western blotting,A syngenic graft tumor model was generated by subcutaneous injection of VNTR+ LLC cells into C57/BL6 mice and tumor size increased rapidly with time and in a cell number dependent manner,VNTR mRNA expression in the tumor formed was confirmed by RT-PCR.After the third immunization mice were challenged subcutaneously with 5x10 5 VNTR* LLC cells,a significant reduction of subcutaneous tumor growth was observed in the groups immunized with VNTR plasmid DNA compared with that in the groups immunized with the vector DNA alone,Thus,the suppression of subcutaneous tumor was antigen-specific,This model is useful for the development of tumor vaccines targeting MUCI VNTRs.

  7. Drug treatment of cancer cell lines: a way to select for cancer stem cells? (United States)

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A Ivana; Mondello, Chiara


    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.

  8. Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

    Directory of Open Access Journals (Sweden)

    Ilaria Chiodi


    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.

  9. 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: [Institute of Molecular Genetics, CNR, via Abbiategrasso 207, 27100 Pavia (Italy)


    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.

  10. Photodynamic therapy-induced programmed cell death in carcinoma cell lines (United States)

    He, Xiao-Yan; Sikes, Robert A.; Thomsen, Sharon L.; Chung, L.; Jacques, Steven L.


    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.

  11. 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.


    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.

  12. 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.


    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.

  13. TIMP-1 overexpression does not affect sensitivity to HER2-targeting drugs in the HER2-gene-amplified SK-BR-3 human breast cancer cell line. (United States)

    Deng, Xiaohong; Fogh, Louise; Lademann, Ulrik; Jensen, Vibeke; Stenvang, Jan; Yang, Huanming; Brünner, Nils; Schrohl, Anne-Sofie


    Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been suggested as a marker of prognosis and response to treatment in breast cancer. In vitro, TIMP-1 can regulate shedding of the extracellular domain of HER2 and signalling via the Akt pathway, and we hypothesize that TIMP-1 therefore can affect sensitivity to the HER2-targeting drugs trastuzumab and lapatinib. SK-BR-3 human breast cancer cells were stably transfected with TIMP-1, characterized with regard to TIMP-1 protein expression, proliferation, and functionality of the secreted TIMP-1, and the sensitivity to trastuzumab and lapatinib was studied in five selected single-cell subclones expressing TIMP-1 protein at various levels plus the parental SK-BR-3 cell line. Both trastuzumab and lapatinib reduced cell viability, as determined by MTT assay, but the sensitivity to the drugs was not associated with the expression level of TIMP-1 protein. Western blotting showed that the activation of Akt, PTEN, and HER2 as well as ADAM10 was similar in all clones. In conclusion, in this model, TIMP-1 overexpression does not affect HER2 cleavage by ADAM10 or signalling via the Akt pathway, and TIMP-1 does not influence sensitivity to trastuzumab and lapatinib.

  14. Evaluation of Stem Cell Markers, CD44/CD24 in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Masoud Hashemi Arabi


    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

  15. Notch reporter activity in breast cancer cell lines identifies a subset of cells with stem cell activity


    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


    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 ...

  16. 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;


    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....

  17. Comparative In Vitro Immune Stimulation Analysis of Primary Human B Cells and B Cell Lines (United States)

    Van Belle, Kristien; Herman, Jean; Boon, Louis; Waer, Mark


    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

  18. Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)


    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...

  19. 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


    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.

  20. 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


    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.

  1. Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines (United States)

    Serfozo, Peter; Schlarman, Maggie S; Pierret, Chris; Maria, Bernard L; Kirk, Mark D


    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

  2. Establishment and characterization of a cell line (OMC-9) originating from a human endometrial stromal sarcoma. (United States)

    Kakuno, Yoshiteru; Yamada, Takashi; Mori, Hiroshi; Narabayashi, Isamu


    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 cells exhibit a human karyotype with a modal chromosomal number in the diploid range. The cells were able to be transplanted into the subcutis of nude mice and produced tumors resembling the original tumor. OMC-9 cells produced tissue polypeptide antigen. Both CD10, a sensitive and diagnostically useful marker of endometrial stromal neoplasms, and vimentin were identified immunohistochemically in the original tumor and the heterotransplanted tumor. The cells were sensitive to actinomycin D, doxorubicin, carboplatin, cisplatin and etoposide, drugs used commonly in the treatment of gynecologic cancer. Only three reports of uterine endometrial stromal sarcoma cell lines have thus far been reported in the literature. OMC-9 is the first endometrial stromal sarcoma cell line in which CD10 expression and chemosensitivity have been identified.

  3. Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines. (United States)

    Gardell, Alison M; Qin, Qin; Rice, Robert H; Li, Johnathan; Kültz, Dietmar


    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

  4. Gene probes to detect cross-culture contamination in hormone producing cell lines

    DEFF Research Database (Denmark)

    Matsuba, I; Lernmark, A; Madsen, Ole Dragsbæk;


    Cross-culture contamination of cell lines propagated in continuous culture is a frequent event and particularly difficult to resolve in cells expressing similar phenotypes. We demonstrate that DNA-DNA hybridization to blotted endonuclease-digested cell DNA effectively detects cross-culture...... the effective use of gene probes to control the origin of cell cultures....... contamination to monitor inter-species as well as intra-species cross contamination. An insulin-producing cell-line, Clone-16, originally cloned from a human fetal endocrine pancreatic cell line did not produce human c-peptide as anticipated. DNA from these cells showed no hybridization to the human ALU...

  5. Effect of arginase II on L-arginine depletion and cell growth in murine cell lines of renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Patterson John R


    Full Text Available Abstract Background L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested. Methods Three murine renal cell carcinoma (mRCC cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC. Results Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01 reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity. The depletion of L-arginine by mRCC induced the decrease expression of CD3ζ a key element for T-cell function. Conclusion The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3ζ. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell

  6. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. (United States)

    Martin, G R


    This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

  7. Network signatures of cellular immortalization in human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of); Jeon, Jae-Pil, E-mail: [Division of Brain Diseases, Center for Biomedical Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of)


    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  8. Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro. (United States)

    Matsumoto, Asako; Harada, Hidemitsu; Saito, Masahiro; Taniguchi, Akiyoshi


    Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.

  9. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines. (United States)

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


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

  10. Electroporation enhances mitomycin C cytotoxicity on T24 bladder cancer cell line

    DEFF Research Database (Denmark)

    Vasquez, Juan Luis; Gehl, Julie; Hermann, Gregers G


    improves the cytotoxicity of mitomycin. In two cell lines, T24 (bladder cancer cell line) and DC3F (Chinese hamster fibroblast), exposure to different concentrations of mitomycin (0.01-2000μM) was tested with and without electroporation (6 pulses of 1kV/cm, duration: 99μs, frequency: 1Hz). Cell viability...

  11. Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia

    DEFF Research Database (Denmark)

    Lanemo Myhrinder, Anna; Hellqvist, Eva; Bergh, Ann-Charlotte;


    /short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA....

  12. A suspended cell line from Trichoplusia ni (Lepidoptera):Characterization and expression of recombinant proteins

    Institute of Scientific and Technical Information of China (English)

    Min-Juan Meng; Tian-Long Li; Chang-You Li; Guo-Xun Li


    A suspended cell line from Trichoplusia ni embryos was established, and its susceptibility to Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)infection was investigated. This cell line had characteristics distinct from the BTI-Tn5B 14 cell line (Tn5B 1-4) from T. ni in growth, and showed approximately the same responses to AcMNPV infection, production of occlusion bodies, and levels of recombinant protein expression. No clumps were observed at maximum cell density at late-log phase in shakeflask or T-flask cultures, and thus the cells represent a useful new contribution for baculovirus research. The cells consist of two major morphological types: approximately 70% spindle-shaped cells and 30% round cells. The cell line was highly susceptible to virus infection and produced around 107 AcMNPV occlusion bodies per cell, on average.Production of β-galactosidase and secreted alkaline phosphatase was high with 3.97 + 0.13×104 IU/mL and 3.48±0.40 IU/mL, respectively. This cell line may be applicable for studies of scale-up production of viruses or baculovirus-insect cell expression. We also believe the new line can be a source for cell clones with higher production of virus and recombinant proteins compared to the parent or other existing cell lines such as Tn5B 1-4.

  13. Prediction of epigenetically regulated genes in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro A; Sadanandam, Anguraj; Durinck, Steffen; Nautiyal, Shivani; Flaucher, Diane; Carlton, Victoria EH; Moorhead, Martin; Lu, Yontao; Gray, Joe W; Faham, Malek; Spellman, Paul; Parvin, Bahram


    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the

  14. Prediction of epigenetically regulated genes in breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Lu Yontao


    Full Text Available Abstract Background Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP. The pipeline (i reduces the dimensionality of the methylation data, (ii associates the reduced methylation data with gene expression data, and (iii ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i methylation sites are grouped across the genome to identify regions of interest, and (ii methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Results Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between

  15. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    DEFF Research Database (Denmark)

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna


    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines...... based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted...... antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors...


    Institute of Scientific and Technical Information of China (English)

    陈建敏; 章扬培; 吴英


    O6-methylguanine-DNA methltransferase(MGMT) gene expression in 6 Mer+(HeLa S3,SMMC-7721,SGC-7901,B-239,AGZY83-a,and Cc801)and 2Mer-(SHG-44,AND HeLa MR) haman tumor cell lines was examined.Southern blot analysis revealed no deletion,amplification,or rearrangement of the MGMT gene in these cell lines.However,-1.0kb transcripts were detected in the 6 Mer+ cell lines but not in the 2 Mer- cell lines by Northern blot analysis.Furthermore,a rough correlation between MGMT activity and mRNA level in these cell lines was observed.These results suggest that transcriptional regulation of the MGMT gene is the molecular basis of the absence of MGMT activity in Mer- cell lines.

  17. The Importance of Physiologically Relevant Cell Lines for Studying Virus–Host Interactions

    Directory of Open Access Journals (Sweden)

    David Hare


    Full Text Available Viruses interact intimately with the host cell at nearly every stage of replication, and the cell model that is chosen to study virus infection is critically important. Although primary cells reflect the phenotype of healthy cells in vivo better than cell lines, their limited lifespan makes experimental manipulation challenging. However, many tumor-derived and artificially immortalized cell lines have defects in induction of interferon-stimulated genes and other antiviral defenses. These defects can affect virus replication, especially when cells are infected at lower, more physiologically relevant, multiplicities of infection. Understanding the selective pressures and mechanisms underlying the loss of innate signaling pathways is helpful to choose immortalized cell lines without impaired antiviral defense. We describe the trials and tribulations we encountered while searching for an immortalized cell line with intact innate signaling, and how directed immortalization of primary cells avoids many of the pitfalls of spontaneous immortalization.


    NARCIS (Netherlands)



    Cisplatin (CDDP) resistance mechanisms were studied in a model of three germ cell tumour and three colon carcinoma cell lines representing intrinsically CDDP-sensitive and -resistant tumours respectively. The CDDP sensitivity of the cell lines mimicked the clinical situation. The glutathione levels

  19. A comparative study of the FcepsilonRI molecule on human mast cell and basophil cell lines

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Dissing, S; Skov, P S;


    Mast cells and basophils express the high-affinity IgE receptor FcepsilonRI. We have analysed the human mast cell line LAD2 and four subclones of the basophil cell line KU812 in order to reveal possible differences concerning the FcepsilonRI surface regulation, anti-IgE-triggered activation...

  20. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells. (United States)

    Radna, R L; Foellmer, B; Feldman, L A; Francke, U; Ozer, H L


    We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

  1. Experimental impact of aspirin exposure on rat intestinal bacteria, epithelial cells and cell line. (United States)

    Upreti, Raj K; Kannan, A; Pant, A B


    Aspirin, a commonly used therapeutic non-steroidal anti-inflammatory drug (NSAID) is known to cause gastric mucosal damage. Intestinal bacteria having a regulatory effect on intestinal homeostasis play significant role in NSAID-induced intestinal injury. Bacteria and specific cell lines are considered to be suitable for toxicity screening and testing of chemicals. Therefore, to evaluate and compare in vitro toxicity, cultures of rat intestinal epithelial cells (IEC), isolated bacteria and IEC-6 cell line were assessed for viability, morphometric analysis, membrane transport enzymes and structural constituents for membrane damage, dehydrogenase activity test for respiratory and energy producing processes and esterase activity test for intra- and extra-cellular degradation, following the post exposure to aspirin (0-50 µg mL(- 1)). Similar pattern of dose-dependent changes in these parameters were observed in three types of cells. Similar in situ effects on IEC validated the in vitro findings. These findings indicate that higher aspirin concentrations may alter cellular functions of IEC and gut bacteria. Furthermore, results suggest that gut bacteria and IEC-6 cell line can be used for the initial screening of gastrointestinal cellular toxicity caused by NSAIDs.

  2. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology. (United States)

    Shaharuddin, Bakiah; Ahmad, Sajjad; Md Latar, Nani; Ali, Simi; Meeson, Annette


    : Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue.

  3. Rapid selection and proliferation of CD133+ cells from cancer cell lines: chemotherapeutic implications.

    Directory of Open Access Journals (Sweden)

    Sarah E Kelly

    Full Text Available Cancer stem cells (CSCs are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133+] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB (Celdyne, Houston, TX. For comparison, another bioreactor, the rotary cell culture system (RCCS manufactured by Synthecon (Houston, TX was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133+ cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a +15-fold proliferation of the CD133+ cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (-4.8-fold decrease in the CD133+cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133+ cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates.

  4. Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

    Directory of Open Access Journals (Sweden)

    Alexa Kiss

    Full Text Available Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns--thereby forced into a bipolar morphology--displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.

  5. Micro-RNA expression in cisplatin resistant germ cell tumor cell lines

    Directory of Open Access Journals (Sweden)

    Meineke Viktor


    Full Text Available Abstract Background We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance. Methods Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP. Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738 known microRNA species of human origin. Results Altogether 72 of 738 (9.8% microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15 of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%. The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79% and NCCIT-R/NCCIT (64%, and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%. Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency, as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21 were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up

  6. Effects of Dioscin Extracted from Polygonatum Zanlanscianense Pamp on Several Human Tumor Cell Lines

    Institute of Scientific and Technical Information of China (English)

    王钊; 周江兵; 巨勇; 姚沈勤; 张洪钧


    Dioscin was extracted and isolated from Polygonatum Zanlanscianense Pamp. The effects of dioscin on HL60, HeLa, H14, and MDA-MB-435 cell lines were studied with the results showing that dioscin dramatically inhibited the growth of the MDA-MB-435, H14, HL60, and HeLa cell lines. The IC50 of dioscin on these cell lines were 2.6, 0.8, 7. 5, and 4.5 μ mol/L respectively.

  7. A bovine cell line that can be infected by natural sheep scrapie prions. (United States)

    Oelschlegel, Anja M; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schatzl, Herman; Schaetzl, Hermann; Groschup, Martin H


    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  8. A bovine cell line that can be infected by natural sheep scrapie prions.

    Directory of Open Access Journals (Sweden)

    Anja M Oelschlegel

    Full Text Available Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice. We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  9. Comparison of the effect of interferon on two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Crespi, M.; Schoub, B.D.; Lyons, S.F.; Chiu, M.N. (University of the Witwatersrand, Johannesburg (South Africa). Dept. of Virology)


    Two human hepatoma cell lines, the PLC/PRF/5 and the Mahlavu cells, which differ in their production of the hepatitis B surface antigen (HBsAg), responded differently to interferon (IFN). After IFN treatment both cell lines were able to inhibit Sindbis virus replication. Oligo A synthetase (E enzyme) could be activated in the PLC/PRF/5 cells although they were not sensitive to exogenous 2 - 5 oligoadenylic acid (2 - 5 A). In contrast, the Mahlavu cells were sensitive to exogenous 2 - 5 A, but unable to activate the E enzyme. Both cell lines were unable to stimulate phosphorylation of the exogenous initiator factor eIF-2.

  10. Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Ageberg, Malin, E-mail: [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden); Rydstroem, Karin, E-mail: [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Linden, Ola, E-mail: [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Linderoth, Johan, E-mail: [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Jerkeman, Mats, E-mail: [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Drott, Kristina, E-mail: [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden)


    Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.

  11. Tumor suppressors status in cancer cell line Encyclopedia. (United States)

    Sonkin, Dmitriy; Hassan, Mehedi; Murphy, Denis J; Tatarinova, Tatiana V


    Tumor suppressors play a major role in the etiology of human cancer, and typically achieve a tumor-promoting effect upon complete functional inactivation. Bi-allelic inactivation of tumor suppressors may occur through genetic mechanisms (such as loss of function mutation, copy number (CN) loss, or loss of heterozygosity (LOH)), epigenetic mechanisms (such as promoter methylation or histone modification), or a combination of the two. We report systematically derived status of 69 known or putative tumor suppressors, across 799 samples of the Cancer Cell Line Encyclopedia. In order to generate such resource we constructed a novel comprehensive computational framework for the assessment of tumor suppressor functional "status". This approach utilizes several orthogonal genomic data types, including mutation data, copy number, LOH and expression. Through correlation with additional data types (compound sensitivity and gene set activity) we show that this integrative method provides a more accurate assessment of tumor suppressor status than can be inferred by expression, copy number, or mutation alone. This approach has the potential for a more realistic assessment of tumor suppressor genes for both basic and translational oncology research.

  12. NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery (United States)

    Santana-Filho, Arquimedes Paixão de; Jacomasso, Thiago; Riter, Daniel Suss; Barison, Andersson; Iacomini, Marcello; Winnischofer, Sheila Maria Brochado; Sassaki, Guilherme Lanzi


    Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of 1H NMR data was able not only to differentiate the melanocyte cell line from the tumorigenic ones but also distinguish among the 3 tumorigenic cell lines. We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines. PMID:28198377

  13. Glioma cells on the run – the migratory transcriptome of 10 human glioma cell lines

    Directory of Open Access Journals (Sweden)

    Holz David


    Full Text Available Abstract Background Glioblastoma multiforme (GBM is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. Results Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA revealed two discriminating patterns between migrating and stationary glioma cells: i global down-regulation and ii global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF. siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. Conclusion Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected

  14. Tick cell lines for study of Crimean-Congo hemorrhagic fever virus and other arboviruses. (United States)

    Bell-Sakyi, Lesley; Kohl, Alain; Bente, Dennis A; Fazakerley, John K


    Continuous cell lines derived from many of the vectors of tick-borne arboviruses of medical and veterinary importance are now available. Their role as tools in arbovirus research to date is reviewed and their potential application in studies of tick cell responses to virus infection is explored, by comparison with recent progress in understanding mosquito immunity to arbovirus infection. A preliminary study of propagation of the human pathogen Crimean-Congo hemorrhagic fever virus (CCHFV) in tick cell lines is reported; CCHFV replicated in seven cell lines derived from the ticks Hyalomma anatolicum (a known vector), Amblyomma variegatum, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, and Ixodes ricinus, but not in three cell lines derived from Rhipicephalus appendiculatus and Ornithodoros moubata. This indicates that tick cell lines can be used to study growth of CCHFV in arthropod cells and that there may be species-specific restriction in permissive CCHFV infection at the cellular level.

  15. Phytoestrogens regulate the proliferation and expression of stem cell factors in cell lines of malignant testicular germ cell tumors. (United States)

    Hasibeder, Astrid; Venkataramani, Vivek; Thelen, Paul; Radzun, Heinz-Joachim; Schweyer, Stefan


    Phytoestrogens have been shown to exert anti-proliferative effects on different cancer cells. In addition it could be demonstrated that inhibition of proliferation is associated with downregulation of the known stem cell factors NANOG, POU5F1 and SOX2 in tumor cells. We demonstrate the potential of Belamcanda chinensis extract (BCE) and tectorigenin as anticancer drugs in cell lines of malignant testicular germ cell tumor cells (TGCT) by inhibition of proliferation and regulating the expression of stem cell factors. The TGCT cell lines TCam-2 and NTera-2 were treated with BCE or tectorigenin and MTT assay was used to measure the proliferation of tumor cells. In addition, the expression of stem cell factors was analyzed by quantitative PCR and western blot analysis. Furthermore, global expression analysis was performed by microarray technique. BCE and tectorigenin inhibited proliferation and downregulated the stem cell factors NANOG and POU5F1 in TGCT cells. In addition, gene expression profiling revealed induction of genes important for the differentiation and inhibition of oncogenes. Utilizing connectivity map in an attempt to elucidate mechanism underlying BCE treatments we found highly positive association to histone deacetylase inhibitors (HDACi) amongst others. Causing no histone deacetylase inhibition, the effects of BCE on proliferation and stem cell factors may be based on histone-independent mechanisms such as direct hyperacetylation of transcription factors. Based on these findings, phytoestrogens may be useful as new agents in the treatment of TGCT.

  16. An evaluation and parameterization of stably stratified turbulence: Insights on the atmospheric boundary layer and implications for wind energy (United States)

    Wilson, Jordan M.

    of mean shear and buoyancy frequency, S and N, respectively. Length scale estimates for LM are given by LkS ≡ k1/2/S and LkN ≡ k1/2/N, where LkS provides an accurate estimate for eddy viscosity, nut, under neutral to strongly stable conditions for SABL data. The relative influence of shear and buoyancy are given by the ratio of the respective time scales, S--1 and N--1, with the pertinent time scale of the large-scale motions, TP ≡ k/P, through the parameters STP and NTP. L kS's range of applicability is further assessed in a STP-NTP parameter space. In developing these parameterizations, the stress-intensity ratio, c2 , is evaluated using high-Re stably stratified data and is shown to exhibit a near constant value (c2 ≈ 0.25) for stably stratified geophysical turbulence. These findings provide a clear trajectory for numerical modeling of stably stratified geophysical shear turbulence without reliance on stability or damping functions, tuning parameters, or artificial parameterizations. An initial modeling study of moderate-Re channel and Ekman layer flows using the proposed parameterizations confirms this supposition. Finally, it is in this new light that large-scale implications of wind energy can now be considered. As a first step in this process, computational fluid dynamics (CFD) studies of wind turbine interactions are carried out under neutrally stratified conditions. Simulations clearly show that actuator line models provide efficacy in wake generation, interaction, and restoration and highlight model requirements for stably stratified conditions. Results suggest that standard horizontal spacings of 5--10 rotor diameters yield significant reductions in power output and increases turbulence intensity and fatigue loading.

  17. Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer. (United States)

    Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong


    This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

  18. Identification of transporters associated with Etoposide sensitivity of stomach cancer cell lines and methotrexate sensitivity of breast cancer cell lines by quantitative targeted absolute proteomics. (United States)

    Obuchi, Wataru; Ohtsuki, Sumio; Uchida, Yasuo; Ohmine, Ken; Yamori, Takao; Terasaki, Tetsuya


    Membrane transporter proteins may influence the sensitivity of cancer cells to anticancer drugs that can be recognized as substrates. The purpose of this study was to identify proteins that play a key role in the drug sensitivity of stomach and breast cancer cell lines by measuring the absolute protein expression levels of multiple transporters and other membrane proteins and examining their correlation to drug sensitivity. Absolute protein expression levels of 90 membrane proteins were examined by quantitative targeted absolute proteomics using liquid chromatography-linked tandem mass spectrometry. Among them, 11 and 14 membrane proteins, including transporters, were present in quantifiable amounts in membrane fraction of stomach cancer and breast cancer cell lines, respectively. In stomach cancer cell lines, the protein expression level of multidrug resistance-associated protein 1 (MRP1) was inversely correlated with etoposide sensitivity. MK571, an MRP inhibitor, increased both the cell-to-medium ratio of etoposide and the etoposide sensitivity of MRP1-expressing stomach cancer cell lines. In breast cancer cell lines, the protein expression level of reduced folate carrier 1 (RFC1) was directly correlated with methotrexate (MTX) sensitivity. Initial uptake rate and steady-state cell-to-medium ratio of [(3)H]MTX were correlated with both RFC1 expression level and MTX sensitivity. These results suggest that MRP1 modulates the etoposide sensitivity of stomach cancer cell lines and RFC1 modulates the MTX sensitivity of breast cancer cell lines. Our results indicate that absolute quantification of multiple membrane proteins could be a useful strategy for identification of candidate proteins involved in drug sensitivity.

  19. Expression of hepatitis C virus envelope protein 2 induces apoptosis in cultured mammalian cells

    Institute of Scientific and Technical Information of China (English)

    Li-Xin Zhu; Jing Liu; You-Hua Xie; Yu-Ying Kong; Ye Ye; Chun-Lin Wang; Guang-Di Li; Yuan Wang


    AIM: To explore the role of hepatitis C virus (HCV) envelope protein 2 (E2) in the induction of apoptosis.METHODS: A carboxyterminal truncated E2 (E2-661) was transiently expressed in several cultured mammalian cell lines or stably expressed in Chinese hamster ovary (CHO)cell line. Cell proliferation was assessed by 3H thymidine uptake. Apoptosis was examined by Hoechst 33258staining, flow cytometry and DNA fragmentation analysis.RESULTS: Reduced proliferation was readily observed in the E2-661 expressing cells. These cells manifested the typical features of apoptosis, including cell shrinkage,chromatin condensation and hypodiploid genomic DNA content. Similar apoptotic cell death was observed in an E2-661 stably expressing cell line.CONCLUSION: HCV E2 can induce apoptosis in cultured mammalian cells.

  20. Generation of breast cancer stem cells by steroid hormones in irradiated human mammary cell lines.

    Directory of Open Access Journals (Sweden)

    Guillaume Vares

    Full Text Available Exposure to ionizing radiation was shown to result in an increased risk of breast cancer. There is strong evidence that steroid hormones influence radiosensitivity and breast cancer risk. Tumors may be initiated by a small subpopulation of cancer stem cells (CSCs. In order to assess whether the modulation of radiation-induced breast cancer risk by steroid hormones could involve CSCs, we measured by flow cytometry the proportion of CSCs in irradiated breast cancer cell lines after progesterone and estrogen treatment. Progesterone stimulated the expansion of the CSC compartment both in progesterone receptor (PR-positive breast cancer cells and in PR-negative normal cells. In MCF10A normal epithelial PR-negative cells, progesterone-treatment and irradiation triggered cancer and stemness-associated microRNA regulations (such as the downregulation of miR-22 and miR-29c expression, which resulted in increased proportions of radiation-resistant tumor-initiating CSCs.

  1. Ethanolic Extract Cytotoxic Effect of Zingiber Afficinale in Breast Cancer (MCF7 Cell Line

    Directory of Open Access Journals (Sweden)

    J Tavakkol Afshari


    Full Text Available Introduction & Objective: Biological activities of Zingiber afficieale plants have been reported as possessing anticancer, antibacterial, anti ulcer, antifungal, and insecticidal properties. However, its antitumor effects haven't been studied in cancer cell lines. The aim of this study was to investigate the antitumor effect of zingiber afficieale on breast cancer cell lines. Materials & Methods: This experimental study was conducted in 2010 at Mashhad University of medical Sciences. Breast cancer cell line (MCF7 and normal connective tissue cell line (L929 were cultured in DMEM medium. Ethanolic extract of Zingiber afficinale was prepared and cell lines were treated with different concentration of extract (5000 to 78 µg. Cell viability was measured by MTT assay after 24, 48, and 72 hours. The collected data were statistically analyzed by SPSS software. Results: The effects of Zingiber afficinale on cell viability were observed after 48 hours on cell lines. Ginger doses in 2500 µg concentration inhibited 50% of cell growth (IC50 in cell lines after 48 hours. Conclusion: Our study revealed that fresh ginger extract has cytotoxic effects on tumor cells, but it doesn’t have any cytotoxic effect on normal cells. It seems that ginger could be considered as a promising chemotherapeutic agent in cancer treatment.

  2. Check your cultures! A list of cross-contaminated or misidentified cell lines. (United States)

    Capes-Davis, Amanda; Theodosopoulos, George; Atkin, Isobel; Drexler, Hans G; Kohara, Arihiro; MacLeod, Roderick A F; Masters, John R; Nakamura, Yukio; Reid, Yvonne A; Reddel, Roger R; Freshney, R Ian


    Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handler's knowledge. Cross-contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response-how can a researcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination for that cell line, it is essential to check the sample itself by performing authentication testing.

  3. Whole-exome characterization of pancreatic neuroendocrine tumor cell lines BON-1 and QGP-1. (United States)

    Vandamme, Timon; Peeters, Marc; Dogan, Fadime; Pauwels, Patrick; Van Assche, Elvire; Beyens, Matthias; Mortier, Geert; Vandeweyer, Geert; de Herder, Wouter; Van Camp, Guy; Hofland, Leo J; Op de Beeck, Ken


    The human BON-1 and QGP-1 cell lines are two frequently used models in pancreatic neuroendocrine tumor (PNET) research. Data on the whole-exome genetic constitution of these cell lines is largely lacking. This study presents, to our knowledge, the first whole-exome profile of the BON-1 and QGP-1 cell lines. Cell line identity was confirmed by short tandem repeat profiling. Using GTG-banding and a CytoSNP-12v2 Beadchip array, cell line ploidy and chromosomal alterations were determined in BON-1 and QGP-1. The exomes of both cell lines were sequenced on Ilumina's HiSeq next-generation sequencing (NGS) platform. Single-nucleotide variants (SNVs) and insertions and deletions (indels) were detected using the Genome Analysis ToolKit. SNVs were validated by Sanger sequencing. Ploidy of BON-1 and QGP-1 was 3 and 4 respectively, with long stretches of loss of heterozygosity across multiple chromosomes, which is associated with aggressive tumor behavior. In BON-1, 57 frameshift indels and 1725 possible protein-altering SNVs were identified in the NGS data. In the QGP-1 cell line, 56 frameshift indels and 1095 SNVs were identified. ATRX, a PNET-associated gene, was mutated in both cell lines, while mutation of TSC2 was detected in BON-1. A mutation in NRAS was detected in BON-1, while KRAS was mutated in QGP-1, implicating aberrations in the RAS pathway in both cell lines. Homozygous mutations in TP53 with possible loss of function were identified in both cell lines. Various MUC genes, implicated in cell signaling, lubrication and chemical barriers, which are frequently expressed in PNET tissue samples, showed homozygous protein-altering SNVs in the BON-1 and QGP-1 cell lines.

  4. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line. (United States)

    Sluch, Valentin M; Davis, Chung-ha O; Ranganathan, Vinod; Kerr, Justin M; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S; Mao, Hai-Quan; Zack, Donald J


    Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.

  5. Capsaicin-induced cell death in a human gastric adenocarcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Yi-Ching Lo; Yuan-Chen Yang; I-Chieh Wu; Fu-Chen Kuo; Chi-Ming Liu; Hao-Wei Wang; Chao-Hung Kuo; Jeng-Yi Wu; Deng-Chyang Wu


    AIM: Capsaicin, a pungent ingredient found in red pepper,has long been used in spices, food additives, and drugs.Cell death induced by the binding of capsaicin was examined in a human gastric adenocarcinoma cell line (AGS cells).METHODS: By using XTT-based cytotoxicityassay, flow cytometry using the TUNEL method, and quantitation of DNA fragmentation, both cell death and DNA fragmentation were detected in AGS cells treated with capsaicin. By using Western blotting methods, capsaicin reduced the expression of Bcl-2, the antiapoptotic protein, in AGS cells in a concentration-dependent manner.RESULTS: After incubation of AGS cells with capsaicin for 24 h, cell viability decreased significantly in a dose-dependent manner. After incubation of AGS cells with capsaicin for 24 h, apoptotic bodies also significantly increased, and were again correlated with the dose of capsaicin. When the concentration of capsaicin was 1 mmol/L, the amount of DNA fragments also increased. Similar results werealso in the lower traces.CONCLUSION: These results suggest that capsaicininduced cell death might be via a Bcl-2 sensitive apoptotic pathway. Therefore, capsaicin might induce protection from gastric cancer.

  6. FTIR characterization of animal lung cells: normal and precancerous modified e10 cell line (United States)

    Zezell, D. M.; Pereira, T. M.; Mennecier, G.; Bachmann, L.; Govone, A. B.; Dagli, M. L. Z.


    The chemical carcinogens from tobacco are related to over 90% of lung cancers around the world. The risk of death of this kind of cancer is high because the diagnosis usually is made only in advanced stages. Therefore, it is necessary to develop new diagnostic methods for detecting the lung cancer in earlier stages. The Fourier Transform Infrared Spectroscopy (FTIR) can offer high sensibility and accuracy to detect the minimal chemical changes into the biological sample. The aim of this study is to evaluate the differences on infrared spectra between normal lung cells and precancerous lung cells transformed by NNK. Non-cancerous lung cell line e10 (ATCC) and NNK-transformed e10 cell lines were maintained in complete culture medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 [DMEM/Ham's F12], supplemented with 100 ng/ml cholera enterotoxin, 10 lg/ml insulin, 0.5 lg/ml. hydrocortisol, 20 ng/ml epidermal growth factor, and 5% horse serum. The cultures were maintained in alcohol 70%. The infrared spectra were acquired on ATR-FTIR Nicolet 6700 spectrophotometer at 4 cm-1 resolution, 30 scans, in the 1800-900 cm-1 spectral range. Each sample had 3 spectra recorded, 30 infrared spectra were obtained from each cell line. The second derivate of spectra indicates that there are displacement in 1646 cm-1 (amine I) and 1255 cm-1(DNA), allowing the possibility to differentiate the two king of cells, with accuracy of 89,9%. These preliminary results indicate that ATR-FTIR is useful to differentiate normal e10 lung cells from precancerous e10 transformed by NNK.

  7. The culture of cancer cell lines as tumorspheres does not systematically result in cancer stem cell enrichment. (United States)

    Calvet, Christophe Y; André, Franck M; Mir, Lluis M


    Cancer stem cells (CSC) have raised great excitement during the last decade and are promising targets for an efficient treatment of tumors without relapses and metastases. Among the various methods that enable to enrich cancer cell lines in CSC, tumorspheres culture has been predominantly used. In this report, we attempted to generate tumorspheres from several murine and human cancer cell lines: B16-F10, HT-29, MCF-7 and MDA-MB-231 cells. Tumorspheres were obtained with variable efficiencies from all cell lines except from MDA-MB-231 cells. Then, we studied several CSC characteristics in both tumorspheres and adherent cultures of the B16-F10, HT-29 and MCF-7 cells. Unexpectedly, tumorspheres-forming cells were less clonogenic and, in the case of B16-F10, less proliferative than attached cells. In addition, we did not observe any enrichment in the population expressing CSC surface markers in tumorspheres from B16-F10 (CD133, CD44 and CD24 markers) or MCF-7 (CD44 and CD24 markers) cells. On the contrary, tumorspheres culture of HT-29 cells appeared to enrich in cells expressing colon CSC markers, i.e. CD133 and CD44 proteins. For the B16-F10 cell line, when 1 000 cells were injected in syngenic C57BL/6 mice, tumorspheres-forming cells displayed a significantly lower tumorigenic potential than adherent cells. Finally, tumorspheres culture of B16-F10 cells induced a down-regulation of vimentin which could explain, at least partially, the lower tumorigenicity of tumorspheres-forming cells. All these results, along with the literature, indicate that tumorspheres culture of cancer cell lines can induce an enrichment in CSC but in a cell line-dependent manner. In conclusion, extensive characterization of CSC properties in tumorspheres derived from any cancer cell line or cancer tissue must be performed in order to ensure that the generated tumorspheres are actually enriched in CSC.

  8. Power-laws and the use of pluripotent stem cell lines

    NARCIS (Netherlands)

    Schuldt, Bernhard M; Guhr, Anke; Lenz, Michael; Kobold, Sabine; MacArthur, Ben D; Schuppert, Andreas; Löser, Peter; Müller, Franz-Josef


    It is widely accepted that the (now reversed) Bush administration's decision to restrict federal funding for human embryonic stem cell (hESC) research to a few "eligible" hESC lines is responsible for the sustained preferential use of a small subset of hESC lines (principally the H1 and H9 lines) in

  9. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses. (United States)

    Kuno, G; Gubler, D J; Vélez, M; Oliver, A


    Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability.

  10. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses* (United States)

    Kuno, G.; Gubler, D. J.; Vélez, M.; Oliver, A.


    Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability. PMID:2861916

  11. Morphological and molecular characterization of an undifferentiated soft tissue sarcoma cell line and derivative clones. (United States)

    Taubert, H; Schmidt, H; Wurl, P; Hinze, R; Meye, A; Bache, M; Berger, D; Holzhausen, H; Dralle, H; Rath, F


    From an undifferentiated soft tissue sarcoma (STS) a cell line designated US8-93 has been established. At subcloning the cell line US8-93 three different lines (US8-93A, B and C) could be set up. In a subsequent study characteristics for ultrastructure, growth, cell cycle distribution, karyotype, protein overexpression detected by immunohistochemistry (IHC) and p53 mutational status were determined. The cell line US8-93 as well as subclones contain mainly bipolar spindle-shaped cells and additionally some polygonal and multinucleated cells. Cells possess the characteristics of primitive mesenchymal cells based on their positive reactions with anti-vimentin and negative reactions for desmin, cytokeratin, myoglobin, S100, and NSE, implying a classification as an undifferentiated STS. Cytogenetic analysis revealed nearly diploid cells with several structural and numerical aberrations for chromosomes 1, 3, 4, 6, 9, 10, 12, 13, 15 and 18. IHC positivity was found for the tumor suppressor proteins p53 and Rb, the oncogene products Bcl-2, K-ras, N-ras, P-glycoprotein Mdr-1 and MDM-2. In the p53 gene a nonsense mutation in exon 4 was detected, that was confirmed in the original primary tumor and in three derivative clonal lines. The described STS cell line represents a valuable supplementation to the relatively small number of human STS cell lines currently available and may also provide a good in vitro model for studies of STS tumorigenesis in respect to a mutated p53 gene.

  12. Proteomics of cancer cell lines resistant to microtubule-stabilizing agents

    DEFF Research Database (Denmark)

    Albrethsen, Jakob; Angeletti, Ruth H; Horwitz, Susan Band


    resistance to the class of MIAs known as microtubule-stabilizing agents (MSA). The human lung cancer cell line A549 was compared with two drug-resistant daughter cell lines, a taxol-resistant cell line (AT12) and an epothilone B (EpoB)-resistant cell line (EpoB40). The ovarian cancer cell line Hey......-resistant cells. Differential abundance of 14-3-3σ, galectin-1 and phosphorylation of stathmin are worthy of further studies as candidate predictive biomarkers for MSAs. This is especially true for galectin-1, a β-galactose-binding lectin that mediates tumor invasion and metastasis. Galectin-1 was greatly...... in resistance to MSAs....

  13. Effect of sirolimus on urinary bladder cancer T24 cell line

    Directory of Open Access Journals (Sweden)

    Oliveira Paula A


    Full Text Available Abstract Background Sirolimus is recently reported to have antitumour effects on a large variety of cancers. The present study was performed to investigate sirolimus's ability to inhibit growth in T24 bladder cancer cells. Methods T24 bladder cancer cells were treated with various concentrations of sirolimus. MTT assay was used to evaluate the proliferation inhibitory effect on T24 cell line. The viability of T24 cell line was determined by Trypan blue exclusion analysis. Results Sirolimus inhibits the growth of bladder carcinoma cells and decreases their viability. Significant correlations were found between cell proliferation and sirolimus concentration (r = 0.830; p Conclusion Sirolimus has an anti-proliferation effect on the T24 bladder carcinoma cell line. The information from our results is useful for a better understanding sirolimus's anti-proliferative activity in the T24 bladder cancer cell line.

  14. Selective COX-2 inhibitor, NS-398, suppresses cellular proliferation in human hepatocellular carcinoma cell lines via cell cycle arrest

    Institute of Scientific and Technical Information of China (English)

    Ji Yeon Baek; Wonhee Hur; Jin Sang Wang; Si Hyun Bae; Seung Kew Yoon


    AIM: To investigate the growth inhibitory mechanism of NS-398, a selective cyclooxygenase-2 (COX-2) inhibitor,in two hepatocellular carcinoma (HCC) cell lines (HepG2and Huh7).METHODS: HepG2 and Huh7 cells were treated with NS-398. Its effects on cell viability, cell proliferation,cell cycles, and gene expression were respectively evaluated by water-soluble tetrazolium salt (WST-1)assay, 4'-6-diamidino-2-phenylindole (DAPI) staining,flow cytometer analysis, and Western blotting,with dimethyl sulfoxide (DMSO) as positive control.RESULTS: NS-398 showed dose- and time-dependent growth-inhibitory effects on the two cell lines.Proliferating cell nuclear antigen (PCNA) expressions in HepG2 and Huh7 cells, particularly in Huh7 cells were inhibited in a time- and dose-independent manner.NS-398 caused cell cycle arrest in the G1 phase with cell accumulation in the sub-G1 phase in HepG2 and Huh7cell lines. No evidence of apoptosis was observed in two cell lines.CONCLUSION: NS-398 reduces cell proliferation by inducing cell cycle arrest in HepG2 and Huh7 cell lines,and COX-2 inhibitors may have potent chemoprevention effects on human hepatocellular carcinoma.

  15. Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells. (United States)

    Rosas, Marcela; Osorio, Fabiola; Robinson, Matthew J; Davies, Luke C; Dierkes, Nicola; Jones, Simon A; Reis e Sousa, Caetano; Taylor, Philip R


    We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM-MØP to effectively present antigen to a T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-γ responses from naïve CD4(+) T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of inflammatory monocyte-like cells, with important differences from bone marrow-derived dendritic cells, which will facilitate functional studies relating to the many 'sub-phenotypes' of inflammatory monocytes.

  16. Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research

    Directory of Open Access Journals (Sweden)

    Silvia A Fuertes Marraco


    Full Text Available Research in vitro facilitates discovery, screening and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice.In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.

  17. Berberine induces cell cycle arrest and apoptosis in human gastric carcinoma SNU-5 cell line

    Institute of Scientific and Technical Information of China (English)

    Jing-Pin Lin; Jai-Sing Yang; Jau-Hong Lee; Wen-Tsong Hsieh; Jing-Gung Chung


    AIM: To investigate the relationship between the inhibited growth (cytotoxic activity) of berberine and apoptotic pathway with its molecular mechanism of action.METHODS: The in vitro cytotoxic techniques were complemented by cell cycle analysis and determination of sub-G1 for apoptosis in human gastric carcinoma SNU-5 cells. Percentage of viable cells, cell cycle, and sub-G1 group (apoptosis) were examined and determined by the flow cytometric methods. The associated proteins for cell cycle arrest and apoptosis were examined by Western blotting.RESULTS: For SNU-5 cell line, the IC (50) was found to be 48 μmol/L of berberine. In SNU-5 cells treated with 25-200 μmol/L berberine, G2/M cell cycle arrest was observed which was associated with a marked increment of the expression of p53, Wee1 and CDk1 proteins and decreased cyclin B. A concentration-dependent decrease of cells in G0/G1 phase and an increase in G2/M phase were detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved in 25-200 μmol/L berberine-treated cells by monitoring the apoptotic pathway. Apoptosis was identified by sub-G0 cell population, and upregulation of Bax, downregulation of Bcl-2, release of Ca2+, decreased the mitochondrial membrane potential and then led to the release of mitochondrial cytochrome C into the cytoplasm and caused the activation of caspase-3, and finally led to the occurrence of apoptosis.CONCLUSION: Berberine induces p53 expression and leads to the decrease of the mitochondrial membrane potential, Cytochrome C release and activation of caspase-3 for the induction of apoptosis.


    Institute of Scientific and Technical Information of China (English)


    Objective To determine whether transforming growth factor betal ( TGF-β1 )/Smad signaling pathway mediates p53-dependent apoptosis in hepatoma cell lines. Methods Three human hepatic carcinoma cell lines, HepG2, Huh-7, and Hep3B, were used in this study. TGF-β31-induced apoptosis in hepatic carcinoma cell lines was analyzed using TUNEL assay. For identifying the mechanism of apoptosis induced by TGF-β1, cell lines were transfected with a TGF-β1-inducible luciferase reportor plasmid containing Smad4 binding elements. After transfection, cells were treated with TGF-β1, then assayed for luciferase activity. Results The apoptosis rate of HepG2 cell lines (48.51% ± 8.21% ) was significantly higher than control (12. 72% ±2. 18%, P <0. 05 ). But TGF-β1 was not able to induce apoptosis of Huh-7 and Hep3B cell lines. The relative luciferase activity of TGF-β1-treated HepG2 cell lines (4. 38) was significantly higher than control (1.00, P <0. 05). But the relative luciferase activity of TGF-β1-treated Huh-7 and Hep3B cell lines less increased compared with control. Conclusions HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh-7 cell lines. Smad4 is a central mediator of TGF-β1 signaling transdution pathway. TGF-β1/Smad signaling pathway might mediate p53-dependent apoptosis in hepatoma cell lines.

  19. Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells

    Institute of Scientific and Technical Information of China (English)

    SHI Cheng; SHEN Huan; JIANG Wei; SONG Zhi-hua; WANG Cheng-yan; WEI Li-hui


    Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background,which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use,especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.Methods Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propogate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells.Results We generated a new Chinese human embryonic stem cells line,CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines:normal morphology,karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.Conclusions This newly established Chinese cell line,CH1,which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells,will provide

  20. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning. (United States)

    Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff


    Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line.

  1. The Tribolium castaneum cell line TcA: a new tool kit for cell biology. (United States)

    Silver, Kristopher; Jiang, Hongbo; Fu, Jinping; Phillips, Thomas W; Beeman, Richard W; Park, Yoonseong


    The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100 pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions.

  2. Human B cell activating factor (BCAF): production by a human T cell tumor line. (United States)

    Fevrier, M; Diu, A; Mollier, P; Abadie, A; Olive, D; Mawas, C; Theze, J


    In a previous study, we demonstrated that supernatants from human T cell clones stimulated by a pair of anti-CD2 monoclonal antibodies cause resting human B cells to become activated and to proliferate in the absence of any other signals. The activity responsible for these effects was shown to be different from already characterized lymphokines and in particular from IL-2 and IL-4, and was named B Cell Activating Factor or BCAF. In this paper, we describe the production of BCAF by a human T cell tumor line T687 after phorbol myristate acetate (PMA) stimulation; this production can be potentiated by phytohemagglutinin (PHA). We further show that the stimulatory phase can be separated from the secretory phase thereby avoiding contamination of BCAF-containing supernatant by PMA and PHA. Supernatants produced under these conditions do not contain either IL-4 or IFN but contain traces of lymphotoxin and 2 to 10 ng/ml of IL-2. The T687 cell line will allow us to obtain a large volume of supernatant for biochemical study and purification of the molecule(s) responsible for BCAF activity.

  3. Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines (United States)

    Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E.; Krishnan, Aswini R.; Tsui, Tzuhan; Aguilera, Joseph A.; Advani, Sunil; Crotty Alexander, Laura E.; Brumund, Kevin T.; Wang-Rodriguez, Jessica


    Objectives Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. Materials and Methods HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 hours to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. Results E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. Conclusion E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. PMID:26547127

  4. Generation of mouse ES cell lines engineered for the forced induction of transcription factors


    Correa-Cerro, Lina S.; Piao, Yulan; Sharov, Alexei A; Nishiyama, Akira; Cadet, Jean S.; Yu, Hong; Sharova, Lioudmila V.; Xin, Li; Hoang, Hien G.; Thomas, Marshall; Qian, Yong; Dudekula, Dawood B.; Meyers, Emily; Binder, Bernard Y.; Mowrer, Gregory


    Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7–10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a ...

  5. Generation of mouse ES cell lines engineered for the forced induction of transcription factors (United States)

    Correa-Cerro, Lina S.; Piao, Yulan; Sharov, Alexei A.; Nishiyama, Akira; Cadet, Jean S.; Yu, Hong; Sharova, Lioudmila V.; Xin, Li; Hoang, Hien G.; Thomas, Marshall; Qian, Yong; Dudekula, Dawood B.; Meyers, Emily; Binder, Bernard Y.; Mowrer, Gregory; Bassey, Uwem; Longo, Dan L.; Schlessinger, David; Ko, Minoru S. H.


    Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7–10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a variety of future biomedical research applications as a community resource. PMID:22355682

  6. Establishment and culture optimization of a new type of pituitary immortalized cell line

    Energy Technology Data Exchange (ETDEWEB)

    Kokubu, Yuko [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Asashima, Makoto [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Life Science Center of TARA, The University of Tsukuba, Ibaraki-ken 305-8577 (Japan); Kurisaki, Akira, E-mail: [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562 (Japan)


    The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells under sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. - Highlights: • Mouse pituitary cell lines were immortalized by introducing TERT, E6, and E7. • The immortalized cell lines mainly expressed thyroid stimulating hormone beta. • The cell lines responded to PKA or PKC pathway activators, and induced Tshb.

  7. Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias. (United States)

    Parton, Angela; Forest, David; Kobayashi, Hiroshi; Dowell, Lori; Bayne, Christopher; Barnes, David


    Cartilaginous fish, primarily sharks, rays and skates (elasmobranchs), appeared 450 million years ago. They are the most primitive vertebrates, exhibiting jaws and teeth, adaptive immunity, a pressurized circulatory system, thymus, spleen, and a liver comparable to that of humans. The most used elasmobranch in biomedical research is the spiny dogfish shark, Squalus acanthias. Comparative genomic analysis of the dogfish shark, the little skate (Leucoraja erincea), and other elasmobranchs have yielded insights into conserved functional domains of genes associated with human liver function, multidrug resistance, cystic fibrosis, and other biomedically relevant processes. While genomic information from these animals is informative in an evolutionary framework, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. We have derived the first multipassage, continuously proliferating cell line of a cartilaginous fish. The line was initiated from embryos of the spiny dogfish shark. The cells were maintained in a medium modified for fish species and supplemented with cell type-specific hormones, other proteins and sera, and plated on a collagen substrate. SAE cells have been cultured continuously for three years. These cells can be transfected by plasmids and have been cryopreserved. Expressed Sequence Tags generated from a normalized SAE cDNA library included a number of markers for cartilage and muscle, as well as proteins influencing tissue differentiation and development, suggesting that SAE cells may be of mesenchymal stem cell origin. Examination of SAE EST sequences also revealed a cartilaginous fish-specific repetitive sequence that may be evidence of an ancient mobile genetic element that most likely was introduced into the cartilaginous fish lineage after divergence from the lineage leading to teleosts.

  8. Mustard NPR1, a mammalian I{kappa}B homologue inhibits NF-{kappa}B activation in human GBM cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Kesanakurti, Divya [Department of Plant Sciences, University of Hyderabad, Hyderabad (India); Sareddy, Gangadhara Reddy [Department of Bio-technology and Animal Sciences, University of Hyderabad, Hyderabad (India); Babu, Phanithi Prakash, E-mail: [Department of Bio-technology and Animal Sciences, University of Hyderabad, Hyderabad (India); Kirti, Pulugurtha Bharadwaja, E-mail: [Department of Plant Sciences, University of Hyderabad, Hyderabad (India)


    NF-{kappa}B activity is tightly regulated by I{kappa}B class of proteins. I{kappa}B proteins possess ankyrin repeats for binding to and inhibiting NF-{kappa}B. The regulatory protein, NPR1 from Brassica juncea possesses ankyrin repeats with sequence similarity to I{kappa}B{alpha} subgroup. Therefore, we examined whether stably expressed BjNPR1 could function as I{kappa}B in inhibiting NF-{kappa}B in human glioblastoma cell lines. We observed that BjNPR1 bound to NF-{kappa}B and inhibited its nuclear translocation. Further, BjNPR1 expression down-regulated the NF-{kappa}B target genes iNOS, Cox-2, c-Myc and cyclin D1 and reduced the proliferation rate of U373 cells. Finally, BjNPR1 decreased the levels of pERK, pJNK and PKC{alpha} and increased the Caspase-3 and Caspase-8 activities. These results suggested that inhibition of NF-{kappa}B activation by BjNPR1 can be a promising therapy in NF-{kappa}B dependent pathologies.

  9. Cellular radiosensitivity of primary and metastatic human uveal melanoma cell lines

    NARCIS (Netherlands)

    G.J.M.J. van den Aardweg (Gerard J. M.); N.C. Naus (Nicole); A.C. Verhoeven; J.E.M.M. de Klein (Annelies); G.P.M. Luyten (Gré)


    textabstractPURPOSE: To investigate the radiosensitivity of uveal melanoma cell lines by a clonogenic survival assay, to improve the efficiency of the radiation regimen. METHODS: Four primary and four metastatic human uveal melanoma cell lines were cultured in the presence of condi

  10. Autophagy is involved in doxorubicin induced resistance of human myeloma cell line RP-MI8226

    Institute of Scientific and Technical Information of China (English)



    Objective To explore the role of autophagy in doxorubicin (DOX) -induced resistance of human myeloma cell line RPMI8226.Methods We established doxorubicin induced resistant subline of myeloma cell line RPMI8226/DOX by drug concentration step-elevation method.Resistant index of DOX was measured by MTT

  11. 9 CFR 113.52 - Requirements for cell lines used for production of biologics. (United States)


    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for cell lines used for production of biologics. 113.52 Section 113.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production...

  12. Chromatin structure and cellular radiosensitivity : A comparison of two human tumour cell lines

    NARCIS (Netherlands)

    Woudstra, EC; Roesink, JM; Rosemann, M; Brunsting, JF; Driessen, C; Orta, T; Konings, AWT; Peacock, JH; Kampinga, HH


    The role of variation in susceptibility to DNA damage induction was studied as a determinant for cellular radiosensitivity. Comparison of the radiosensitive HX142 and radioresistant RT112 cell lines previously revealed higher susceptibility to X-ray-induced DNA damage in the sensitive cell line usin

  13. Epithelial progenitor cell lines as models of normal breast morphogenesis and neoplasia

    DEFF Research Database (Denmark)

    Petersen, Ole William; Gudjonsson, Thorarinn; Villadsen, René;


    epithelial or the myoepithelial cell phenotype in primary cultures. Having succeeded in continuous propagation presumably without loss of markers, we could show that a subset of the luminal epithelial cells could convert to myoepithelial cells, signifying the possible existence of a progenitor cell...... cell lines. This suprabasal-derived epithelial cell line is able to generate both itself and differentiated luminal epithelial and myoepithelial cells, and in addition, is able to form elaborate terminal duct lobular unit (TDLU)-like structures within a reconstituted basement membrane. As more than 90...

  14. Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells. (United States)

    Gale, Zoe; Cooper, Paul R; Scheven, Ben A


    Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (AlP) activity showing a significant dose-dependent inhibition of relative AlP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated that GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFRα1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology.