WorldWideScience

Sample records for cell line undergoing

  1. Quantitative Membrane Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteogenic Differentiation

    DEFF Research Database (Denmark)

    Christiansen, Helle

    -transform mass spectrometry was used due to the high resolution and sensitivity provided by this strategy enabling very high sensitivity data and thus increased possibility of finding specific marker candidates for these cells. We have chosen to employ quantitative mass spectrometry on SILAC-labeled (Stable...... of proteins during several timepoints in osteogenesis and find that nearly all proteins quantified increase in level as osteogenesis progresses. This study expands beyond qualitative identification of markers candidates and into the quantitative profiling of membrane proteome changes over time in osteogenic....... The objectives of this project were to identify and quantify such surface protein markers of human MSCs using quantitative mass spectrometry-based proteomics and to monitor the quantitative changes of these surface markers during timepoints in osteogenic differentiation. Quantitative proteomics by fourier...

  2. Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation

    DEFF Research Database (Denmark)

    Foster, Leonard J; Zeemann, Patricia A; Li, Chen;

    2005-01-01

    One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely...... in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane...... or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another...

  3. Technical advance: Langerhans cells derived from a human cell line in a full-thickness skin equivalent undergo allergen-induced maturation and migration.

    Science.gov (United States)

    Ouwehand, Krista; Spiekstra, Sander W; Waaijman, Taco; Scheper, Rik J; de Gruijl, Tanja D; Gibbs, Susan

    2011-11-01

    In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a(+)Langerin(+)HLA-DR(+) MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO(4) and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

  4. The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture

    Science.gov (United States)

    Schatten, H.; Hedrick, J.; Chakrabarti, A.

    1998-01-01

    Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

  5. Storage of cell lines.

    Science.gov (United States)

    Parker, Katharine A

    2011-01-01

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

  6. The use of ovarian cancer cells from patients undergoing surgery to generate primary cultures capable of undergoing functional analysis.

    Science.gov (United States)

    O Donnell, Rachel L; McCormick, Aiste; Mukhopadhyay, Asima; Woodhouse, Laura C; Moat, Madeleine; Grundy, Anna; Dixon, Michelle; Kaufman, Angelika; Soohoo, San; Elattar, Ahmed; Curtin, Nicola J; Edmondson, Richard J

    2014-01-01

    The use of cell lines or animal models has significant disadvantages when dealing with a set of heterogeneous diseases such as epithelial ovarian cancer. This has clinical relevance in that biomarkers developed using cell line or animal models are often not transferable to the clinical setting. In this study, we describe the development of a robust protocol for developing primary cultures of ovarian cancer which will overcome some of these difficulties. Women undergoing surgery for ovarian cancer were recruited and samples of ascites and solid tumour deposits were used to develop primary cultures. Cells were characterised using a panel of immunofluorescent antibodies prior to use in a variety of assays including functional assessment of DNA repair pathways. During the four year study period, viable cultures, confirmed to be epithelial in origin were generated from 156 of 172 (91%) cases recruited. Characterisation was carried out using a panel of antibodies including pancytokeratin, CA125, EpCAM, MOC-31, D2-40 and vimentin. Senescence occurred between the 2nd and 8th passages in all cultures except one in which spontaneous immortalization occurred. Cells could be successfully cultured even after a period of storage at 4°C and cultured cells were capable of being used for a variety of applications including functional assays. Upon functional assessment there was minimal intra-tumour heterogeneity. It is therefore possible to derive viable ovarian cancer cell cultures in the majority of patients undergoing surgery. Cells cultured directly from patient cancers provide an accurate and highly diverse model.

  7. The use of ovarian cancer cells from patients undergoing surgery to generate primary cultures capable of undergoing functional analysis.

    Directory of Open Access Journals (Sweden)

    Rachel L O Donnell

    Full Text Available The use of cell lines or animal models has significant disadvantages when dealing with a set of heterogeneous diseases such as epithelial ovarian cancer. This has clinical relevance in that biomarkers developed using cell line or animal models are often not transferable to the clinical setting. In this study, we describe the development of a robust protocol for developing primary cultures of ovarian cancer which will overcome some of these difficulties. Women undergoing surgery for ovarian cancer were recruited and samples of ascites and solid tumour deposits were used to develop primary cultures. Cells were characterised using a panel of immunofluorescent antibodies prior to use in a variety of assays including functional assessment of DNA repair pathways. During the four year study period, viable cultures, confirmed to be epithelial in origin were generated from 156 of 172 (91% cases recruited. Characterisation was carried out using a panel of antibodies including pancytokeratin, CA125, EpCAM, MOC-31, D2-40 and vimentin. Senescence occurred between the 2nd and 8th passages in all cultures except one in which spontaneous immortalization occurred. Cells could be successfully cultured even after a period of storage at 4°C and cultured cells were capable of being used for a variety of applications including functional assays. Upon functional assessment there was minimal intra-tumour heterogeneity. It is therefore possible to derive viable ovarian cancer cell cultures in the majority of patients undergoing surgery. Cells cultured directly from patient cancers provide an accurate and highly diverse model.

  8. CLO : The cell line ontology

    NARCIS (Netherlands)

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

    2014-01-01

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

  9. Derivation of the human embryonic stem cell line RCM1

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

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

  10. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

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

    1991-12-01

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

  11. Can children undergoing ophthalmologic examinations under anesthesia be safely anesthetized without using an IV line?

    Directory of Open Access Journals (Sweden)

    Vigoda M

    2011-04-01

    Full Text Available Michael M Vigoda, Azeema Latiff, Timothy G Murray, Jacqueline L Tutiven, Audina M Berrocal, Steven GayerBascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USAPurpose: To document that with proper patient and procedure selection, children undergoing general inhalational anesthesia for ophthalmologic exams (with or without photos, ultrasound, laser treatment, peri-ocular injection of chemotherapy, suture removal, and/or replacement of ocular prosthesis can be safely anesthetized without the use of an intravenous (IV line. Children are rarely anesthetized without IV access placement. We performed a retrospective study to determine our incidence of IV access placement during examinations under anesthesia (EUA and the incidence of adverse events that required intraoperative IV access placement.Methods: Data collected from our operating room (OR information system includes but is not limited to diagnosis, anesthesiologist, surgeon, and location of IV catheter (if applicable, patient’s date of birth, actual procedure, and anesthesia/procedure times. We reviewed the OR and anesthetic records of children (>1 month and <10 years who underwent EUAs between January 1, 2003 and May 31, 2009. We determined the percentage of children who were anesthetized without IV access placement, as well as the incidence of any adverse events that required IV access placement, intraoperatively.Results: We analyzed data from 3196 procedures performed during a 77-month period. Patients’ ages ranged from 1 month to 9 years. Overall, 92% of procedures were performed without IV access placement. Procedure duration ranged from 1–39 minutes. Reasons for IV access placement included parental preference for antinausea medication and/or attending preference for IV access placement. No child who underwent anesthesia without an IV line had an intraoperative adverse event requiring insertion of an IV line.Conclusion: Our data suggest that for

  12. Oral changes in individuals undergoing hematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Regina Haddad Barrach

    2015-04-01

    Full Text Available INTRODUCTION: Patients undergoing hematopoietic stem cell transplantation receive high doses of chemotherapy and radiotherapy, which cause severe immunosuppression.OBJECTIVE: To report an oral disease management protocol before and after hematopoietic stem cell transplantation.METHODS: A prospective study was carried out with 65 patients aged > 18 years, with hematological diseases, who were allocated into two groups: A (allogeneic transplant, 34 patients; B (autologous transplant, 31 patients. A total of three dental status assessments were performed: in the pre-transplantation period (moment 1, one week after stem cell infusion (moment 2, and 100 days after transplantation (moment 3. In each moment, oral changes were assigned scores and classified as mild, moderate, and severe risks.RESULTS: The most frequent pathological conditions were gingivitis, pericoronitis in the third molar region, and ulcers at the third moment assessments. However, at moments 2 and 3, the most common disease was mucositis associated with toxicity from the drugs used in the immunosuppression.CONCLUSION: Mucositis accounted for the increased score and potential risk of clinical complications. Gingivitis, ulcers, and pericoronitis were other changes identified as potential risk factors for clinical complications.

  13. Reduction of the loads on a cylinder undergoing harmonic in-line motion

    Science.gov (United States)

    Marzouk, Osama A.; Nayfeh, Ali H.

    2009-08-01

    We use the finite-difference computational fluid dynamics method to study in detail the flow field around a circular cylinder in a uniform stream while undergoing in-line harmonic motion. For a given motion amplitude, there exists a critical forcing frequency below which the lift and drag can be period-n, quasiperiodic, or chaotic. Similarly, for a given frequency, there exists a critical amplitude below which the lift and drag can be period-n, quasiperiodic, or chaotic. Above these critical conditions, the lift and drag are synchronous with the forcing. The lift nearly vanishes and the mean drag drops and saturates at a value that is independent of the driving frequency, whereas the oscillatory drag quadratically depends on it. We relate these features to changes in the wake and the surface-pressure distribution. We examine the influence of the Reynolds number on these critical frequency and amplitude. Second- and higher-order spectral analyses show remarkable changes in the linear and quadratic coupling between the lift and drag when synchronization takes place; it destroys the two-to-one coupling between them in the cases of no motion and synchronization due to cross-flow motion.

  14. Beryllium-stimulated apoptosis in macrophage cell lines.

    Science.gov (United States)

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

    2000-08-21

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

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

    Science.gov (United States)

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

    2013-06-01

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

  16. Activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress.

    Science.gov (United States)

    Lu, Jun; Chen, Jian; Xu, Nianjun; Wu, Jun; Kang, Yani; Shen, Tingting; Kong, Hualei; Ma, Chao; Cheng, Ming; Shao, Zhifeng; Xu, Ling; Zhao, Xiaodong

    2016-09-01

    Application of cisplatin (DDP) for treating lung cancer is restricted due to its toxicity and lung cancer's drug resistance. In this study, we examined the effect of Jinfukang (JFK), an effective herbal medicine against lung cancer, on DDP-induced cytotoxicity in lung cancer cells. Morphologically, we observed that JFK increases DDP-induced pro-apoptosis in A549 cells in a synergistic manner. Transcriptome profiling analysis indicated that the combination of JFK and DDP regulates genes involved in apoptosis-related signaling pathways. Moreover, we found that the combination of JFK and DDP produces synergistic pro-apoptosis effect in other lung cancer cell lines, such as NCI-H1975, NCI-H1650, and NCI-H2228. Particularly, we demonstrated that AIFM2 is activated by the combined treatment of JFK and DDP and partially mediates the synergistic pro-apoptosis effect. Collectively, this study not only offered the first evidence that JFK promotes DDP-induced cytotoxicity, and activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress, but also provided a novel insight for improving cytotoxicity by combining JFK with DDP to treat lung cancer cells.

  17. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shuxian Jiang

    Full Text Available BACKGROUND: Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo. METHODOLOGY/PRINCIPAL FINDINGS: To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo. CONCLUSIONS/SIGNIFICANCE: Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

  18. Pollen tube reuses intracellular components of nucellar cells undergoing programmed cell death in Pinus densiflora.

    Science.gov (United States)

    Hiratsuka, Rie; Terasaka, Osamu

    2011-04-01

    Through the process known as programmed cell death (PCD), nucelli of Pinus densiflora serve as the transmitting tissue for growth of the pollen tube. We sought to clarify the processes of degradation of nucellar cell components and their transport to the pollen tube during PCD in response to pollen tube penetration of such nucelli. Stimulated by pollination, synthesis of large amounts of starch grains occurred in cells in a wide region of the nucellus, but as the pollen tube penetrated the nucellus, starch grains were degraded in amyloplasts of nucellar cells. In cells undergoing PCD, electron-dense vacuoles with high membrane contrast appeared, assumed a variety of autophagic structures, expanded, and ultimately collapsed and disappeared. Vesicles and electron-dense amorphous materials were released inside the thickened walls of cells undergoing PCD, and those vesicles and materials reaching the pollen tube after passing through the extracellular matrix were taken into the tube by endocytosis. These results show that in PCD of nucellar cells, intracellular materials are degraded in amyloplasts and vacuoles, and some of the degraded material is supplied to the pollen tube by vesicular transport to support tube growth.

  19. Can children undergoing ophthalmologic examinations under anesthesia be safely anesthetized without using an IV line?

    OpenAIRE

    Vigoda M; Latiff A; Murray T; Tutiven J; Berrocal A; Gayer S

    2011-01-01

    Michael M Vigoda, Azeema Latiff, Timothy G Murray, Jacqueline L Tutiven, Audina M Berrocal, Steven GayerBascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL, USAPurpose: To document that with proper patient and procedure selection, children undergoing general inhalational anesthesia for ophthalmologic exams (with or without photos, ultrasound, laser treatment, peri-ocular injection of chemotherapy, suture removal, and/or replacement of ocular prosthesis) can b...

  20. Immature hematopoietic stem cells undergo maturation in the fetal liver.

    Science.gov (United States)

    Kieusseian, Aurelie; Brunet de la Grange, Philippe; Burlen-Defranoux, Odile; Godin, Isabelle; Cumano, Ana

    2012-10-01

    Hematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2γc(-/-) mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.

  1. Melanoma cells undergo aggressive coalescence in a 3D Matrigel model that is repressed by anti-CD44

    Science.gov (United States)

    Voss, Edward; Kuhl, Spencer; Buchele, Emma C.; Klemme, Michael R.; Russell, Kanoe B.; Ambrose, Joseph; Soll, Benjamin A.; Bossler, Aaron; Milhem, Mohammed; Goldman, Charles

    2017-01-01

    Using unique computer-assisted 3D reconstruction software, it was previously demonstrated that tumorigenic cell lines derived from breast tumors, when seeded in a 3D Matrigel model, grew as clonal aggregates which, after approximately 100 hours, underwent coalescence mediated by specialized cells, eventually forming a highly structured large spheroid. Non-tumorigenic cells did not undergo coalescence. Because histological sections of melanomas forming in patients suggest that melanoma cells migrate and coalesce to form tumors, we tested whether they also underwent coalescence in a 3D Matrigel model. Melanoma cells exiting fragments of three independent melanomas or from secondary cultures derived from them, and cells from the melanoma line HTB-66, all underwent coalescence mediated by specialized cells in the 3D model. Normal melanocytes did not. However, coalescence of melanoma cells differed from that of breast-derived tumorigenic cell lines in that they 1) coalesced immediately, 2) underwent coalescence as individual cells as well as aggregates, 3) underwent coalescence far faster and 4) ultimately formed long, flat, fenestrated aggregates that were extremely dynamic. A screen of 51 purified monoclonal antibodies (mAbs) targeting cell surface-associated molecules revealed that two mAbs, anti-beta 1 integrin/(CD29) and anti-CD44, blocked melanoma cell coalescence. They also blocked coalescence of tumorigenic cells derived from a breast tumor. These results add weight to the commonality of coalescence as a characteristic of tumorigenic cells, as well as the usefulness of the 3D Matrigel model and software for both investigating the mechanisms regulating tumorigenesis and screening for potential anti-tumorigenesis mAbs. PMID:28264026

  2. Modelling a tethered mammalian sperm cell undergoing hyperactivation

    KAUST Repository

    Curtis, M.P.

    2012-09-01

    The beat patterns of mammalian sperm flagella can be categorised into two different types. The first involves symmetric waves propagating down the flagellum with a net linear propulsion of the sperm cell. The second, hyperactive, waveform is classified by vigorous asymmetric waves of higher amplitude, lower wavenumber and frequency propagating down the flagellum resulting in highly curved trajectories. The latter beat pattern is part of the capacitation process whereby sperm prepare for the prospective penetration of the zona pellucida and fusion with the egg. Hyperactivation is often observed to initiate as sperm escape from epithelial and ciliary bindings formed within the isthmic regions of the female oviducts, leading to a conjecture in the literature that this waveform is mechanically important for sperm escape. Hence, we explore the mechanical effects of hyperactivation on a tethered sperm, focussing on a Newtonian fluid. Using a resistive force theory model we demonstrate that hyperactivation can indeed generate forces that pull the sperm away from a tethering point and consequently a hyperactivated sperm cell bound to an epithelial surface need not always be pushed by its flagellum. More generally, directions of the forces generated by tethered flagella are insensitive to reductions in beat frequency and the detailed flagellar responses depend on the nature of the binding at the tethering point. Furthermore, waveform asymmetry and amplitude increases enhance the tendency for a tethered flagellum to start tugging on its binding. The same is generally predicted to be true for reductions in the wavenumber of the flagellum beat, but not universally so, emphasising the dynamical complexity of flagellar force generation. Finally, qualitative observations drawn from experimental data of human sperm bound to excised female reproductive tract are also presented and are found to be consistent with the theoretical predictions. © 2012 Elsevier Ltd.

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Science.gov (United States)

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

    2003-02-01

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

  5. Virus Discovery Using Tick Cell Lines

    Science.gov (United States)

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

  7. A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

    OpenAIRE

    Domínguez, Fernándo; Moreno Onorato, Francisco Javier; Cejudo Fernández, Francisco Javier

    2003-01-01

    The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl trans...

  8. NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery

    Science.gov (United States)

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

    2017-01-01

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

  9. In vivo formation of complex microvessels lined by human endothelial cells in an immunodeficient mouse

    OpenAIRE

    2000-01-01

    We have identified conditions for forming cultured human umbilical vein endothelial cells (HUVEC) into tubes within a three-dimensional gel that on implantation into immunoincompetent mice undergo remodeling into complex microvessels lined by human endothelium. HUVEC suspended in mixed collagen/fibronectin gels organize into cords with early lumena by 24 h and then apoptose. Twenty-hour constructs, s.c. implanted in immunodeficient mice, display HUVEC-lined thin-walled microvessels within the...

  10. Successful afatinib treatment of advanced non-small-cell lung cancer patients undergoing hemodialysis.

    Science.gov (United States)

    Imai, Hisao; Kaira, Kyoichi; Naruse, Ichiro; Hayashi, Hideki; Iihara, Hirotoshi; Kita, Yutaro; Mizusaki, Naoki; Asao, Takayuki; Itoh, Yoshinori; Sugiyama, Tadashi; Minato, Koichi; Yamada, Masanobu

    2017-01-01

    The treatment for patients with lung cancer undergoing hemodialysis, who are frequently elderly and have poor performance status, becomes a more important subject. However, the feasibility of afatinib in patients with chronic renal failure undergoing hemodialysis has not, so far, been reported. Here, afatinib was administered to three patients with NSCLC harboring EGFR mutation and chronic renal failure undergoing hemodialysis. Pharmacokinetic (PK) data of afatinib supported the safety of afatinib treatment. After receiving their written informed consent from all patients, they were administered 30 mg afatinib daily with HD three times a week. We performed PK analyses of afatinib on days 1, 2, 10, and 11 after initial administration of afatinib. All three patients exhibited a partial response without any serious adverse events during the administration of afatinib. These PK data were similar to those of patients with normal organ function, which were previously reported. Our findings may be particularly useful given the current opportunity to use afatinib as a first-line treatment for EGFR-mutated NSCLC patients, providing an additional option for patients with impaired renal function.

  11. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-07-30

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

  12. Stem cell characteristics in prostate cancer cell lines.

    NARCIS (Netherlands)

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

    2010-01-01

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

  13. Postoperative infection and natural killer cell function following blood transfusion in patients undergoing elective colorectal surgery

    DEFF Research Database (Denmark)

    Jensen, L S; Andersen, A J; Christiansen, P M

    1992-01-01

    The frequency of infection in 197 patients undergoing elective colorectal surgery and having either no blood transfusion, transfusion with whole blood, or filtered blood free from leucocytes and platelets was investigated in a prospective randomized trial. Natural killer cell function was measured...... before operation and 3, 7 and 30 days after surgery in 60 consecutive patients. Of the patients 104 required blood transfusion; 48 received filtered blood and 56 underwent whole blood transfusion. Postoperative infections developed in 13 patients transfused with whole blood (23 per cent, 95 per cent...... function was significantly (P less than 0.001) impaired up to 30 days after surgery in patients transfused with whole blood. These data provide a strong case against the use of whole blood transfusion in patients undergoing elective colorectal surgery. Udgivelsesdato: 1992-Jun...

  14. Modelling the damage potential of fluid flows for animal cells undergoing cultivation in bioreactors.

    Science.gov (United States)

    Stanford Keen, Giles

    1996-11-01

    Mechanical disruption and injury sustained by animal cells undergoing cultivation in bioreactors is an important problem in biotechnology. Damage to cells is thought to be caused primarily by bubbles bursting at the free surface of the culture medium. Here we present computational studies applying a mathematical model for the cell damage rates experienced by cells in laminar flow. Two fluid dynamical systems are considered - namely a converging channel and a single bursting bubble. The flows are calculated using a fourth-order finite difference technique on a stretched grid, and a boundary integral method respectively. It is possible to obtain an estimate for the number of cells in a particular population which are likely to be disrupted by the forces they experience in the flow. This is done by calculating the maximum rate of strain experienced by fluid particles, and combining this with experimental data on the strength and size of cells, obtained by micromanipulation techniques. The resulting information is then used together with the cell damage model to produce a cell damage prediction. The computational results are compared with experimental measurements of cell death, to validate the model for cell damage.

  15. Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine.

    Directory of Open Access Journals (Sweden)

    Alex F Thompson

    Full Text Available RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype.661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology.Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested.661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.

  16. Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro

    Institute of Scientific and Technical Information of China (English)

    TongmingLiu; HaobinZhao; WeijiaWang; RongLiu; TianshengChen; JiaorongDeng; JianfangGui

    2005-01-01

    Spermatogonia are the male germ stem cells that continuously produce sperm for the next generation. Spermatogenesis is a complicated process that proceeds through mitotic phase of stem cell renewal and differentiation, meiotic phase, and postmeiotic phase of spermiogenesis. Full recapitulation of spermatogenesis in vitro has been impossible, as generation of normal spermatogonial stem cell lines without immortalization and production of motile sperm from these cells after long-term culture have not been achieved. Here we report the derivation of a normal spermatogonial cell line from a mature medakafish testis without immortalization. After 140 passages during 2 years of culture, this cell line retains stable but growth factor-dependent proliferation, a diploid karyotype, and the phenotype and gene expression pattern of spermatogonial stem cells. Furthermore, we show that this cell line can undergo meiosis and spermiogenesis to generate motile sperm.Therefore, the ability of continuous proliferation and sperm production in culture is an intrinsic property of medaka spermatogonial stem cells, and immortalization apparently is not necessary to derive male germ cell cultures. Our findings and cell line will offer a unique opportunity to study and recapitulate spe rmatogenesis in vitro and to develop approaches for germ-line transmission.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

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

  19. Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation.

    Directory of Open Access Journals (Sweden)

    Rafaela C Sartore

    Full Text Available The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC cells, embryonic stem (ES cells and induced pluripotent stem (iPS cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal

  20. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2007-12-21

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

  2. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.

    Science.gov (United States)

    Koopman, G; Reutelingsperger, C P; Kuijten, G A; Keehnen, R M; Pals, S T; van Oers, M H

    1994-09-01

    Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.

  3. Successful rescue of pure red cell aplasia in two aged patients undergoing pancrease-kindey transplantation

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yin-fu; YANG Tong-han; WANG Qing-yu; Wang Ping-xian; Fan Ming-qi; Feng Jia-yu

    2001-01-01

    To explore the correlation between hypoimmunity and the occurrence of pure red cell aplasia (PRCA) in senile patients undergoing combined pancrease-kindey transplantation. Methods: PRCA occurred in 2 patients out of 5 who were performed combined pancrease-kindey transplantation.The general scheme of treatment mainly included selective administration of immunosuppressants and antivirus drugs, infusion of red blood cells on the basis of surveillance of parvorirus B19 and the ratio of T4 and T8 as well as the changes of the myelogram.Results: The myelogram of the patients returned to normal in 2 and 3 weeks after operation, respectively, and subsequent follow-up revealed no recurrence. Conclusion: This series illustrate the point that advanced age, hypo immunity, parvorirus B19 and immunosuppressants are vulnerable to PRCA. Combined treatment is an effectiveremedy for these patients.

  4. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

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

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

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

  6. Radiosensitivity of Human Melanoma Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-07-01

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

  7. Red cell distribution width in anemic patients undergoing transcatheter aortic valve implantation

    Institute of Scientific and Technical Information of China (English)

    Katharina Hellhammer; Tobias Zeus; Pablo E Verde; Verena Veulemanns; Lisa Kahlstadt; Georg Wolff; Ralf Erkens; Ralf Westenfeld; Eliano P Navarese; Marc W Merx; Tienush Rassaf; Malte Kelm

    2016-01-01

    AIM: To determine the impact of red blood cell distribution width on outcome in anemic patients undergoing transcatheter aortic valve implantation(TAVI).METHODS: In a retrospective single center cohort study we determined the impact of baseline red cell distribution width(RDW) and anemia on outcome in 376 patients with aortic stenosis undergoing TAVI. All patients were discussed in the institutional heart team and declined for surgical aortic valve replacement due to high operative risk. Collected data included patient characteristics, imaging findings, periprocedural in hospital data, laboratory results and follow up data. Blood samples for hematology and biochemistry analysis were taken from every patient before and at fixed intervals up to 72 h after TAVI including blood count and creatinine. Descriptive statistics were used for patient’s characteristics. KaplanMeier survival curves were used for time to event outcomes. A recursive partitioning regression and classification was used to investigate the association between potential risk factors and outcome variables.RESULTS: Mean age in our study population was 81 ± 6.1 years. Anemia was prevalent in 63.6%(n = 239) of our patients. Age and creatinine were identified as risk factors for anemia. In our study population, anemia per se did influence 30-d mortality but did not predict longterm mortality. In contrast, a RDW > 14% showed to be highly predictable for a reduced short- and longterm survival in patients with aortic valve disease after TAVI procedure.CONCLUSION: Age and kidney function determine the degree of anemia. The anisocytosis of red blood cells in anemic patients supplements prognostic information in addition to that derived from the WHO-based definition of anemia.

  8. Iron overload in patients with acute leukemia or MDS undergoing myeloablative stem cell transplantation.

    Science.gov (United States)

    Armand, Philippe; Kim, Haesook T; Rhodes, Joanna; Sainvil, Marie-Michele; Cutler, Corey; Ho, Vincent T; Koreth, John; Alyea, Edwin P; Hearsey, Doreen; Neufeld, Ellis J; Fleming, Mark D; Steen, Hanno; Anderson, Damon; Kwong, Raymond Y; Soiffer, Robert J; Antin, Joseph H

    2011-06-01

    Patients with hematologic malignancies undergoing allogeneic stem cell transplantation (HSCT) commonly have an elevated serum ferritin prior to HSCT, which has been associated with increased mortality after transplantation. This has led to the suggestion that iron overload is common and deleterious in this patient population. However, the relationship between serum ferritin and parenchymal iron overload in such patients is unknown. We report a prospective study of 48 patients with acute leukemia (AL) or myelodysplastic syndromes (MDS) undergoing myeloablative HSCT, using magnetic resonance imaging (MRI) to estimate liver iron content (LIC) and cardiac iron. The median (and range) pre-HSCT value of serum ferritin was 1549 ng/mL (20-6989); serum hepcidin, 59 ng/mL (10-468); labile plasma iron, 0 LPI units (0.0-0.9). Eighty-five percent of patients had hepatic iron overload (HIO), and 42% had significant HIO (LIC ≥5.0 mg/gdw). Only 1 patient had cardiac iron overload. There was a strong correlation between pre-HSCT serum ferritin and estimated LIC (r = .75), which was mostly dependent on prior transfusion history. Serum hepcidin was appropriately elevated in patients with HIO. Labile plasma iron elevation was rare. A regression calibration analysis supported the hypothesis that elevated pre-HSCT LIC is significantly associated with inferior post-HSCT survival. These results contribute to our understanding of the prevalence, mechanism, and consequences of iron overload in HSCT.

  9. Kinetics of circulating endothelial progenitor cells in patients undergoing carotid artery surgery

    Directory of Open Access Journals (Sweden)

    Kalender G

    2016-12-01

    Full Text Available G Kalender,1 A Kornberger,2 M Lisy,1 Andres Beiras-Fernandez,2 UA Stock2 1Deparment of General, Thoracic and Vascular Surgery, Hoechst Hospital, 2Department of Thoracic and Cardiovascular Surgery, University Hospital Frankfurt, Frankfurt am Main, Germany Aim: Endothelial progenitor cells (EPCs are primitive cells found in the bone marrow and peripheral blood (PB. In particular, the potential of EPCs to differentiate into mature endothelial cells remains of high interest for clinical applications such as bio-functionalized patches for autologous seeding after implantation. The objective of this study was to determine EPCs’ kinetics in patients undergoing carotid artery thromboendarterectomy (CTEA and patch angioplasty. Methods: Twenty CTEA patients were included (15 male, mean age 76 years. PB samples were taken at 1 day preoperatively, and at 1, 3, and 5 days postoperatively. Flow cytometric analysis was performed for CD34, CD133, KDR, and CD45. Expression of KDR, SDF-1α, and G-CSF was analyzed by means of enzyme-linked immunosorbent assay. Results: Fluorescence-activated cell sorting analysis revealed 0.031%±0.016% (% of PB mononuclear cells KDR+ cells and 0.052%±0.022% CD45-/CD34+/CD133+ cells, preoperatively. A 33% decrease of CD45–/CD34+/CD133+ cells was observed at day 1 after surgery. However, a relative number (compared to initial preoperative values of CD45-/CD34+/CD133+ cells was found on day 3 (82% and on day 5 (94% postoperatively. More profound upregulated levels of CD45–CD34+/CD133+ cells were observed for diabetic (+47% compared to nondiabetic and male (+38% compared to female patients. No significant postoperative time-dependent differences were found in numbers of KDR+ cells and the concentrations of the cytokines KDR and G-CSF. However, the SDF-1α levels decreased significantly on day 1 postoperatively but returned to preoperative levels by day 3. Conclusion: CTEA results in short-term downregulation of circulating

  10. Transfusion rate and prevalence of unexpected red blood cell alloantibodies in women undergoing hysterectomy for benign disease

    DEFF Research Database (Denmark)

    Thoestesen, Lisbeth M; Rasmussen, Kjeld L; Lauszus, Finn F;

    2011-01-01

    To determine transfusion rates, risk factors for transfusion and the prevalence of unexpected red blood cell alloantibodies in women undergoing hysterectomy for benign disease. In addition, we aimed to evaluate the necessity of the pretransfusion testing for red blood cell alloantibodies....

  11. Evidence for progenitors of chronic lymphocytic leukemia B cells that undergo intraclonal differentiation and diversification.

    Science.gov (United States)

    Dono, M; Hashimoto, S; Fais, F; Trejo, V; Allen, S L; Lichtman, S M; Schulman, P; Vinciguerra, V P; Sellars, B; Gregersen, P K; Ferrarini, M; Chiorazzi, N

    1996-02-15

    Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha-expressing cDNA were present in greater amounts that unrelated (non-CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

  12. Evaluation of febrile neutropenia in patients undergoing hematopoietic stem cell transplantation.

    Directory of Open Access Journals (Sweden)

    Shahideh Amini

    2014-01-01

    Full Text Available The aim of this study was to determine the incidence and causes of fever as a major problem contributing to transplantation related mortality among patients undergoing hematopoietic stem cell transplantation (HSCT and evaluation of antibiotic use, according to reliable guidelines.We retrospectively reviewed hospital records of 195 adult patients who underwent HSCT between 2009-2011 at hematology-oncology and bone marrow transplantation research center. Baseline information and also data related to fever and neutropenia, patient's outcomes, duration of hospitalization and antibiotic use pattern were documented.A total of 195 patients were analyzed and a total of 268 febrile episodes in 180 patients were recorded (mean 1.5 episodes per patient. About 222 episodes (82% were associated with neutropenia which one-fourth of them were without any documented infection sources. Microbiologic documents showed that the relative frequencies of gram positive and gram negative bacteria were 62.5% and 37.5%, respectively. The hospital stay duration was directly related to the numbers of fever episodes (P<0.0001.The rate of febrile episodes in autologous stem cell transplantation was significantly higher compared to allogeneic type (P<0.05.It is necessary to determine not only the local profile of microbiologic pattern, but also antibiotic sensitivities in febrile neutropenic patients following hematopoietic stem cell transplantation, and reassess response to antibiotic treatment to establish any necessity for modifications to treatment guidelines in order to prevent any fatal complications from infection.

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

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

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

  14. Biological characteristics of cell lines of human dental alveolus

    Institute of Scientific and Technical Information of China (English)

    陈世璋; 黄靖香; 孙明学; 赵斌

    2003-01-01

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

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

    OpenAIRE

    2013-01-01

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

  16. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-05-01

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

  17. Caspofungin as antifungal prophylaxis in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation: a retrospective analysis

    Directory of Open Access Journals (Sweden)

    Döring Michaela

    2012-07-01

    Full Text Available Abstract Background Pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT often receive intravenous liposomal amphotericin B (L-AmB as antifungal prophylaxis. There are no guidelines for antifungal prophylaxis in children in this situation. Caspofungin (CAS, a broad-spectrum echinocandin, could be an effective alternative with lower nephrotoxicity than L-AmB. Methods We retrospectively analyzed the safety, feasibility, and efficacy of CAS in our center, and compared the results with L-AmB as antifungal monoprophylaxis in pediatric patients undergoing HSCT. 60 pediatric patients received L-AmB (1 or 3 mg/kg bw/day and another 60 patients received CAS (50 mg/m2/day as antifungal monoprophylaxis starting on day one after HSCT. The median ages of patients receiving L-AmB and CAS were 7.5 years and 9.5 years, respectively. Results No proven breakthrough fungal infection occurred in either group during the median treatment period of 23 days in the L-AmB group and 24 days in the CAS group. One patient receiving CAS developed probable invasive aspergillosis. During L-AmB treatment, potassium levels significantly decreased below normal values. Patients treated with L-AmB had more drug-related side effects and an increased need for oral supplementation with potassium, sodium bicarbonate and calcium upon discharge as compared with the CAS group. CAS was well-tolerated and safe in this cohort of immunocompromised pediatric patients, who underwent high-dose chemotherapy and HSCT. Conclusion Prophylactic CAS and L-AmB showed similar efficacy in this biggest cohort of pediatric patients after allogeneic HSCT reported, so far. A prospective randomized trial in children is warranted to allow for standardized guidelines.

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

  19. Different glycosylation of cadherins from human bladder non-malignant and cancer cell lines

    Directory of Open Access Journals (Sweden)

    Lityńska Anna

    2002-06-01

    Full Text Available Abstract Background The aim of the present study was to determine whether stage of invasiveness of bladder cancer cell lines contributes to alterations in glycan pattern of their cadherins. Results Human non-malignant epithelial cell of ureter HCV29, v-raf transfected HCV29 line (BC3726 and transitional cell cancers of urine bladder Hu456 and T24 were grown in cell culture. Equal amounts of protein from each cell extracts were separated by SDS-PAGE electrophoresis and were blotted on an Immobilon P membrane. Cadherins were immunodetected using anti-pan cadherin mAb and lectin blotting assays were performed, in parallel. N-oligosaccharides were analysed by specific reaction with Galanthus nivalis agglutinin (GNA, Sambucus nigra agglutinin (SNA, Maackia amurensis agglutinin (MAA, Datura stramonium agglutinin (DSA, Aleuria aurantia agglutinin (AAA, Phaseolus vulgaris agglutinin (PHA-L and wheat germ agglutinin (WGA. The cadherin from HCV29 cell line possessed bi- and/or 2,4-branched triantennary complex type glycans, some of which were α2,6-sialylated. The cadherin from BC3726 cell line exhibited exclusively high mannose type glycans. Cadherins from Hu456 and T24 cell lines expressed high mannose type glycans as well as β1,6-branched oligosaccharides with poly-N-acetyllactosamine structures and α2,3-linked sialic acid residues. Additionally, the presence of fucose and α2,6-sialic acid residues on the cadherin from T24 cell line was detected. Conclusions These results indicate that N-glycosylation pattern of cadherin from bladder cancer cell line undergoes modification during carcinogenesis.

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  1. Constitutive activation of extracellular signal-regulated kinase predisposes diffuse large B-cell lymphoma cell lines to CD40-mediated cell death

    DEFF Research Database (Denmark)

    Hollmann, C Annette; Owens, Trevor; Nalbantoglu, Josephine;

    2006-01-01

    CD40 promotes survival, proliferation, and differentiation of normal B cells but can cause activation-induced cell death in malignant B lymphocytes. CD40 ligand and anti-CD40 antibodies have been used successfully to induce apoptosis in lymphoma lines both in vitro and in xenograft tumor models...... a specific cell line or tumor will undergo apoptosis when stimulated with CD40 and to identify targets downstream of CD40 that affect only the apoptotic arm of CD40 signaling. We have analyzed gene expression patterns in CD40-sensitive and CD40-resistant diffuse large B-cell lymphoma (DLBCL) cell lines...... and no increase in ERK activity in response to CD40 stimulation. Our results suggest that constitutive activation of ERK may be required for death signaling by CD40....

  2. Presence of base excision repair enzymes in the wheat aleurone and their activation in cells undergoing programmed cell death.

    Science.gov (United States)

    Bissenbaev, Amangeldy K; Ishchenko, Alexander A; Taipakova, Sabira M; Saparbaev, Murat K

    2011-10-01

    Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the β-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.

  3. POTENTIAL CELL LINE TOXICITY OF ENVIRONMENTAL NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Mohan Durga

    2012-01-01

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

  4. A human follicular lymphoma B cell line hypermutates its functional immunoglobulin genes in vitro.

    Science.gov (United States)

    Wu, H; Pelkonen, E; Knuutila, S; Kaartinen, M

    1995-12-01

    The functional immunoglobulin (Ig) genes of B lymphocytes undergo somatic mutations during immune responses. These mutations modify the antigen binding site of the immunoglobulins, thereby enhancing the average affinity of the antibodies produced. The molecular mechanism underlying these B cell hypermutations remains unresolved, partly because it is difficult to grow normal B cells in long-term cell cultures and because there is no suitable transformed or malignant B cell line which generates mutations in its immunoglobulin genes in vitro. Here, we show that the recently established follicular lymphoma line HF-1.3.4 generates somatic hypermutations in vitro at a high frequency of 0.7 x 10(-6) mutations per base pair per generation in standard cell cultures (RPMI 1640 + 5% fetal calf serum). This shows for the first time that B cell hypermutation can occur without T cells or T cell factors. The mutation frequency increased approximately tenfold to 1 x 10(-5) mutations/base pair/generation with B cell-specific growth factors (interleukins-2 and -4 and three antibodies stimulatory to HF-1.3.4 cells). This HF-1.3.4 lymphoma line may help to elucidate the molecular mechanism of Ig gene hypermutation.

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

    Science.gov (United States)

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

    2016-01-01

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

  6. The impact of preparation and support procedures for children with sickle cell disease undergoing MRI

    Energy Technology Data Exchange (ETDEWEB)

    Cejda, Katherine R. [St. Jude Children' s Research Hospital, Child Life Program, Memphis, TN (United States); Smeltzer, Matthew P. [St. Jude Children' s Research Hospital, Department of Biostatistics, Memphis, TN (United States); Hansbury, Eileen N. [Baylor International Hematology Center of Excellence and the Texas Children' s Center for Global Health, Houston, TX (United States); McCarville, Mary Elizabeth; Helton, Kathleen J. [St. Jude Children' s Research Hospital, Department of Radiological Sciences, Memphis, TN (United States); Hankins, Jane S. [St. Jude Children' s Research Hospital, Department of Hematology, Memphis, TN (United States)

    2012-10-15

    Children with sickle cell disease (SCD) often undergo MRI studies to assess brain injury or to quantify hepatic iron. MRI requires the child to lie motionless for 30-60 min, thus sedation/anesthesia might be used to facilitate successful completion of exams, but this poses additional risks for SCD patients. To improve children's ability to cope with MRI examinations and avoid sedation, our institution established preparation and support procedures (PSP). To investigate the impact of PSP in reducing the need for sedation during MRI exams among children with SCD. Data on successful completion of MRI testing were compared among 5- to 12-year-olds who underwent brain MRI or liver R2*MRI with or without receiving PSP. Seventy-one children with SCD (median age 9.85 years, range 5.57-12.99 years) underwent a brain MRI (n = 60) or liver R2*MRI (n = 11). Children who received PSP were more likely to complete an interpretable MRI exam than those who did not 30 of 33; 91% vs. 27 of 38; 71%, unadjusted OR = 4.1 (P = 0.04) and OR = 8.5 (P < 0.01) when adjusting for age. PSP can help young children with SCD complete clinically interpretable, nonsedated MRI exams, avoiding the risks of sedation/anesthesia. (orig.)

  7. Stomatitis-related pain in women with breast cancer undergoing autologous hematopoietic stem cell transplant.

    Science.gov (United States)

    Fall-Dickson, Jane M; Mock, Victoria; Berk, Ronald A; Grimm, Patricia M; Davidson, Nancy; Gaston-Johansson, Fannie

    2008-01-01

    The purpose of this cross-sectional, correlational study was to describe stomatitis-related pain in women with breast cancer undergoing autologous hematopoietic stem cell transplant. The hypotheses that significant, positive relationships would exist between oral pain and stomatitis, state anxiety, depression, and alteration in swallowing were tested. Stomatitis, sensory dimension of oral pain, and state anxiety were hypothesized to most accurately predict oral pain overall intensity. Thirty-two women were recruited at 2 East Coast comprehensive cancer centers. Data were collected on bone marrow transplantation day +7 +/- 24 hours using Painometer, Oral Mucositis Index-20, Oral Assessment Guide, State-Trait Anxiety Inventory, and Beck Depression Inventory. Data analysis included descriptive statistics, correlations, and stepwise multiple regression. All participants had stomatitis; 47% had oral pain, with a subset reporting continuous moderate to severe oral pain despite pain management algorithms. Significant, positive associations were seen between oral pain, stomatitis, and alteration in swallowing and between oral pain with swallowing and alteration in swallowing. Oral pain was not significantly correlated with state anxiety and depression. Oral sensory and affective pain intensity most accurately predicted oral pain overall intensity. Future research needs to explore factors that affect perception and response to stomatitis-related oropharyngeal pain and individual patient response to opioid treatment.

  8. Ovarian tissue cryopreservation in girls undergoing haematopoietic stem cell transplant: experience of a single centre.

    Science.gov (United States)

    Biasin, E; Salvagno, F; Berger, M; Nesi, F; Quarello, P; Vassallo, E; Evangelista, F; Marchino, G L; Revelli, A; Benedetto, C; Fagioli, F

    2015-09-01

    Fertility after childhood haemopoietic stem cell transplant (HSCT) is a major concern. Conditioning regimens before HSCT present a high risk (>80%) of ovarian failure. Since 2000, we have proposed cryopreservation of ovarian tissue to female patients undergoing HSCT at our centre, to preserve future fertility. After clinical and haematological evaluation, the patients underwent ovarian tissue collection by laparoscopy. The tissue was analysed by histologic examination to detect any tumour contamination and then frozen following the slow freezing procedure and cryopreserved in liquid nitrogen. From August 2000 to September 2013, 47 patients planned to receive HSCT, underwent ovarian tissue cryopreservation. The median age at diagnosis was 11.1 years and at the time of procedure it was 13 years, respectively. Twenty-four patients were not pubertal at the time of storage, whereas 23 patients had already experienced menarche. The median time between laparoscopy and HSCT was 25 days. Twenty-six out of 28 evaluable patients (93%) developed hypergonadotropic hypogonadism at a median time of 23.3 months after HSCT. One patient required autologous orthotopic transplantation that resulted in one live birth. Results show a very high rate of iatrogenic hypergonadotropic hypogonadism, highlighting the need for fertility preservation in these patients.

  9. Investigation of the selenium metabolism in cancer cell lines

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  10. Physical exercise for patients undergoing hematopoietic stem cell transplantation: systematic review and meta-analyses of randomized controlled trials

    NARCIS (Netherlands)

    Haren, I.E.P.M.; Timmerman, H.; Potting, C.M.J.; Blijlevens, N.M.A.; Staal, J.B.; Nijhuis-Van der Sanden, M.W.G.

    2013-01-01

    BACKGROUND: The treatment-related burden for patients undergoing hematopoietic stem cell transplantation (HSCT) may be relieved by physical exercises. PURPOSE: The purpose of this study was to summarize and analyze the evidence provided by randomized controlled trials (RCTs) on physical exercise int

  11. Circulating Tumor Cells Identify Early Recurrence in Patients with Non-Small Cell Lung Cancer Undergoing Radical Resection.

    Directory of Open Access Journals (Sweden)

    Clara Bayarri-Lara

    Full Text Available Surgery is the treatment of choice for patients with non-small cell lung cancer (NSCLC stages I-IIIA. However, more than 20% of these patients develop recurrence and die due to their disease. The release of tumor cells into peripheral blood (CTCs is one of the main causes of recurrence of cancer. The objectives of this study are to identify the prognostic value of the presence and characterization of CTCs in peripheral blood in patients undergoing radical resection for NSCLC.56 patients who underwent radical surgery for previously untreated NSCLC were enrolled in this prospective study. Peripheral blood samples for CTC analysis were obtained before and one month after surgery. In addition CTCs were phenotypically characterized by epidermal growth factor receptor (EGFR expression.51.8% of the patients evaluated were positive with the presence of CTCs at baseline. A decrease in the detection rate of CTCs was observed in these patients one month after surgery (32.1% (p = 0.035. The mean number of CTCs was 3.16 per 10 ml (range 0-84 preoperatively and 0.66 (range 0-3 in postoperative determination. EGFR expression was found in 89.7% of the patients at baseline and in 38.9% patients one month after surgery. The presence of CTCs after surgery was significantly associated with early recurrence (p = 0.018 and a shorter disease free survival (DFS (p = .008. In multivariate analysis CTC presence after surgery (HR = 5.750, 95% CI: 1.50-21.946, p = 0.010 and N status (HR = 0.296, 95% CI: 0.091-0.961, p = 0.043 were independent prognostic factors for DFS.CTCs can be detected and characterized in patients undergoing radical resection for non-small cell lung cancer. Their presence might be used to identify patients with increased risk of early recurrence.

  12. Circulating Tumor Cells Identify Early Recurrence in Patients with Non-Small Cell Lung Cancer Undergoing Radical Resection

    Science.gov (United States)

    Cueto Ladrón de Guevara, Antonio; Puche, Jose L.; Ruiz Zafra, Javier; de Miguel-Pérez, Diego; Ramos, Abel Sánchez-Palencia; Giraldo-Ospina, Carlos Fernando; Navajas Gómez, Juan A.; Delgado-Rodriguez, Miguel; Lorente, Jose A.; Serrano, María Jose

    2016-01-01

    Background Surgery is the treatment of choice for patients with non-small cell lung cancer (NSCLC) stages I-IIIA. However, more than 20% of these patients develop recurrence and die due to their disease. The release of tumor cells into peripheral blood (CTCs) is one of the main causes of recurrence of cancer. The objectives of this study are to identify the prognostic value of the presence and characterization of CTCs in peripheral blood in patients undergoing radical resection for NSCLC. Patients and Methods 56 patients who underwent radical surgery for previously untreated NSCLC were enrolled in this prospective study. Peripheral blood samples for CTC analysis were obtained before and one month after surgery. In addition CTCs were phenotypically characterized by epidermal growth factor receptor (EGFR) expression. Results 51.8% of the patients evaluated were positive with the presence of CTCs at baseline. A decrease in the detection rate of CTCs was observed in these patients one month after surgery (32.1%) (p = 0.035). The mean number of CTCs was 3.16 per 10 ml (range 0–84) preoperatively and 0.66 (range 0–3) in postoperative determination. EGFR expression was found in 89.7% of the patients at baseline and in 38.9% patients one month after surgery. The presence of CTCs after surgery was significantly associated with early recurrence (p = 0.018) and a shorter disease free survival (DFS) (p = .008). In multivariate analysis CTC presence after surgery (HR = 5.750, 95% CI: 1.50–21.946, p = 0.010) and N status (HR = 0.296, 95% CI: 0.091–0.961, p = 0.043) were independent prognostic factors for DFS. Conclusion CTCs can be detected and characterized in patients undergoing radical resection for non-small cell lung cancer. Their presence might be used to identify patients with increased risk of early recurrence. PMID:26913536

  13. Perioperative iloprost and endothelial progenitor cells in uremic patients with severe limb ischemia undergoing peripheral revascularization.

    Science.gov (United States)

    Coppolino, Giuseppe; Buemi, Antoine; Bolignano, Davide; Lacquaniti, Antonio; La Spada, Michele; Stilo, Francesco; De Caridi, Giovanni; Benedetto, Francesco; Loddo, Saverio; Buemi, Michele; Spinelli, Francesco

    2009-11-01

    The incidence of severe limb ischemia (SLI) is high among haemodialysis (HD) patients. Limb rescue rate after surgical revascularization is relatively poor compared with patients with normal renal function. Prostanoids are an interesting category as adjuvants to revascularization. New vessel growth develops not exclusively by proliferation of endothelial cells in vascular extremities but also by cells mobilized from the bone marrow (HSC), transformed into endothelial progenitor cells (EPC) contributing to both re-endothelialization and neovascularization. Basal number of HSC and EPC is significantly reduced in HD patients and correlated with a subsequent defective neovascularization. The aim of this study was to evaluate the effects of perioperative treatment with iloprost in uremic patients with acute ischemia of lower limbs, undergoing surgical revascularization, on endothelial progenitor cells, hypothesizing a possible biological mechanism induced by the prostanoids. A search was also made for vascular remodeling processes through the analysis of the concentrations of soluble adhesion molecules (i-CAM, v-CAM, e-selectin), biochemical markers of endothelial activation. Thirty HD patients with SLI undergoing peripheral revascularization were enrolled (15 were treated with iloprost and 15 with a placebo). Iloprost was administered as an intra-arterial bolus of 3000 ng over 1 to 3 min immediately after revascularization and in the same affected artery. Serum samples were taken before revascularization (T0), at 6 (T6) and 24 h (T24) after infusion to measure sICAM-1, sE-selectin, and sVCAM-1, and for quantification of HSC and EPC. Progenitors were identified by specific surface markers CD34+, CD133+ and VEGFR2+. Count was conducted using PROCOUNT performed in a TRUCOUNT tube and with a FACSort flow cytometer. Before revascularization, all patients showed a decreased number of HSC and EPC. After 6 h, HSC augmented significantly compared with T0 in both groups. The

  14. Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

    Directory of Open Access Journals (Sweden)

    Lijuan Han

    2016-05-01

    Full Text Available Abstract Background Somatic calreticulin (CALR, Janus kinase 2 (JAK2, and thrombopoietin receptor (MPL mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN, suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated. Methods The murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment. Results The present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation. Conclusions These

  15. Involvement of soluble Fas Ligand in germ cell apoptosis in testis of rats undergoing autoimmune orchitis.

    Science.gov (United States)

    Jacobo, Patricia Verónica; Fass, Mónica; Pérez, Cecilia Valeria; Jarazo-Dietrich, Sabrina; Lustig, Livia; Theas, María Susana

    2012-11-01

    Experimental autoimmune orchitis (EAO) is a model of chronic inflammation and infertility useful for studying immune and germ cell (GC) interactions. EAO is characterized by severe damage of seminiferous tubules (STs) with GCs that undergo apoptosis and sloughing. Based on previous results showing that Fas-Fas Ligand (L) system is one of the main mediators of apoptosis in EAO, in the present work we studied the involvement of Fas and the soluble form of FasL (sFasL) in GC death induction. EAO was induced in rats by immunization with testis homogenate and adjuvants; control (C) rats were injected with adjuvants; a group of non-immunized normal (N) rats was also studied. Activation of Fas employing an anti-Fas antibody decreased viability (trypan blue exclusion test) and induced apoptosis (TUNEL) of GCs from STs of N and EAO rats, an effect more pronounced on GCs from EAO STs. By Western blot we detected an increase in sFasL content in the testicular fluid of rats with severe EAO compared to N and C rats. By intratesticular injection of FasL conjugated to Strep-Tag molecule (FasL-Strep, BioTAGnology) and its immunofluorescent localization, we demonstrated that sFasL is able to enter the adluminal compartment of the STs. Moreover, FasL-Strep induced GC apoptosis in testicular fragments of N rats. By flow cytometry, we detected an increase in the number of membrane FasL-expressing CD4+ and CD8+ T cells in testis during EAO development but no expression of FasL by macrophages. Our results demonstrate that sFasL is locally produced in the chronically inflamed testis and that this molecule is able to enter the adluminal compartment of STs and induce apoptosis of Fas-bearing GCs.

  16. Defibrotide for the management of sinusoidal obstruction syndrome in patients who undergo haemopoietic stem cell transplantation.

    Science.gov (United States)

    Coutsouvelis, John; Avery, Sharon; Dooley, Michael; Kirkpatrick, Carl; Spencer, Andrew

    2016-11-01

    Sinusoidal obstruction syndrome, previously known as veno-occlusive disease (VOD/SOS), is a complication in patients undergoing haemopoietic stem cell transplantation (HSCT). Severe VOD/SOS, including progression to multi-organ failure, has resulted in a mortality of greater than 80%. Defibrotide's varying pharmacological actions, particularly on endothelial cells, make it is a useful agent to consider for prophylaxis and treatment of VOD/SOS. Barriers to its routine use include the high acquisition cost and the fact that neither the oral or parenteral formulations are licensed products in many countries at this time. This review summarises available literature on the use of defibrotide in the management of VOD/SOS. Publications consist predominantly of single centre cohort studies and case series. Available evidence indicates that defibrotide is effective in the management of VOD/SOS. Using defibrotide prophylaxis should also be considered, especially in the paediatric setting, where there are available results from a large, open label, randomized controlled trial. Patient outcome data from the larger studies and compassionate programs can inform consensus recommendations on dosing regimen and criteria for the treatment of VOD/SOS with defibrotide in the adult population. The reviewed literature indicates an effective and safe dose for treatment is 25mg/kg/day, continued for at least 14days or until complete response is achieved. Further studies are required to determine the optimal dose and duration of treatment in both paediatric patients and adults. Recent recommendations and a phase 3 trial using historical controls indicate that defibrotide should be included as a pharmacotherapy option in protocols guiding management of VOD/SOS.

  17. Identification of unsafe human induced pluripotent stem cell lines using a robust surrogate assay for pluripotency.

    Science.gov (United States)

    Polanco, Juan Carlos; Ho, Mirabelle S H; Wang, Bei; Zhou, Qi; Wolvetang, Ernst; Mason, Elizabeth; Wells, Christine A; Kolle, Gabriel; Grimmond, Sean M; Bertoncello, Ivan; O'Brien, Carmel; Laslett, Andrew L

    2013-08-01

    Human induced pluripotent stem cells (hiPSC) have the potential to generate healthy cells and tissues for the study and medical treatment of a large number of diseases. The utility of putative hiPSC-based therapies is constrained by a lack of robust quality-control assays that address the stability of the cells or their capacity to form teratomas after differentiation. Here we report that virally derived hiPSC, but not human embryonic stem cells (hESC) or hiPSC derived using episomal nonintegrating vectors, exhibit a propensity to revert to a pluripotent phenotype following differentiation. This instability was revealed using our published method to identify pluripotent cells undergoing very early-stage differentiation in standard hESC cultures, by fluorescence activated cell sorting (FACS) based on expression of the cell surface markers TG30 (CD9) and GCTM-2. Differentiated cells cultured post-FACS fractionation from virally derived hiPSC lines reacquired immunoreactivity to TG30 (CD9) and GCTM-2, formed stem cell-like colonies, and re-expressed canonical pluripotency markers. Furthermore, differentiated cells from pluripotency-reverting hiPSC lines generated teratomas in immunocompromised mice, raising concerns about their safety in downstream applications. In contrast, differentiated cell populations from hESC and episomally derived hiPSC did not show any of these abnormalities. Our assays may be used to identify "unsafe" hiPSC cell lines and this information should be considered when selecting hiPSC lines for clinical use and indicate that experiments using these "unsafe" hiPSC lines should be interpreted carefully.

  18. Detection of RUNX2 gene expression in cumulus cells in women undergoing controlled ovarian stimulation

    Directory of Open Access Journals (Sweden)

    Papamentzelopoulou Myrto

    2012-11-01

    Full Text Available Abstract Background RUNX2 is a transcription factor, whose expression has been recently identified in the mouse ovary. Regulation of RUNX2 expression and its function in the human ovary have not been determined yet. The aim of the present study is the investigation of the possible correlation between RUNX2 gene expression in cumulus cells and controlled ovarian stimulation and pregnancy outcomes after ART treatment. Methods A total of 41 patients undergoing ICSI treatment for male factor infertility were enrolled into a specific ART program, during which cumulus cells were collected. The expression of RUNX2 gene in cumulus cells was examined by real-time PCR. Results Concerning RUNX2 gene expression, 12 out of 41 women were detected with RUNX2 expression, with ratios ranging from 0.84 to 1.00, while 28 out of 41 women had no expression (ratio = 0. Only 1 woman presented a weak RUNX2 gene expression (ratio = 0.52. From 8 women that proceeded to pregnancy, 7 of them did not express RUNX2 gene in cumulus cells, while one was the woman with weak gene expression that also achieved pregnancy. The group of women without RUNX2 expression presented higher number of follicles (p = 0.013, higher number of retrieved oocytes (p = 0.016, higher basal LH serum levels (p = 0.016 and higher peak estradiol levels (p = 0.013, while the number of fertilized oocytes differed marginally between the two groups (p = 0.089. Moreover, RUNX2 expression was negatively associated with LH levels (OR = 0.22, p = 0.021 and E2 levels (OR = 0.25, p = 0.026. Conclusions Consequently, based on the preliminary findings of the present pilot study a potential inhibitory mechanism of RUNX2 gene is observed in the ovary when high mRNA levels are detected, suggesting that RUNX2 could possibly be used as a candidate genetic marker in the monitoring of the outcome of an ART treatment.

  19. Emp is a component of the nuclear matrix of mammalian cells and undergoes dynamic rearrangements during cell division.

    Science.gov (United States)

    Bala, Shashi; Kumar, Ajay; Soni, Shivani; Sinha, Sudha; Hanspal, Manjit

    2006-04-21

    Emp, originally detected in erythroblastic islands, is expressed in numerous cell types and tissues suggesting a functionality not limited to hematopoiesis. To study the function of Emp in non-hematopoietic cells, an epitope-tagged recombinant human Emp was expressed in HEK cells. Preliminary studies revealed that Emp partitioned into both the nuclear and Triton X-100-insoluble cytoskeletal fractions in approximately a 4:1 ratio. In this study, we report investigations of Emp in the nucleus. Sequential extractions of interphase nuclei showed that recombinant Emp was present predominantly in the nuclear matrix. Immunofluorescence microscopy showed that Emp was present in typical nuclear speckles enriched with the spliceosome assembly factor SC35 and partially co-localized with actin staining. Coimmunoprecipitation and GST-pull-down assays confirmed the apparent close association of Emp with nuclear actin. During mitosis, Emp was detected at the mitotic spindle/spindle poles, as well as in the contractile ring during cytokinesis. These results suggest that Emp undergoes dynamic rearrangements within the nuclear architecture that are correlated with cell division.

  20. Authentication of the R06E Fruit Bat Cell Line

    Directory of Open Access Journals (Sweden)

    Ingo Jordan

    2012-05-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Chang-Wen Yu; Bi-Sheng Zhu

    2016-01-01

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

  2. Cancer and inflammation studies using zebrafish cell lines

    NARCIS (Netherlands)

    He, Shuning

    2010-01-01

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

  3. Trichloroethylene toxicity in a human hepatoma cell line

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-31

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

  4. Authentication of newly established human esophageal squamous cell carcinoma cell line (YM-1) using short tandem repeat (STR) profiling method.

    Science.gov (United States)

    Ayyoob, Khosravi; Masoud, Khoshnia; Vahideh, Kazeminejad; Jahanbakhsh, Asadi

    2016-03-01

    Cross-contamination during or early after establishment of a new cell line could result in the worldwide spread of a misidentified cell line. Therefore, newly established cell lines need to be authenticated by a reference standard method. This study was conducted to investigate the authenticity of a newly established epithelial cell line of human esophageal squamous cell carcinoma (ESCC) called YM-1 using short tandem repeat (STR) DNA profiling method. Primary human ESCC epithelial cells were cultured from the fresh tumor tissue of an adult female patient. Growth characteristics and epithelial originality of YM-1 cells were studied. Genomic DNA was isolated from YM-1 cells harvested at passage 22 and ESCC donor tumor sample on two different days to prevent probable DNA contamination. STR profiling was performed using AmpFℓSTR® Identifiler® Plus PCR Amplification Kit. To address whether YM-1 cells undergo genetic alteration as the passage number increases, STR profiling was performed again on harvested cells at passage 51. YM-1 cells grew as a monolayer with a population doubling time of 40.66 h. Epithelial originality of YM-1 cells was confirmed using ICC/IF staining of cytokeratins AE1/AE3. The STR profile of the ESCC donor tumor sample was the same with YM-1 cells at passage 22. However, STR profile of the donor tumor sample showed an off-ladder (OL) allele in their D7S820 locus. Also, re-profiling of YM-1 cells at passage 51 showed a loss of heterozygosity (LOH) at D18S51 locus. This suggests that long-term culture of cell lines may alter their DNA profile. Comparison of the DNA fingerprinting results in DSMZ, and ATCC STR profiling databases confirmed unique identity of YM-1 cell line. This study provides an easy, fast, and reliable procedure for authentication of newly established cell lines, which helps in preventing the spread of misidentified cells and improving the reproducibility and validity of experiments, consequently.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-11-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  7. Extramedullary Relapse Following Total Marrow and Lymphoid Irradiation in Patients Undergoing Allogeneic Hematopoietic Cell Transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Hyun [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States); Stein, Anthony [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States); Tsai, Nicole [Department of Biostatistics, City of Hope National Medical Center, Duarte, California (United States); Schultheiss, Timothy E. [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States); Palmer, Joycelynne [Department of Biostatistics, City of Hope National Medical Center, Duarte, California (United States); Liu, An [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States); Rosenthal, Joseph [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States); Department of Pediatrics, City of Hope National Medical Center, Duarte, California (United States); Forman, Stephen J. [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States); Wong, Jeffrey Y.C., E-mail: jwong@coh.org [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States)

    2014-05-01

    Purpose: Approximately 5% to 20% of patients who undergo total body irradiation (TBI) in preparation for hematopoietic cell transplantation (HCT) can develop extramedullary (EM) relapse. Whereas total marrow and lymphoid irradiation (TMLI) provides a more conformally targeted radiation therapy for patients, organ sparing has the potential to place the patient at a higher risk for EM relapse than TBI. This study evaluated EM relapse in patients treated with TMLI at our institution. Methods and Materials: Patients eligible for analysis had been enrolled in 1 of 3 prospective TMLI trials between 2006 and 2012. The TMLI targeted bones, major lymph node chains, liver, spleen, testes, and brain, using image-guided tomotherapy with total dose ranging from 12 to 15 Gy. Results: A total of 101 patients with a median age of 47 years were studied. The median follow-up was 12.8 months. Incidence of EM relapse and bone marrow (BM) relapse were 12.9% and 25.7%, respectively. Of the 13 patients who had EM relapse, 4 also had BM relapse, and 7 had EM disease prior to HCT. There were a total of 19 EM relapse sites as the site of initial recurrence: 11 soft tissue, 6 lymph node, 2 skin. Nine of these sites were within the target region and received ≥12 Gy. Ten initial EM relapse sites were outside of the target region: 5 sites received 10.1 to 11.4 Gy while 5 sites received <10 Gy. Pretransplantation EM was the only significant predictor of subsequent EM relapse. The cumulative incidence of EM relapse was 4% at 1 year and 11.4% at 2 years. Conclusions: EM relapse incidence was as frequent in regions receiving ≥10 Gy as those receiving <10 Gy. EM relapse rates following TMLI that included HCT regimens were comparable to published results with regimens including TBI and suggest that TMLI is not associated with an increased EM relapse risk.

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

    Science.gov (United States)

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

    2000-02-01

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

  9. Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors

    Directory of Open Access Journals (Sweden)

    Clare Judith Stones

    2013-05-01

    Full Text Available The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signalling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001. IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98 with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment melanomas of with activated BRAF. The high sensitivity to trametinib of some lines with wild-type BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents.

  10. Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors.

    Science.gov (United States)

    Stones, Clare J; Kim, Ji Eun; Joseph, Wayne R; Leung, Euphemia; Marshall, Elaine S; Finlay, Graeme J; Shelling, Andrew N; Baguley, Bruce C

    2013-01-01

    The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signaling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations, and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition) for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001). IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98) with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment of melanomas with activated BRAF. The high sensitivity to trametinib of some lines with wildtype BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents.

  11. Derivation of human embryonic stem cell line Genea022

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

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

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

    Science.gov (United States)

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

    2008-03-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  15. Deriving cell lines from zebrafish embryos and tumors.

    Science.gov (United States)

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

    2013-09-01

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

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

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Khordadmehr, M.,

    2014-05-01

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

  19. KIR2DS2 and KIR2DS4 Promoter hypomethylation patterns in patients undergoing hematopoietic cell transplantation (HCT)

    OpenAIRE

    Gallez-Hawkins, Ghislaine M.; Li, Xiuli; Franck, Anne E.; Gendzekhadze, Ketevan; Nakamura, Ryotaro; Forman, Stephen J.; Senitzer, David; Zaia, John A.

    2012-01-01

    The killer cell Ig-like receptor (KIR)-MHC class I pathway is an integral part of natural killer cell immunity, and its role in host protection from both cancer and infection is important In addition, we have shown elevated KIR2DS2 and 2DS4 expression in PBMCs of patients undergoing hematopoietic cell transplantation (HCT) [1]. Since all inhibitory KIR promoters are known to be heavily methylated, the question asked here is how and when KIR2DS2 and 2DS4 promoters had changed their methylation...

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

    Science.gov (United States)

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

    2007-08-01

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

  1. 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.

  2. Functional changes of dendritic cells derived from allogeneic partial liver graft undergoing acute rejection in rats

    Institute of Scientific and Technical Information of China (English)

    Ming-Qing Xu; Zhen-Xiang Yao

    2003-01-01

    AIM: To investigate functional change of dendritic cells (DCs)derived from allogeneic partial liver graft undergoing acuterejection in rats.METHODS: Allogeneic (SD rat to LEW rat) whole and 50 %partial liver transplantation were performed. DCs from livergrafts 0 hr and 4 days after transplantation were isolated andpropagated in the presence of GM-CSFin vitro. Morphologicalcharacteristics of DCs propagated for 4 days and 10 dayswere observed by electron rmicroscopy. Phenotypical featuresof DCs propagated for 10 days were analyzed by flowcytometry. Expression of IL-12 protein and IL-12 receptormRNA in DCs propagated for 10 days was also measured byWestern blotting and semiquantitative RT-PCR, respectively.Histological grading of rejection were determined.RESULTS: Allogeneic whole liver grafts showed no featuresof rejection at day 4 after transplantation. In contrast,allogeneic partial liver grafts demonstrated moderate tosevere rejection at day 4 after transplantation. DCs derivedfrom allogeneic partial liver graft 4 days after transplantationexhibited typical morphological characteristics of DC after 4days' culture in the presence of GM-CSF. DCs from allogeneicwhole liver graft 0 hr and 4 days after transplantation didnot exhibit typical morphological characteristics of DC untilafter 10 days' culture in the presence of GM-CSF. After 10days' propagationin vitro, DCs derived from allogeneic wholeliver graft exhibited features of immature DC, with absenceof CD40, CD80 and CD86 surface expression, and low levelsof IL-12 proteins (IL-12 p35 and IL-12 p40) and IL-12receptor (IL-12Rβ1 and IL-12Rβ2) mRNA, whereas DCs fromallogeneic partial liver graft 4 days after transplantationdisplayed features of mature DC, with high levels of CD40,CD80 and CD86 surface expression, and as a consequence,higher expression of IL-12 proteins (IL-12 p35 and IL-12 p40)and IL-12 receptors (IL-12Rβ1 and IL-12Rβ2) mRNA thanthose of DCs both from partial liver graft 0 hr and whole livergraft

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

    Science.gov (United States)

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

    2013-10-01

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

  4. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  5. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

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

  8. Differential effects of bisphosphonates on breast cancer cell lines

    NARCIS (Netherlands)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2011-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

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

  11. 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

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

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

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

  13. Determinants of intrinsic radiosensitivity of mammalian cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1998-12-31

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

  14. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

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

  15. Cold storage and cryopreservation of tick cell lines

    Directory of Open Access Journals (Sweden)

    Lallinger Gertrud

    2010-04-01

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

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

    Science.gov (United States)

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

    2004-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Slavc Irene

    2004-12-01

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

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

    Science.gov (United States)

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

    2013-10-01

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

  19. In vitro radiosensitivity of human leukemia cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-05-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-01-01

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

  1. Efficacy of ribavirin against malignant glioma cell lines

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ritter Peter R

    2010-03-01

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

  3. Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant

    Directory of Open Access Journals (Sweden)

    Nicolas Lecland

    2013-01-01

    In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

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

    Science.gov (United States)

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

    2015-08-21

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

  5. Biobanking human embryonic stem cell lines

    DEFF Research Database (Denmark)

    Holm, Søren

    2016-01-01

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

  6. Characterisation of thyroid medullary carcinoma TT cell line.

    Science.gov (United States)

    Zabel, M; Grzeszkowiak, J

    1997-01-01

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

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

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

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

  8. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2008-03-01

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

  10. Transportation characteristics of nolatrexed in three tumor cell lines

    Institute of Scientific and Technical Information of China (English)

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

    2002-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Sherwood, J.B.; Shouval, D.

    1986-01-01

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

  12. Infectious complications associated with the use of central venous catheters in patients undergoing hematopoietic stem cell transplantation.

    Science.gov (United States)

    Martinho, Gláucia Helena; Romanelli, Roberta M C; Teixeira, Gustavo Machado; Macedo, Antonio V; Chaia, Juliana M C; Nobre, Vandack

    2013-07-01

    In this prospective, observational study, we sought to investigate the incidence, risk factors, and outcomes of central venous catheter-associated infection in 56 patients admitted for hematopoietic stem cell transplantation. In multivariate analysis, we found a 7-fold higher risk of central line-associated bloodstream infection with central venous catheter insertion in the internal jugular vein as compared with the subclavian access. Patients with central line-associated bloodstream infection had a higher incidence of acute renal failure.

  13. Retrotransposition-Competent Human LINE-1 Induces Apoptosis in Cancer Cells With Intact p53

    Directory of Open Access Journals (Sweden)

    Abdelali Haoudi

    2004-01-01

    Full Text Available Retrotransposition of human LINE-1 (L1 element, a major representative non-LTR retrotransposon in the human genome, is known to be a source of insertional mutagenesis. However, nothing is known about effects of L1 retrotransposition on cell growth and differentiation. To investigate the potential for such biological effects and the impact that human L1 retrotransposition has upon cancer cell growth, we examined a panel of human L1 transformed cell lines following a complete retrotransposition process. The results demonstrated that transposition of L1 leads to the activation of the p53-mediated apoptotic pathway in human cancer cells that possess a wild-type p53. In addition, we found that inactivation of p53 in cells, where L1 was undergoing retrotransposition, inhibited the induction of apoptosis. This suggests an association between active retrotransposition and a competent p53 response in which induction of apoptosis is a major outcome. These data are consistent with a model in which human retrotransposition is sensed by the cell as a “genetic damaging event” and that massive retrotransposition triggers signaling pathways resulting in apoptosis.

  14. Derivation of human embryonic stem cell line Genea019

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

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

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

    Science.gov (United States)

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

    2014-09-09

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

  16. [Prophylaxis against tuberculosis in pediatric and adult patients undergoing solid organ and hematopoietic stem cells transplantation].

    Science.gov (United States)

    Lafourcade, Mónica

    2012-09-01

    Recipients of SOT and HSCT constitute a risk group for becoming ill with tuberculosis (TB). The prevalence of active TB in patients undergoing TOS is higher than in patients undergoing HSCT, probably for the shortest period of immunosuppression of the latter. Most TB cases occur in transplant patients by reactivation of latent infection after immunosuppression, which occurs most often within the first year post-transplant, causing graft loss and in some cases death. Relevant variables to assess the risk of TB infection in a transplant recipient are the medical history of donor and recipient, images, microbiology and tuberculin tests and interferon gamma levels. PPD is routinely performed in the donor and in the recipient before transplantation. If PPD is > 5 mm in the recipient or > 10 mm in the donor, it is neccesary to exclude active TB (pulmonary and renal) (A2). It is recommended che-moprophylaxis in recipients PPD (+) and in recipients with recent seroconversion (B3), if the donor has a history of untreated TB, there was contact to someone with active TB (B3), the radiological imeges are suspect (A2) and interferon gamma release assays is (+) (B2). The selected drug is isoniazid (C3).

  17. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  18. 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.

  19. 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.

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

    Science.gov (United States)

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

    1985-01-01

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

  1. Effects of Electro-acupuncture on T Cell Subpopulations, NK Activity,Humoral Immunity and Leukocyte Count in Patients Undergoing Chemotherapy

    Institute of Scientific and Technical Information of China (English)

    Ye Fang; Liu Deshan; Wang Shuli; Xu Lan; Wang Xinzhong

    2007-01-01

    Objective: To observe the effects of electro-acupuncture on T cell subpopulations, natural killer cell (NK)activity, humoral immunity and leukocyte count in patients undergoing chemotherapy. Methods:Electro-acupuncture was added for patients undergoing chemotherapy. Tests were done on T cell subpopulations, NK activity, humoral immunity and leukocyte count before treatment and after 4 courses of treatment. Results: After 4 courses of treatment with chemotherapy and electro-acupuncture, no obvious changes were found in T cell subpopulations, NK activity, humoral immunity and leukocyte count (P > 0.05) as compared with those before treatment. Patients undergoing chemotherapy combined with electro-acupuncture showed obviously higher leukocyte count than that of the control group given no leukogenic drugs (P < 0.01). Conclusion: Electro-acupuncture may reduce immunologic damage caused by chemotherapy, thus it can be used as the auxiliary therapy for patients undergoing chemotherapy.

  2. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    Science.gov (United States)

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

    2011-01-01

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

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

    OpenAIRE

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-01-01

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

  4. Endothelial cells undergo morphological, biomechanical, and dynamic changes in response to tumor necrosis factor-α

    OpenAIRE

    Stroka, Kimberly M.; Vaitkus, Janina A.; Aranda-Espinoza, Helim

    2012-01-01

    The immune response triggers a complicated sequence of events, one of which is release of the cytokine tumor necrosis factor-α (TNF-α) from stromal cells such as monocytes and macrophages. In this work we explored the biophysical effects of TNF-α on endothelial cells (ECs), including changes in cell morphology, biomechanics, migration, and cytoskeletal dynamics. We found that TNF-α induces a wide distribution of cell area and aspect ratio, with these properties increasing on average during tr...

  5. Mechanical Stimulation in Preventing Bone Density Loss in Patients Undergoing Donor Stem Cell Transplant

    Science.gov (United States)

    2012-07-05

    Accelerated Phase Chronic Myelogenous Leukemia; Adult Acute Lymphoblastic Leukemia in Remission; Adult Acute Myeloid Leukemia in Remission; Adult Acute Myeloid Leukemia With 11q23 (MLL) Abnormalities; Adult Acute Myeloid Leukemia With Del(5q); Adult Acute Myeloid Leukemia With Inv(16)(p13;q22); Adult Acute Myeloid Leukemia With t(15;17)(q22;q12); Adult Acute Myeloid Leukemia With t(16;16)(p13;q22); Adult Acute Myeloid Leukemia With t(8;21)(q22;q22); Atypical Chronic Myeloid Leukemia, BCR-ABL1 Negative; Blastic Phase Chronic Myelogenous Leukemia; Chronic Eosinophilic Leukemia; Chronic Myelomonocytic Leukemia; Chronic Neutrophilic Leukemia; Chronic Phase Chronic Myelogenous Leukemia; de Novo Myelodysplastic Syndromes; Disseminated Neuroblastoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable; Nodal Marginal Zone B-cell Lymphoma; Noncontiguous Stage II Adult Burkitt Lymphoma; Noncontiguous Stage II Adult Diffuse Large Cell Lymphoma; Noncontiguous Stage II Adult Diffuse Mixed Cell Lymphoma; Noncontiguous Stage II Adult Diffuse Small Cleaved Cell Lymphoma; Noncontiguous Stage II Adult Immunoblastic Large Cell Lymphoma; Noncontiguous Stage II Adult Lymphoblastic Lymphoma; Noncontiguous Stage II Grade 1 Follicular Lymphoma; Noncontiguous Stage II Grade 2 Follicular Lymphoma; Noncontiguous Stage II Grade 3 Follicular Lymphoma; Noncontiguous Stage II Mantle Cell Lymphoma; Noncontiguous Stage II Marginal Zone Lymphoma; Noncontiguous Stage II Small Lymphocytic Lymphoma; Plasma Cell Neoplasm; Poor Prognosis Metastatic Gestational Trophoblastic Tumor; Previously Treated Myelodysplastic Syndromes; Primary Myelofibrosis; Prolymphocytic Leukemia; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Acute Myeloid Leukemia; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved

  6. Skin Biopsy and Patient-Specific Stem Cell Lines

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2016-05-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-09-01

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

  10. Judging diatoms by their cover: variability in local elasticity of Lithodesmium undulatum undergoing cell division.

    Directory of Open Access Journals (Sweden)

    Lee Karp-Boss

    Full Text Available Unique features of diatoms are their intricate cell covers (frustules made out of hydrated, amorphous silica. The frustule defines and maintains cell shape and protects cells against grazers and pathogens, yet it must allow for cell expansion during growth and division. Other siliceous structures have also evolved in some chain-forming species as means for holding neighboring cells together. Characterization and quantification of mechanical properties of these structures are crucial for the understanding of the relationship between form and function in diatoms, but thus far only a handful of studies have addressed this issue. We conducted micro-indentation experiments, using atomic force microscopy (AFM, to examine local variations in elastic (Young's moduli of cells and linking structures in the marine, chain-forming diatom Lithodesmium undulatum. Using a fluorescent tracer that is incorporated into new cell wall components we tested the hypothesis that new siliceous structures differ in elastic modulus from their older counterparts. Results show that the local elastic modulus is a highly dynamic property. Elastic modulus of stained regions was significantly lower than that of unstained regions, suggesting that newly formed cell wall components are generally softer than the ones inherited from the parent cells. This study provides the first evidence of differentiation in local elastic properties in the course of the cell cycle. Hardening of newly formed regions may involve incorporation of additional, possibly organic, material but further studies are needed to elucidate the processes that regulate mechanical properties of the frustule during the cell cycle.

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

    Science.gov (United States)

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

    2012-01-01

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

  12. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

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

  14. Ionizing radiation predisposes non-malignant human mammaryepithelial cells to undergo TGF beta-induced epithelial to mesenchymaltransition

    Energy Technology Data Exchange (ETDEWEB)

    Andarawewa, Kumari L.; Erickson, Anna C.; Chou, William S.; Costes, Sylvain; Gascard, Philippe; Mott, Joni D.; Bissell, Mina J.; Barcellos-Hoff, Mary Helen

    2007-04-06

    Transforming growth factor {beta}1 (TGF{beta}) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGF{beta}, activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGF{beta}-mediated epithelial to mesenchymal transition (EMT). Non-malignant HMEC (MCF10A, HMT3522 S1 and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture, or treated with a low concentration of TGF{beta} (0.4 ng/ml), or double-treated. All double-treated (IR+TGF{beta}) HMEC underwent a morphological shift from cuboidal to spindle-shaped. This phenotype was accompanied by decreased expression of epithelial markers E-cadherin, {beta}-catenin and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin and vimentin. Furthermore, double-treatment increased cell motility, promoted invasion and disrupted acinar morphogenesis of cells subsequently plated in Matrigel{trademark}. Neither radiation nor TGF{beta} alone elicited EMT, even though IR increased chronic TGF{beta} signaling and activity. Gene expression profiling revealed that double treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes non-malignant HMEC to undergo TGF{beta}-mediated EMT. Consistent with this, Erk phosphorylation were transiently induced by irradiation, persisted in irradiated cells treated with TGF{beta}, and treatment with U0126, a Mek inhibitor, blocked the EMT phenotype. Together, these data demonstrate that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression.

  15. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

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

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

    Science.gov (United States)

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

    2005-01-01

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

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Ismail Noorliza M

    2010-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Valentina Pozzi

    2015-05-01

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

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

    Science.gov (United States)

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

    2009-12-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-12-01

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

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

    Science.gov (United States)

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

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

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

    Science.gov (United States)

    Krampe, Britta; Al-Rubeai, Mohamed

    2010-07-01

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

  4. Human B cells have an active phagocytic capability and undergo immune activation upon phagocytosis of Mycobacterium tuberculosis.

    Science.gov (United States)

    Zhu, Qi; Zhang, Min; Shi, Ming; Liu, Yang; Zhao, Qing; Wang, Wenjing; Zhang, Guangyun; Yang, Longxiu; Zhi, Jin; Zhang, Lin; Hu, Gengyao; Chen, Pin; Yang, Yining; Dai, Wen; Liu, Tingting; He, Ying; Feng, Guodong; Zhao, Gang

    2016-04-01

    The paradigm that B cells are nonphagocytic was taken for granted for a long time until phagocytic B cells were found in early vertebrate animals. Thereafter, limited evidence has shown that human B cells may also internalize bacteria. However, whether human B cells can actively phagocytose bacteria has been less extensively investigated; in particular, the mechanisms and significance of the phagocytosis require clarification. Here, we show that the human Raji B cell line can phagocytose both live and dead Mycobacterium tuberculosis (Mtb), and the phagocytosed Mtb in turn affects the immune functions of the B cells. After incubation of Raji cells with Mtb, our confocal microscopy, electron microscopy and flow cytometry data showed that Raji cells effectively engulfed Mtb as well as latex beads. The phagocytic rate was proportional to the incubation time and the amount of Mtb or beads added. Additionally, we found that normal human serum could enhance the ability of Raji cells to phagocytose Mtb, while heat-inactivated serum reversed this promoting effect. The phagocytic process of B cells could partially be inhibited by cytochalasin B, an actin inhibitor. Importantly, the phagocytosed Mtb could regulate B cell immune functions, such as stimulating IgM production and upregulating the expression of the antigen-presenting costimulatory molecules CD80 and CD86. Therefore, our results provide the first evidence that human B cells can phagocytose Mtb in an active manner that is independent of bacterial viability, and phagocytosed Mtb can in turn regulate the immune activation of B cells.

  5. Oral lorazepam prevents seizure during high-dose busulfan in children undergoing hematopoietic stem cell transplantation: a prospective study.

    Science.gov (United States)

    Hamidieh, Amir Ali; Hamedani, Ravak; Hadjibabaie, Molouk; Amini, Mohsen; Sadrai, Sima; Ghavamzadeh, Ardeshir

    2010-10-01

    High-dose Busulfan in combination chemotherapy has been used commonly for hematopoietic stem cell transplantation. It crosses the blood-brain barrier and could cause seizure. Benzodiazepines have been used as anticonvulsant prophylaxis. This is a prospective study using oral lorazepam together with busulfan-based conditioning regimen in 30 children undergoing hematopoietic stem cell transplantation. The dose of lorazepam used ranged from 0.017 to 0.039 mg/kg (median = 0.026 mg/kg) per dose. None of the patients developed seizure while receiving oral lorazepam or within 72 hours of the last dose of Busulfan. Oral lorazepam was tolerated by the patients, but all patients needed dose reduction due to some adverse effects. In the authors' experience, oral lorazepam is a useful anticonvulsant prophylaxis for children receiving high-dose busulfan.

  6. Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines

    Directory of Open Access Journals (Sweden)

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

    2011-01-01

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

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

    CERN Document Server

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

    2015-01-01

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

  8. LRP5 Signaling in Osteosarcomagenesis: a Cautionary Tale of Translation from Cell Lines to Tumors

    Directory of Open Access Journals (Sweden)

    Logan Horne

    2016-10-01

    Full Text Available Previous reports document expression of low-density lipoprotein receptor-related protein 5 (LRP5 in osteosarcoma (OS tissue. Expression of this Wnt receptor correlated with metastatic disease and poor disease-free survival. Forced expression of dominant-negative LRP5 (dnLRP5, which lacks the membrane binding domain of the native protein and therefore functions as a soluble receptor-sponge for Wnt ligands, reduced in vitro cellular invasion and in vivo xenograft tumor growth for osteosarcoma cell lines. Here, we use a genetically engineered mouse model of osteosarcomagenesis with and without expression of dnLRP5 to assess to what degree tumorigenesis is affected and whether Wnt/β-catenin signaling is circumvented or maintained. Each cohort of mice developed osteosarcoma at a similar ultimate prevalence, but after a slightly increased latency in those also expressing dnLRP5. On histology, there was no difference between groups, despite previous reports that the dnLRP5 osteosarcoma cells specifically undergo a mesenchymal-to-epithelial transition in vitro. Finally, immunohistochemistry showed the presence of cytosolic and nuclear β-catenin and nuclear Cyclin D1, markers consistent with preserved Wnt/β-catenin signaling despite constitutive blockade of the cell surface receipt of Wnt signaling ligand. These data suggest that canonical Wnt signaling plays a role in OS progression and that while blockade of singular nodes in signaling pathways can have dramatic effects on individual cell lines, real tumors readily evade such focused attacks.

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

    Science.gov (United States)

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

    2012-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-01

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

  11. Auditory hair cell centrioles undergo confined Brownian motion throughout the developmental migration of the kinocilium.

    OpenAIRE

    Lepelletier, Léa; de Monvel, Jacques Boutet; Buisson, Johanna; Desdouets, Chantal; Petit, Christine

    2013-01-01

    Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements o...

  12. Comparative expression profiling of distinct T cell subsets undergoing oxidative stress.

    Directory of Open Access Journals (Sweden)

    Rudolf Lichtenfels

    Full Text Available The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA(+ and their memory/effector CD45RO(+ T cell counterparts in the presence and absence of low dose hydrogen peroxide (H(2O(2 was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA(+ and CD45RO(+ T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies

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

    Science.gov (United States)

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

  14. Fission yeast cells undergo nuclear division in the absence of spindle microtubules.

    Directory of Open Access Journals (Sweden)

    Stefania Castagnetti

    Full Text Available Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.

  15. Surprisal analysis characterizes the free energy time course of cancer cells undergoing epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Zadran, Sohila; Arumugam, Rameshkumar; Herschman, Harvey; Phelps, Michael E; Levine, R D

    2014-09-09

    The epithelial-to-mesenchymal transition (EMT) initiates the invasive and metastatic behavior of many epithelial cancers. Mechanisms underlying EMT are not fully known. Surprisal analysis of mRNA time course data from lung and pancreatic cancer cells stimulated to undergo TGF-β1-induced EMT identifies two phenotypes. Examination of the time course for these phenotypes reveals that EMT reprogramming is a multistep process characterized by initiation, maturation, and stabilization stages that correlate with changes in cell metabolism. Surprisal analysis characterizes the free energy time course of the expression levels throughout the transition in terms of two state variables. The landscape of the free energy changes during the EMT for the lung cancer cells shows a stable intermediate state. Existing data suggest this is the previously proposed maturation stage. Using a single-cell ATP assay, we demonstrate that the TGF-β1-induced EMT for lung cancer cells, particularly during the maturation stage, coincides with a metabolic shift resulting in increased cytosolic ATP levels. Surprisal analysis also characterizes the absolute expression levels of the mRNAs and thereby examines the homeostasis of the transcription system during EMT.

  16. Third-line chemotherapy for small cell lung cancer

    NARCIS (Netherlands)

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

    2006-01-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    RUANBo; ZHAOJunguo; 等

    1999-01-01

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

  19. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

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

  20. Crude subcellular fractionation of cultured mammalian cell lines

    Directory of Open Access Journals (Sweden)

    Holden Paul

    2009-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Chen Lei

    2011-06-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

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

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

    Science.gov (United States)

    McGuigan, Claire F; Li, Xing-Fang

    2014-06-01

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

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

    Science.gov (United States)

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

    2003-02-01

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

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

    Science.gov (United States)

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

    2000-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  8. Centrosomal dysregulation in human metastatic melanoma cell lines.

    Science.gov (United States)

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

    2011-09-01

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

  9. 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.

  10. Differences in radiosensitivity between three HER2 overexpressing cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-06-15

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

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

    Science.gov (United States)

    Bradbury, Andrew M.

    2012-07-24

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

  13. Optical scatter imaging as an apoptosis assay for cells undergoing ALA-mediated photodynamic therapy in vitro

    Science.gov (United States)

    Rossi, Vincent M.; Baugher, Paige J.; Jacques, Steven L.

    2013-02-01

    Aminolevulinic acid (ALA) is converted to protoporphyrin-IX (PpIX) within mitochondria, causing the assumption that ALA-mediated photodynamic therapy (PDT) results in mitochondrial damage and therefore an apoptotic response. Mitochondria within apoptosing cells swell, forming pores in their outer mitochondrial membranes which release cytochrome-c, triggering apoptosis. Optical scatter imaging (OSI) makes use of scattered fields in order to indicate the morphology of subcellular components, and is used here in order to measure changes in mitochondrial size as a response to ALA-mediated PDT. Two images of the same field of view are spatially filtered in the Fourier plane of a 4-F system. Both spatial filters block directly transmitted light, while accepting different angles of scattered light through an adjustable iris. The optical scatter image ratio (OSIR) of the local intensities of these two spatially filtered images is indicative of scattering particle size. Mie theory is used to calculate the predicted OSIR as a function of scattering particle size. In this fashion, the OSI system is calibrated using polystyrene microspheres of know sizes. Comparison of the measured OSIR from cellular images to theoretical values predicted for mitochondria then serves as an indication as to whether cells are apoptosing. Cells are treated at varying concentrations of ALA and varying exposures of 635 nm light and imaged at varying time points in order to develop a broader understanding of an apoptotic response of cells undergoing ALA mediated PDT.

  14. Cochlear outer hair cells undergo an apical circumference remodeling constrained by the hair bundle shape.

    Science.gov (United States)

    Etournay, Raphaël; Lepelletier, Léa; Boutet de Monvel, Jacques; Michel, Vincent; Cayet, Nadège; Leibovici, Michel; Weil, Dominique; Foucher, Isabelle; Hardelin, Jean-Pierre; Petit, Christine

    2010-04-01

    Epithelial cells acquire diverse shapes relating to their different functions. This is particularly relevant for the cochlear outer hair cells (OHCs), whose apical and basolateral shapes accommodate the functioning of these cells as mechano-electrical and electromechanical transducers, respectively. We uncovered a circumferential shape transition of the apical junctional complex (AJC) of OHCs, which occurs during the early postnatal period in the mouse, prior to hearing onset. Geometric analysis of the OHC apical circumference using immunostaining of the AJC protein ZO1 and Fourier-interpolated contour detection characterizes this transition as a switch from a rounded-hexagon to a non-convex circumference delineating two lateral lobes at the neural side of the cell, with a negative curvature in between. This shape tightly correlates with the 'V'-configuration of the OHC hair bundle, the apical mechanosensitive organelle that converts sound-evoked vibrations into variations in cell membrane potential. The OHC apical circumference remodeling failed or was incomplete in all the mouse mutants affected in hair bundle morphogenesis that we tested. During the normal shape transition, myosin VIIa and myosin II (A and B isoforms) displayed polarized redistributions into and out of the developing lobes, respectively, while Shroom2 and F-actin transiently accumulated in the lobes. Defects in these redistributions were observed in the mutants, paralleling their apical circumference abnormalities. Our results point to a pivotal role for actomyosin cytoskeleton tensions in the reshaping of the OHC apical circumference. We propose that this remodeling contributes to optimize the mechanical coupling between the basal and apical poles of mature OHCs.

  15. Characterization of the estrogen receptor transfected MCF10A breast cell line 139B6.

    Science.gov (United States)

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

    1996-01-01

    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.

  16. The fog-3 gene and regulation of cell fate in the germ line of Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Ellis, R.; Kimble, J. [Univ. of Wisconsin, Madison, WI (United States)

    1995-02-01

    In the nematode Caenorhabditis elegans, germ cells normally adopt one of three fates: mitosis, spermatogenesis or oogenesis. We have identified and characterized the gene fog-3, which is required for germ cells to differentiate as sperm rather than as oocytes. Analysis of double mutants suggests that fog-3 is absolutely required for spermatogenesis and acts at the end of the regulatory hierarchy controlling sex determination for the germ line. By contrast, mutations in fog-3 do not alter the sexual identity of other tissues. We also have characterized the null phenotype of fog-1, another gene required for spermatogenesis; we demonstrate that it too controls the sexual identity of germ cells but not of other tissues. Finally, we have studied the same interaction of these two fog genes with gld-1, a gene required for germ cells to undergo oogenesis rather than mitosis. On the basis of these results, we propose that germ-cell fate might be controlled by a set of inhibitory interactions among genes that specify one of three fates: mitosis, spermatogenesis or oogenesis. Such a regulatory network would link the adoption of one germ-cell fate to the suppression of the other two. 68 refs., 7 figs., 6 tabs.

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

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

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

  18. Arecoline induces HA22T/VGH hepatoma cells to undergo anoikis - involvement of STAT3 and RhoA activation

    Directory of Open Access Journals (Sweden)

    Hung Thu-Ching

    2010-05-01

    Full Text Available Abstract Background Our previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6, increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism. Methods HA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface β1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3 pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined. Results A low concentration of arecoline (≤ 100 μg/ml caused cytoskeletal changes in HA22T/VGH cells, but not hepatocytes, and this was accompanied by decreased β1-integrin expression and followed by apoptosis, indicating that HA22T/VGH cells undergo anoikis after arecoline treatment. IL-6 expression and phosphorylation of STAT3, which provides protection against anoikis, were inhibited and levels of downstream signaling proteins, including Bcl-XL and Bcl-2, were decreased, while Bax expression, mitochondrial cytochrome c release, and caspase-3 activity were increased. In addition, phosphorylation/activation of p190RhoGAP, a RhoA inhibitor, and of its upstream regulator, SHP2, was inhibited by arecoline treatment, while Rho/Rock activation was increased. Addition of the RhoA inhibitor attenuated the effects of arecoline. Conclusions This study demonstrated that arecoline induces anoikis of HA22T/VGH cells involving inhibition of STAT3 and increased Rho

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

    Directory of Open Access Journals (Sweden)

    Michael Iacovos P

    2012-12-01

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

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

    Science.gov (United States)

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

    2016-02-03

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2016-01-01

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

  2. Evaluation of Quality of Life and Care Needs of Turkish Patients Undergoing Hematopoietic Stem Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Neslisah Yasar

    2016-01-01

    Full Text Available This descriptive study explored the quality of life and care needs of Turkish patients who underwent hematopoietic stem cell transplantation. The study sample consisted of 100 hematopoietic stem cell transplant patients. Their quality of life was assessed using Functional Assessment of Cancer Therapy-Bone Marrow Transplant Scale. The mean patient age was 44.99 ± 13.92 years. Changes in sexual functions, loss of hair, loss of taste, loss of appetite, and sleep disturbances were the most common symptoms. The quality of life of transplant patients was moderately affected; the functional well-being and social/family well-being subscales were the most adversely and least negatively affected (12.13 ± 6.88 dimensions, respectively. Being female, being between 50 and 59 years of age, being single, having a chronic disease, and having a history of hospitalization were associated with lower quality of life scores. Interventions to improve functional status, physical well-being, and emotional status of patients during the transplantation process may help patients cope with treatment-related impairments more effectively. Frequent screening and management of patient symptoms in order to help patients adapt to life following allogeneic hematopoietic stem cell transplantation are crucial for meeting care needs and developing strategies to improve their quality of life.

  3. Evaluation of Quality of Life and Care Needs of Turkish Patients Undergoing Hematopoietic Stem Cell Transplantation

    Science.gov (United States)

    Yasar, Neslisah

    2016-01-01

    This descriptive study explored the quality of life and care needs of Turkish patients who underwent hematopoietic stem cell transplantation. The study sample consisted of 100 hematopoietic stem cell transplant patients. Their quality of life was assessed using Functional Assessment of Cancer Therapy-Bone Marrow Transplant Scale. The mean patient age was 44.99 ± 13.92 years. Changes in sexual functions, loss of hair, loss of taste, loss of appetite, and sleep disturbances were the most common symptoms. The quality of life of transplant patients was moderately affected; the functional well-being and social/family well-being subscales were the most adversely and least negatively affected (12.13 ± 6.88) dimensions, respectively. Being female, being between 50 and 59 years of age, being single, having a chronic disease, and having a history of hospitalization were associated with lower quality of life scores. Interventions to improve functional status, physical well-being, and emotional status of patients during the transplantation process may help patients cope with treatment-related impairments more effectively. Frequent screening and management of patient symptoms in order to help patients adapt to life following allogeneic hematopoietic stem cell transplantation are crucial for meeting care needs and developing strategies to improve their quality of life. PMID:28116155

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

    Directory of Open Access Journals (Sweden)

    N. Rahimi-Fard

    2006-06-01

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

  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

    1999-01-01

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

  6. Rituximab in Treating Patients Undergoing Donor Peripheral Blood Stem Cell Transplant for Relapsed or Refractory B-cell Lymphoma

    Science.gov (United States)

    2015-11-23

    B-cell Adult Acute Lymphoblastic Leukemia; B-cell Childhood Acute Lymphoblastic Leukemia; B-cell Chronic Lymphocytic Leukemia; Childhood Burkitt Lymphoma; Childhood Diffuse Large Cell Lymphoma; Childhood Immunoblastic Large Cell Lymphoma; Cutaneous B-cell Non-Hodgkin Lymphoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Intraocular Lymphoma; Nodal Marginal Zone B-cell Lymphoma; Post-transplant Lymphoproliferative Disorder; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Childhood Acute Lymphoblastic Leukemia; Recurrent Childhood Grade III Lymphomatoid Granulomatosis; Recurrent Childhood Large Cell Lymphoma; Recurrent Childhood Lymphoblastic Lymphoma; Recurrent Childhood Small Noncleaved Cell Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Small Lymphocytic Lymphoma; Recurrent/Refractory Childhood Hodgkin Lymphoma; Refractory Chronic Lymphocytic Leukemia; Refractory Hairy Cell Leukemia; Small Intestine Lymphoma; Splenic Marginal Zone Lymphoma; Testicular Lymphoma; Waldenström Macroglobulinemia

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

    Science.gov (United States)

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

  8. Cytolytic replication of echoviruses in colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Gullberg Maria

    2011-10-01

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

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

    DEFF Research Database (Denmark)

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

    1985-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

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

  11. Icing oral mucositis: Oral cryotherapy in multiple myeloma patients undergoing autologous hematopoietic stem cell transplant.

    Science.gov (United States)

    Chen, Joey; Seabrook, Jamie; Fulford, Adrienne; Rajakumar, Irina

    2017-03-01

    Background Up to 70% of patients receiving hematopoietic stem cell transplant develop oral mucositis as a side effect of high-dose melphalan conditioning chemotherapy. Oral cryotherapy has been documented to be potentially effective in reducing oral mucositis. The aim of this study was to examine the effectiveness of the cryotherapy protocol implemented within the hematopoietic stem cell transplant program. Methods A retrospective chart review was conducted of adult multiple myeloma patients who received high-dose melphalan conditioning therapy for autologous hematopoietic stem cell transplant. Primary endpoints were incidence and severity of oral mucositis. Secondary endpoints included duration of oral mucositis, duration of hospital stay, parenteral narcotics use and total parenteral nutrition use. Results One hundred and forty patients were included in the study, 70 patients in both no cryotherapy and cryotherapy groups. Both oral mucositis incidence and severity were found to be significantly lower in the cryotherapy group. Fifty (71.4%) experienced mucositis post cryotherapy compared to 67 (95.7%) in the no cryotherapy group (p < 0.001). The median oral mucositis severity, assessed using the WHO oral toxicity scale from grade 0-4, experienced in the no group was 2.5 vs. 2 in the cryotherapy group (p = 0.03). Oral mucositis duration and use of parenteral narcotics were also significantly reduced. Duration of hospital stay and use of parenteral nutrition were similar between the two groups. Conclusion The cryotherapy protocol resulted in a significantly lower incidence and severity of oral mucositis. These results provide evidence for the continued use of oral cryotherapy, an inexpensive and generally well-tolerated practice.

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

    Science.gov (United States)

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

    1995-12-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  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

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

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

  16. Ni(II) and Cu(II) N(4)-ethylmorpholine citronellalthiosemicarbazonate: a comparative analysis of cytotoxic effects in malignant human cancer cell lines.

    Science.gov (United States)

    Bisceglie, Franco; Alinovi, Rossella; Pinelli, Silvana; Goldoni, Matteo; Buschini, Annamaria; Franzoni, Susanna; Mutti, Antonio; Tarasconi, Pieralberto; Pelosi, Giorgio

    2013-11-01

    In this paper we report a study conducted with two analogous complexes, bis(N(4)-ethylmorpholine citronellalthiosemicarbazonate) nickel(II) and -copper(II) on four tumour cell lines (U937, HL60, SK-N-MC and HT29). All cell lines appear to be sensitive to both metal complexes, but while in U937, HL60 and SK-N-MC, apoptosis is the main mode through which cell death occurs, HT29 cells undergo necrosis. Among the cell lines which undergo apoptosis, SK-N-MC response is characterized by the intrinsic pathway, whereas U937 and HL60 involve both the intrinsic and the extrinsic pathways. The redox activity of the two complexes provides experimental evidence that they can modulate reactive oxygen species (ROS) production as a function of both the metal and the cell line used. Among the four cell lines, HL60 does not seem to give a significant response to exposure to both compounds. In the case of the nickel derivative, ROS generation is a relatively early event, and ROS could be the mediator leading to cellular damage. HT29 shows a remarkable and rapid ROS increase and a significant induction of membrane peroxidation that could be correlated to the onset of necrosis.

  17. PROPHYLACTIC ADMINISTRATION OF DOXYCYCLINE REDUCES CENTRAL VENOUS CATHETER INFECTIONS IN PATIENTS UNDERGOING HEMATOPOIETIC CELL TRANSPLANTATION

    Directory of Open Access Journals (Sweden)

    Mohamed Kharfan-Dabaja

    2013-02-01

    Full Text Available Hematopoietic stem cells are usually transfused through a central venous catheter (CVC, which also facilitates administration of medications and intravenous fluids. We had observed high rate of catheter-related blood-stream infection (CR-BSI at our Bone Marrow Transplantation (BMT unit despite prescribing fluoroquinolones for anti-bacterial prophylaxis. Accordingly, we implemented prophylactic use of a relatively inexpensive broad spectrum antibiotic, namely doxycycline to address this problem. We wanted to investigate whether doxycycline prophylaxis reduces CR-BSI rate. Data was collected retrospectively on 54 consecutive patients, 26 of whom received doxycycline (doxycycline group, and we compared their outcomes to a previous cohort of 28 patients who did not receive doxycycline (comparison group. The groups were comparable in regards to age, gender, hematopoietic cell transplant type, and primary diagnosis. No CVC infection (0% was observed in the doxycycline group, while 5 infection episodes (18.5% occurred in 4 patients in the comparison group (p<0.001. Episodes of CR-BSI were due to: Escherichia-coli (EC=1, coagulase-negative Staphylococcus-spp (CNSS=2, both EC & CNSS=1. Our results demonstrate that CR-BSI was reduced significantly after introducing doxycycline. This finding suggests a beneficial role for systemic use of doxycycline prophylaxis to prevent CR-BSI in adult BMT patients. Nevertheless, a randomized controlled study is warranted to confirm these findings.

  18. Features of transfusion therapy in patients undergoing hematopoietic stem cell transplantation. Review of the literature

    Directory of Open Access Journals (Sweden)

    D. N. Balashov

    2013-01-01

    Full Text Available The indications for transfusion of blood components support after stem cell transplantation (SCT usually do not differ form other clinical situations, but the rules for such therapy have a number of features. One of them is the possibility of inconsistence of AB0 group between donor and recipient of hematopoietic stem cells, which is not only fraught with the development of various alloimmune complications, but also fundamentally changes the standards for the selection of blood components for transfusion. A major problem after HSCT is a secondary immunodeficiency, which is important to consider for ensuring prevention of transfusion-transmitted infections (eg, CMV, as well as to carry out activities aimed for the prevention of transfusion- associated graft-versus-host disease. HSCT is a medical technology today, the effectiveness of which is often dependent on the accuracy and integrity of its implementation. So, serious attitude to various supportive therapy, including transfusions of blood components is an important component which determines the success of the treatment.

  19. Features of transfusion therapy in patients undergoing hematopoietic stem cell transplantation. Review of the literature

    Directory of Open Access Journals (Sweden)

    D. N. Balashov

    2014-07-01

    Full Text Available The indications for transfusion of blood components support after stem cell transplantation (SCT usually do not differ form other clinical situations, but the rules for such therapy have a number of features. One of them is the possibility of inconsistence of AB0 group between donor and recipient of hematopoietic stem cells, which is not only fraught with the development of various alloimmune complications, but also fundamentally changes the standards for the selection of blood components for transfusion. A major problem after HSCT is a secondary immunodeficiency, which is important to consider for ensuring prevention of transfusion-transmitted infections (eg, CMV, as well as to carry out activities aimed for the prevention of transfusion- associated graft-versus-host disease. HSCT is a medical technology today, the effectiveness of which is often dependent on the accuracy and integrity of its implementation. So, serious attitude to various supportive therapy, including transfusions of blood components is an important component which determines the success of the treatment.

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

    Institute of Scientific and Technical Information of China (English)

    李虹; 肖丽英; 李华林; 李婉宜; 蒋中华; 张林; 李明远

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ogura,Hajime

    1978-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Edyta Biskup

    2010-05-01

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

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

    Science.gov (United States)

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

    2007-09-01

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

  4. Statin use is associated with improved survival in patients undergoing surgery for renal cell carcinoma

    DEFF Research Database (Denmark)

    Kaffenberger, Samuel D; Lin-Tsai, Opal; Stratton, Kelly L

    2015-01-01

    ) and disease-specific survival (DSS). Univariable survival analyses were performed using the Kaplan-Meier and the log-rank methods. Multivariable analysis was performed using a Cox proportional hazards model. The predictive discrimination of the models was assessed using the Harrell c-index. RESULTS......: 0.43-0.90; P = 0.011) and DSS (hazard ratio = 0.48; 95% CI: 0.28-0.83; P = 0.009). The multivariable model for DSS had excellent predictive discrimination with a c-index of 0.91. CONCLUSIONS: These data suggest that statin usage at time of surgery is independently associated with improved OS and DSS......PURPOSE: To determine whether statin use at time of surgery is associated with survival following nephrectomy or partial nephrectomy for renal cell carcinoma (RCC). Statins are thought to exhibit a protective effect on cancer incidence and possibly cancer survival in a number of malignancies...

  5. Safety and efficacy of resistance training in germ cell cancer patients undergoing chemotherapy

    DEFF Research Database (Denmark)

    Christensen, Jesper Frank; Jones, L W; Tolver, Anders

    2014-01-01

    Abstract Background: Bleomycin–etoposid–cisplatin (BEP) chemotherapy is curative in most patients with disseminated germ cell cancer (GCC) but also associated with toxic actions and dysfunction in non-targeted tissues. We investigated changes in muscle function during BEP and the safety...... and efficacy of resistance training to modulate these changes. Methods: Thirty GCC patients were randomly assigned to resistance training (resistance training group (INT), n=15) or usual care (CON, n=15) during 9 weeks of BEP therapy. Resistance training consisted of thrice weekly sessions of four exercises, 3...... changes compared with the INT-group (PBEP was associated with significant reduction in lean mass and strength and trends toward unfavourable changes in muscle fibre size and phenotype composition. Resistance training was safe and attenuated dysfunction in selected endpoints, but BEP...

  6. Circulating tumour cells in patients with lung cancer undergoing endobronchial cryotherapy.

    Science.gov (United States)

    Chudasama, Dimple; Rice, Alexandra; Soppa, Gopal; Anikin, Vladimir

    2015-08-01

    Early diagnosis of lung cancer still poses a major issue, with a large proportion of patients diagnosed at late stages. Therapeutic options and treatment remain limited in these patients. In most cases only palliative therapies are available to alleviate any severe symptoms. Endobronchial cryotherapy (EC) is one form of palliative treatment offered to patients with obstructive airway tumours. Although successful, the impact on circulating tumour cell (CTCs) spread has not been investigated in detail. This study recruited 20 patients awaiting EC treatment. Baseline and post EC blood samples were analysed for presence of CTCs. Results showed an increase in CTCs following EC in 75% of patients. Significant increases were noticeable in some cases. Although EC is a well-accepted modality of treatment to alleviate symptoms, it may lead to an increase in CTCs, which in turn may have implications for tumour dissemination and metastatic spread.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2008-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Amanda Simcox

    2008-08-01

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

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

    Science.gov (United States)

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

    1999-07-01

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

  11. Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells.

    Directory of Open Access Journals (Sweden)

    Takaharu Okada

    2005-06-01

    Full Text Available Interactions between B and T cells are essential for most antibody responses, but the dynamics of these interactions are poorly understood. By two-photon microscopy of intact lymph nodes, we show that upon exposure to antigen, B cells migrate with directional preference toward the B-zone-T-zone boundary in a CCR7-dependent manner, through a region that exhibits a CCR7-ligand gradient. Initially the B cells show reduced motility, but after 1 d, motility is increased to approximately 9 microm/min. Antigen-engaged B cells pair with antigen-specific helper T cells for 10 to more than 60 min, whereas non-antigen-specific interactions last less than 10 min. B cell-T cell conjugates are highly dynamic and migrate extensively, being led by B cells. B cells occasionally contact more than one T cell, whereas T cells are strictly monogamous in their interactions. These findings provide evidence of lymphocyte chemotaxis in vivo, and they begin to define the spatiotemporal cellular dynamics associated with T cell-dependent antibody responses.

  12. Myeloma clonotypic B cells are hampered in their ability to undergo B-cell differentiation in vitro

    NARCIS (Netherlands)

    Guikema, JEJ; Vellenga, E; Bakkus, MHC; Bos, NA

    2002-01-01

    In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present that express the identical V( D) J rearrangements as the malignant plasma cells in the bone marrow. In the present study, the proliferative capacity of clonotypic B cells from MM patients (n = 10) and the

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  14. Advances in conditioning regimens for older adults undergoing allogeneic stem cell transplantation to treat hematologic malignancies.

    Science.gov (United States)

    William, Basem M; de Lima, Marcos

    2013-06-01

    Allogeneic stem cell transplantation (SCT) is a potentially curative treatment for patients with hematological malignancies. These diseases, however, have their peak incidence in the sixth to eighth decades of life. Historically, elderly patients have been considered unsuitable candidates for SCT because of high treatment-related mortality (TRM). Over the past 15 years, the use of reduced-intensity conditioning (RIC) regimens before SCT has allowed patients in the sixth and seventh decades of life to be routinely transplanted. Despite major differences among transplant centers in the intensity and composition of the conditioning regimen and immunosuppression, choice of graft source, postgraft immunomodulation, and supportive care, there has been a dramatic decrease in TRM, allowing safer delivery of SCT. Major obstacles to SCT in elderly patients include donor availability, graft-versus-host disease, delayed immune recovery, multiple comorbidities, and chemo refractoriness. Here we review the current results of SCT in elderly patients, focusing on the role of RIC, and using myeloid diseases as the model for discussion.

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

    Science.gov (United States)

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

    2013-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Mozhgan Sedigh-Ardekani

    2013-03-01

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

  17. Off-line test of the KISS gas cell

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-12-15

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

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  20. P2 receptors in human heart: upregulation of P2X6 in patients undergoing heart transplantation, interaction with TNFalpha and potential role in myocardial cell death.

    Science.gov (United States)

    Banfi, Cristina; Ferrario, Silvia; De Vincenti, Ombretta; Ceruti, Stefania; Fumagalli, Marta; Mazzola, Alessia; D' Ambrosi, Nadia; Volontè, Cinzia; Fratto, Pasquale; Vitali, Ettore; Burnstock, Geoffrey; Beltrami, Elena; Parolari, Alessandro; Polvani, GianLuca; Biglioli, Paolo; Tremoli, Elena; Abbracchio, Maria P

    2005-12-01

    ATP acts as a neurotransmitter via seven P2X receptor-channels for Na(+) and Ca(2+), and eight G-protein-coupled P2Y receptors. Despite evidence suggesting roles in human heart, the map of myocardial P2 receptors is incomplete, and their involvement in chronic heart failure (CHF) has never received adequate attention. In left myocardia from five to nine control and 5-12 CHF subjects undergoing heart transplantation, we analyzed the full repertoire of P2 receptors and of 10 "orphan" P2Y-like receptors. All known P2Y receptors (i.e. P2Y(1,2,4,6,11,12,13,14)) and two P2Y-like receptors (GPR91 and GPR17) were detected in all subjects. All known P2X(1-7) receptors were also detected; of these, only P2X(6) was upregulated in CHF, as confirmed by quantitative real time-PCR. The potential significance of this change was studied in primary cardiac fibroblasts freshly isolated from young pigs. Exposure of cardiac fibroblasts to ATP or its hydrolysis-resistant-analog benzoylATP induced apoptosis. TNFalpha (a cytokine implicated in CHF progression) exacerbated cell death. Similar effects were induced by ATP and TNFalpha in a murine cardiomyocytic cell line. In cardiac fibroblasts, TNFalpha inhibited the downregulation of P2X(6) mRNA associated to prolonged agonist exposure, suggesting that, by preventing ATP-induced P2X(6) desensitization, TNFalpha may abolish a defense mechanism meant at avoiding Ca(2+) overload and, ultimately, Ca(2+)-dependent cell death. This may provide a basis for P2X(6) upregulation in CHF. In conclusion, we provide the first characterization of P2 receptors in the human heart and suggest that the interaction between TNFalpha and the upregulated P2X(6) receptor may represent a novel pathogenic mechanism in CHF.

  1. NRS2002 assesses nutritional status of leukemia patients undergoing hematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    Peng Liu; Zhao-Feng Zhang; Jing-Jing Cai; Bo-Shi Wang; Xia Yan

    2012-01-01

    Objective:To discuss whether nutritional risk screening 2002 (NRS2002) is appropriate for nutritional risk screening for leukemia patients before and after hematopoietic stem cell transplantation (HSCT),and whether there are risk differences in other conditions,such as age,gender and matching degree; to find the methods and indicators of nutritional risk screening for these patients before and after HSCT,in order to give timely intervention to guarantee the successful completion of the entire transplantation process.Methods:Nutritional risk of 99 leukemia patients was screened with NRS2002 before and after HSCT.The x2 test was applied to compare the risk differences between groups such as age,gender and matching degree,while the differences of other enumeration data,such as recent (1-3 months) weight loss,reduced food intake within one week and BMI,were compared by continuity correction.Results:Of the 99 leukemia patients,22 cases (22.2%) had nutritional risk before HSCT,while all patients had nutritional risk after HSCT; there is no significant difference in nutritional risk between male and female,and patients of less than 30 years old,not-full matched,recent (1-3 months) weight loss,reduced food intake within a week or BMI <18.5 were more likely to have nutritional risk; and 77 cases (77.8%) had weight loss,among which 49 patients (63.6%) had more than 5% weight loss within one month.Conclusions:This study showed that leukemia patients should receive the nutritional risk screening conventionally before and after HSCT,and NRS2002 was only appropriate for nutritional risk screening before HSCT.More attention should be paid to the patients less than 30 years old or not-full matched.Weight change was one of the important nutritional indicators for patients after HSCT.

  2. Longitudinal analysis of HIV-1 coreceptor tropism by single and triplicate HIV-1 RNA and DNA sequencing in patients undergoing successful first-line antiretroviral therapy

    Science.gov (United States)

    Meini, Genny; Rossetti, Barbara; Bianco, Claudia; Ceccherini-Silberstein, Francesca; Di Giambenedetto, Simona; Sighinolfi, Laura; Monno, Laura; Castagna, Antonella; Rozera, Gabriella; D'Arminio Monforte, Antonella; Zazzi, Maurizio; De Luca, Andrea; Moroni, M.; Angarano, G.; Antinori, A.; Armignacco, O.; d'Arminio Monforte, A.; Castelli, F.; Cauda, R.; Di Perri, G.; Galli, M.; Iardino, R.; Ippolito, G.; Lazzarin, A.; Perno, C. F.; von Schloesser, F.; Viale, P.; d'Arminio Monforte, A.; Antinori, A.; Castagna, A.; Ceccherini-Silberstein, F.; Cozzi-Lepri, A.; Girardi, E.; Lo Caputo, S.; Mussini, C.; Puoti, M.; Andreoni, M.; Ammassari, A.; Antinori, A.; Balotta, C.; Bonfanti, P.; Bonora, S.; Borderi, M.; Capobianchi, M. R.; Castagna, A.; Ceccherini-Silberstein, F.; Cingolani, A.; Cinque, P.; Cozzi-Lepri, A.; d'Arminio Monforte, A; De Luca, A.; Di Biagio, A.; Girardi, E.; Gianotti, N.; Gori, A.; Guaraldi, G.; Lapadula, G.; Lichtner, M.; Lo Caputo, S.; Madeddu, G.; Maggiolo, F.; Marchetti, G.; Marcotullio, S.; Monno, L.; Mussini, C.; Puoti, M.; Quiros Roldan, E.; Rusconi, S.; Cozzi-Lepri, A.; Cicconi, P.; Fanti, I.; Formenti, T.; Galli, L.; Lorenzini, P.; Giacometti, A.; Costantini, A.; Angarano, G.; Monno, L.; Santoro, C.; Maggiolo, F.; Suardi, C.; Viale, P.; Vanino, E.; Verucchi, G.; Castelli, F.; Quiros Roldan, E.; Minardi, C.; Quirino, T.; Abeli, C.; Manconi, P.E.; Piano, P.; Vecchiet, J.; Falasca, K.; Sighinolfi, L.; Segala, D.; Mazzotta, F.; Lo Caputo, S.; Cassola, G.; Viscoli, G.; Alessandrini, A.; Piscopo, R.; Mazzarello, G.; Mastroianni, C.; Belvisi, V.; Bonfanti, P.; Caramma, I.; Castelli, A. P.; Galli, M.; Lazzarin, A.; Rizzardini, G.; Puoti, M.; d'Arminio Monforte, A.; Ridolfo, A. L.; Piolini, R.; Castagna, A.; Salpietro, S.; Carenzi, L.; Moioli, M. C.; Cicconi, P.; Marchetti, G.; Mussini, C.; Puzzolante, C.; Gori, A.; Lapadula, G.; Abrescia, N.; Chirianni, A.; Guida, M. G.; Gargiulo, M.; Baldelli, F.; Francisci, D.; Parruti, G.; Ursini, T.; Magnani, G.; Ursitti, M. A.; Cauda, R.; Andreoni, M.; Antinori, A.; Vullo, V.; Cingolani, A.; d'Avino, A.; Ammassari, A.; Gallo, L.; Nicastri, E.; Acinapura, R.; Capozzi, M.; Libertone, R.; Tebano, G.; Cattelan, A.; Mura, M. S.; Madeddu, G.; Caramello, P.; Di Perri, G.; Orofino, G. C.; Bonora, S.; Sciandra, M.; Pellizzer, G.; Manfrin, V.

    2014-01-01

    Objectives Maraviroc has been shown to be effective in patients harbouring CCR5-tropic HIV-1. While this CCR5 antagonist has initially been used in salvage therapy, its excellent safety profile makes it ideal for antiretroviral treatment simplification strategies in patients with suppressed plasma viraemia. The aim of this study was to compare HIV-1 tropism as detected in baseline plasma RNA and peripheral blood mononuclear cell (PBMC) DNA prior to first-line therapy and to analyse tropism evolution while on successful treatment. Methods HIV-1 tropism was determined using triplicate genotypic testing combined with geno2pheno[coreceptor] analysis at a 10% false positive rate in 42 patients. Paired pre-treatment plasma RNA and PBMC DNA and two subsequent PBMC DNA samples (the first obtained after reaching undetectable plasma HIV-1 RNA and the second after at least 2 years of suppression of plasma viraemia) were evaluated. Results Coreceptor tropism was completely concordant in paired pre-treatment RNA and DNA, with 26.2% of HIV-1 sequences predicted to be non-CCR5-tropic. During follow-up, coreceptor tropism switches were detected in 4 (9.5%) patients without any preferential direction. Although false positive rate discrepancies within triplicates were common, the rate of discordance of coreceptor tropism assignment among triplicate results in this mostly CCR5-tropic dataset was only 2.1%, questioning the added value of triplicate testing compared with single testing. Conclusions HIV-1 coreceptor tropism changes during virologically successful first-line treatment are infrequent. HIV-1 DNA analysis may thus support the choice of a CCR5 antagonist in treatment switch strategies; however, maraviroc treatment outcome data are required to confirm this option. PMID:24155059

  3. HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation

    Science.gov (United States)

    Granelli-Piperno, Angela; Golebiowska, Angelika; Trumpfheller, Christine; Siegal, Frederick P.; Steinman, Ralph M.

    2004-05-01

    Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced.

  4. Stem cell factor and stromal cell co-culture prevent apoptosis in a subculture of the megakaryoblastic cell line, UT-7.

    Science.gov (United States)

    Liesveld, J L; Harbol, A W; Abboud, C N

    1996-07-01

    The megakaryoblastic cell line, UT-7, is dependent for its growth upon interleukin-3 (IL-3), erythropoietin, or granulocyte-macrophage colony stimulating factor (GM-CSF). A subculture of this line can be maintained in recombinant human c-kit ligand [stem cell factor (SCF)] at 100 ng/ml without requirement for other growth factors. Removal of this subculture from SCF results in rapid loss of viability and decreased proliferation. Cells grown in SCF also can be maintained in GM-CSF but not vice versa. In this work, we have characterized the SCF dependence of this UT-7 subculture. Stem cell factor removal results in apoptosis and a decline in viability which can be restored partially by re-addition of SCF, GM-CSF, or co-culture with adherent marrow stromal cells. Apoptosis in the factor-starved UT-7 population has been documented by light microscopy, electron microscopy and DNA analysis, showing the typical 180 base pair laddering characteristic of apoptosis. To quantitate the degree of apoptosis in the cell populations, and to assess whether apoptosis decreased with re-exposure of starved cells to growth factors or stroma, we utilized flow cytometry. This confirmed that exposure of previously factor-starved cells to stroma decreased the percentage of cells undergoing apoptosis. Co-culture with an SCF-deficient murine stromal cell line was also able to prevent apoptosis, suggesting contribution of other stromal cell factors. Experiments performed using trans-well inserts which do not allow cell passage, showed greatest viability of cells in contact with stroma, but viability was also improved in cells cultured in the presence of, but not in contact with, stromal cells compared to those cultured above plastic, suggesting a role for soluble stroma-produced substances. These data demonstrate that SCF alone can prevent apoptosis in cells dependent upon its presence for proliferation. Also, marrow stromal cells can serve as a partial substitute for growth factor in the

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

    Science.gov (United States)

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

    2002-05-01

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

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

    Science.gov (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  11. Chromosome abnormalities in Japanese Burkitt lymphoma cell lines.

    Directory of Open Access Journals (Sweden)

    Hamasaki,Kazuhide

    1982-02-01

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

  12. Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

    Science.gov (United States)

    Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

    2013-07-21

    To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-07-20

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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  15. Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest

    Directory of Open Access Journals (Sweden)

    Helena Carén

    2015-11-01

    Full Text Available Glioblastoma (GBM is an aggressive brain tumor whose growth is driven by stem cell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stem cells (GSCs and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Here we find only a subset of GSC cultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy for GBM.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Growth-inhibitory effects of the chemopreventive agent indole-3-carbinol are increased in combination with the polyamine putrescine in the SW480 colon tumour cell line

    Directory of Open Access Journals (Sweden)

    Gescher Andreas

    2003-01-01

    Full Text Available Abstract Background Many tumours undergo disregulation of polyamine homeostasis and upregulation of ornithine decarboxylase (ODC activity, which can promote carcinogenesis. In animal models of colon carcinogenesis, inhibition of ODC activity by difluoromethylornithine (DFMO has been shown to reduce the number and size of colon adenomas and carcinomas. Indole-3-carbinol (I3C has shown promising chemopreventive activity against a range of human tumour cell types, but little is known about the effect of this agent on colon cell lines. Here, we investigated whether inhibition of ODC by I3C could contribute to a chemopreventive effect in colon cell lines. Methods Cell cycle progression and induction of apoptosis were assessed by flow cytometry. Ornithine decarboxylase activity was determined by liberation of CO2 from 14C-labelled substrate, and polyamine levels were measured by HPLC. Results I3C inhibited proliferation of the human colon tumour cell lines HT29 and SW480, and of the normal tissue-derived HCEC line, and at higher concentrations induced apoptosis in SW480 cells. The agent also caused a decrease in ODC activity in a dose-dependent manner. While administration of exogenous putrescine reversed the growth-inhibitory effect of DFMO, it did not reverse the growth-inhibition following an I3C treatment, and in the case of the SW480 cell line, the effect was actually enhanced. In this cell line, combination treatment caused a slight increase in the proportion of cells in the G2/M phase of the cell cycle, and increased the proportion of cells undergoing necrosis, but did not predispose cells to apoptosis. Indole-3-carbinol also caused an increase in intracellular spermine levels, which was not modulated by putrescine co-administration. Conclusion While indole-3-carbinol decreased ornithine decarboxylase activity in the colon cell lines, it appears unlikely that this constitutes a major mechanism by which the agent exerts its antiproliferative

  18. Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

    Directory of Open Access Journals (Sweden)

    Duensing Stefan

    2008-07-01

    Full Text Available Abstract Background Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX. Results Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300. Conclusion Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-19

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

  20. Internalization of cystatin C in human cell lines.

    Science.gov (United States)

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

    2008-09-01

    Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.

  1. Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Møller, C; Bock, E

    1992-01-01

    characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were...... embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression....

  2. Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts

    Science.gov (United States)

    Zhao, Lei; Shao, Qianqian; Zhang, Yun; Zhang, Lin; He, Ying; Wang, Lijie; Kong, Beihua; Qu, Xun

    2016-01-01

    Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal–placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN+CD14+CD1a− phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4+CD25+Foxp3+ Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal–fetal interface. PMID:26857012

  3. Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection

    Science.gov (United States)

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

    2010-01-01

    The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10–20 fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2 fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors. PMID:20188762

  4. Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines.

    Science.gov (United States)

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

    1979-02-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.

  5. 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.

  6. β-D-Glucan Screening for Detection of Invasive Fungal Disease in Children Undergoing Allogeneic Hematopoietic Stem Cell Transplantation.

    Science.gov (United States)

    Koltze, Antonia; Rath, Peter; Schöning, Stefan; Steinmann, Jörg; Wichelhaus, Thomas A; Bader, Peter; Bochennek, Konrad; Lehrnbecher, Thomas

    2015-08-01

    While the assessment of β-D-glucan (BDG) levels in adults improves the early diagnosis of invasive fungal disease (IFD), data on BDG levels in children are limited. We therefore assessed in a prospective cohort study the value of serial BDG screening for early detection of IFD in children undergoing allogeneic hematopoietic stem cell transplantation (HSCT). IFD was defined according to the revised European Organization for Research and Treatment of Cancer/Mycosis Study Group (EORTC/MSG) criteria, with the necessary modification that BDG was not included as a microbiological criterion. For the analysis, a total of 702 serum samples were obtained in 34 pediatric HSCT recipients. Proven IFD occurred in two patients (fusariosis and Candida sepsis, respectively), and probable invasive aspergillosis was diagnosed in four patients. Analyses including different cutoff values for BDG levels and different definitions of the onset of IFD demonstrated that the BDG assay has a relatively high sensitivity and good negative predictive value, whereas the positive predictive value has major limitations (screening in pediatric HSCT recipients has a low positive predictive value and is therefore of limited usefulness.

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

    Science.gov (United States)

    Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D; Ziller, Michael; Croft, Gist F; Amoroso, Mackenzie W; Oakley, Derek H; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

    2011-02-04

    The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

  8. The effect of a multimodal intervention on treatment-related symptoms in patients undergoing hematopoietic stem cell transplantation: a randomized controlled trial

    DEFF Research Database (Denmark)

    Jarden, Mary; Nelausen, Knud; Hovgaard, Doris;

    2009-01-01

    in patients undergoing myeloablative allogeneic hematopoietic stem cell transplantation (allo-HSCT). Forty-two patients (18-65 years) were randomized either to an intervention or a control group. The intervention group received standard treatment and care, and a supervised four- to six-week structured...

  9. [Role of beta-carotene in the prevention of genotoxic damage in patients undergoing radiotherapy. Monitoring by the micronucleus test in exfoliative cells of the oral cavity].

    Science.gov (United States)

    Oldini, C; Malusardi, G; Grossi, L; Chiarelli, G

    1992-01-01

    Radiotherapic treatment of patients with carcinoma usually causes genotoxis damage. This has been studied recently using the test of micronuclei in esfoliated cells. This test presents methodologic advantages in compared with the classic citogenetic analysis and as it is carried out on esfolieted cells from the oral cavity it faithfully reflects the genotoxic damage undergone by the cells of the basal layer of the epitelium. The preliminary result obtained so far have confirmed the anticlastogenic activity of beta-carotene in fact, the frequence of micronuclei in esfolieted cells from the oral cavity in patients undergoing radiotherapy or undergoing treatment with beta-carotene is inferior to that of patients undergoing treatment with beta-carotene is inferior to that of patients undergoing radiotherapy without the subministration of carotenoids. Treatment with carotenoids does not influence the therapeutic efficiency of radiotherapy treatment. Therefore, the results seem to confirm that indirect ossidaction processes are involved in the mechanism of the clastogenic action of radiotherapia. The carotenoids seem to be able to contrast validly this undesirable effect without interfering with the desirable therapeutic effect.

  10. Fluorouracil selectively enriches stem-like cells in the lung adenocarcinoma cell line SPC.

    Science.gov (United States)

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

    2013-06-01

    Most adult stem cells are in the G0 or quiescent phase of the cell cycle and account for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. This study sought to enrich cancer stem cells and explore cancer stem-like cell clones using 5-fluorouracil (5-FU) in the lung adenocarcinoma cell line, SPC. Proliferation inhibition was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, according to which half maximal inhibitory concentration values were calculated. Expression levels of stem cell markers after treatment with 5-FU were examined using immunofluorescence and Western blotting. Additionally, side population (SP) cells were sorted using FACS. Properties of SP cells were evaluated by using Transwell, colony-forming assays, and tumor formation experiments. 5-FU greatly inhibits proliferation, especially of cells in S phase. SP cells possess greater invasive potential, higher clone-forming potential, and greater tumor-forming ability than non-SP cells. Treatment with 5-FU enriches the SP cells with stem cell properties in human lung adenocarcinoma cell lines.

  11. Isolation and characterization of cancer stem-like cells from MHCC97H Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Shanyong Yi; Kejun Nan; Aihua Yuan; Chuangxin Lu

    2009-01-01

    Objective:To identify and isolate CD133 positive cancer stem-like cells (CD133+ cells) from the highly invasive human hepatocellular carcinoma cell line(MHCC97H), and examine their potential for clonogenicity and tumorigenicity. Methods: CD133+ and CD133- cells were isolated from MHCC97H cell line by magnetic bead cell sorting(MACS), and the potentials of CD133+ cells for colony formation and tumorigenicity were evaluated by soft agar cloning and tumor formation following nude mice inoculation. Results:CD133+ cells represent a minority(0.5-2.0%) of the tumor cell population with a greater colony-forming efficiency and greater tumor production ability. The colony-forming efficiency of CD133+ cells in soft agar was significantly higher than CD133- cells(36.8±1.4 vs 12.9±0.8, P<0.05).After 6 weeks, 3/5 mice inoculated with 1 × 103 CD133+ cells, 4/5 with 1 × 104 CD133+ cells and 5/5 with 1 × 105 CD133+ cells developed detectable tumors at the injection site, while only one tumor was found in mice treated with same numbers of CD133- cells. Conclusion: CD133 may be a hallmark of liver cancer stem cells (CSC) in human hepatocellular carcinoma(HCC), because the CD133+ cells identified and isolated with anti-CD133 labeled magnetic beads from MHCC97H cell line exhibit high potentials for clonogenicity and tumorigenicity. These CD133+ cells might contribute to hepatocarcinogenesis, as well as the growth and recurrence of human HCC, and therefore may be a useful target for anti-cancer therapy.

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

    Directory of Open Access Journals (Sweden)

    Garcia Jose

    2010-06-01

    Full Text Available Abstract Background Fascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC. Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation. Methods We examined whether fascin is a potential target in ESCC using in vitro and in vivo studies utilizing a specific siRNA. We established a stable transfectant with downregulated fascin from KYSE170 cell line. Results The fascin downregulated cell lines showed a slower growth pattern by 40.3% (p In vivo, the tumor size was significantly smaller in the tumor with fascin knockdown cells than in mock cells by 95% at 30 days after inoculation. Conclusions These findings suggest that fascin overexpression plays a role in tumor growth and progression in ESCC and that cell death caused by its downregulation might be induced by cell adhesion loss. This indicates that targeting fascin pathway could be a novel therapeutic strategy for the human ESCC.

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

    Science.gov (United States)

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

    2011-09-01

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

  14. Specific binding of benzodiazepines to human breast cancer cell lines.

    Science.gov (United States)

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

    1999-01-01

    Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.

  15. Proapoptotic Activity of Propolis and Their Components on Human Tongue Squamous Cell Carcinoma Cell Line (CAL-27).

    Science.gov (United States)

    Czyżewska, Urszula; Siemionow, Katarzyna; Zaręba, Ilona; Miltyk, Wojciech

    2016-01-01

    Propolis has been used since ancient times in folk medicine. It is a popular medicine possessing a broad spectrum of biological activities. This material is one of the richest sources of polyphenolic compounds such as flavonoids and phenolic acids. The ethanolic extract of propolis (EEP) evokes antibacterial, antiviral, antifungal and anticancer properties. Due to pharmacological properties it is used in the commercial production of nutritional supplements. In this study, gas chromatography coupled with mass spectrometry (GC-MS) was used to quantify main polyphenols in EEPs. The effect of EEPs, individual EEPs components (chrysin, galangin, pinocembrin, caffeic acid, p-coumaric acid, ferulic acid) and their mixture on viability of human tongue squamous cell carcinoma cell line (CAL-27) as well as the molecular mechanisms of the process were examined. The results of MTTs assay demonstrated that EEP, polyphenols and mixture of polyphenolic compounds were cytotoxic for CAL-27 cells in a dose dependent manner. The mechanism of cytotoxicity induced by these components undergoes through apoptosis as detected by flow cytometry. The ethanolic extracts of propolis activated caspases -3, -8, -9. Mixture of polyphenols was found as the most potent inducer of apoptosis thorough both intrinsic and extrinsic pathway. Therefore, we suggest that anticancer properties of propolis is related to synergistic activity of its main components.

  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

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  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 line...ve.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  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 line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  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 line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  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... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  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... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  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... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.20.AllAg.Lymphoblastoid_cell_line.bed ...

  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... http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Pol.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  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...://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.10.AllAg.Lymphoblastoid_cell_line.bed ...

  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...://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

  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 line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  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 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.50.AllAg.Lymphoblastoid_cell_line.bed ...

  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 line...ciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bld.05.AllAg.Lymphoblastoid_cell_line.bed ...

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

    Science.gov (United States)

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

    2013-01-01

    Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients. We previously demonstrated a relationship between increased oxidative stress and hypomethylation of the transposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressor gene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In this study, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell (UM-UC-3) under oxidative stress conditions, stimulated by challenge with H2O2 for 72 h. Cells were pretreated with an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 and RUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR, respectively. Levels of LINE-1 methylation were significantly decreased in H2O2-treated cells, and reestablished after pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cells exposed to H2O2. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylation of LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally induced by reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotes urothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanistic pathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promoter might differ.

  2. Analysis of G-banding in tumor cell lines derived from human neural stem cells

    Institute of Scientific and Technical Information of China (English)

    Junhua Zou; Yanhui Li

    2006-01-01

    BACKGROUND: The application of neural stem cell (NSC) is restricted because of its tumorigenesis, and the possible pathogenesis needs investigation.OBJECTIVE: To compare the differences of chromosomal G-banding between human NSCs (hNSCs) derived tumor cell line and hNSCs derived normal cell lines.DESIGN: A randomized controlled observation.SETTING: Building of Anatomy, Peking University Health Science Center.MATERIALS: The hNSC lines and hNSC-derived tumor cell lines were provided by the Research Center of Stem Cells, Peking University; DMEM/F12 (1:1) medium, N2 additive, B27 additive epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were produced by GIBCO BRL Company (USA); fetal bovine serum by HYCLONE Company (USA).METHODS: The experiments were carried out in the Department of Genetics, Peking University Health Science Center from February 2003 to July 2004. Human fetal striatal NSCs were inoculated hypodermically on the right scapular of nude mice; Normal human fetal striatal NSCs were cultured to 5-8 passages as controls. Karyotyping was performed on the 5th passage of hNSC-derived tumor cells at 6 weeks after hN-SC transplantation into nude mice (T1) and tumor cells at 15 weeks after transplantation (T2). Metaphase chromosomes were examined with microscope, G-banding cytogenetic analysis and karyotyping were performed according to the Cytoscan Karyotyping FISH and CGH software system (United biotechnology USA Corporation).MAIN OUTCOME MEASURES: G-banded analytical results of human fetal striatal nerve stem cells derived tumor cell lines (T1 and T2) of metaphase chromosomes were observed.RESULTS: ① Chromosome analysis of hNSC-derived tumor cell lines 1 (T1): Twenty-five well-spread metaphases were randomly selected for analysis. The karyotypes were 64, XX (8, 32%); 65, XX (1, 4%); 67,XX (5, 20%); 68, XX (11, 44%). The modal number of chromosomes in this cell lines was 68, which were all hypotriploid. The analysis of 8 G

  3. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade.

    Science.gov (United States)

    Gargett, Tessa; Yu, Wenbo; Dotti, Gianpietro; Yvon, Eric S; Christo, Susan N; Hayball, John D; Lewis, Ian D; Brenner, Malcolm K; Brown, Michael P

    2016-06-01

    Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients.

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

    Directory of Open Access Journals (Sweden)

    Zhang Ping

    2006-09-01

    Full Text Available Abstract Background Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. Methods A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated. Results Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells. Conclusion The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis

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

    NARCIS (Netherlands)

    Mouthaan, Ton; Vertregt, Maarten

    1986-01-01

    A charge injection device has been realized in which charge can be injected on to an MOS-capacitor from a buried layer via an isolated transfer layer. The cell is positioned vertically between word and bit line. LOCOS (local oxidation) is used to isolate the cells and (deep) ion implantation to real

  6. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

    Science.gov (United States)

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

    2010-05-28

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

  7. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Qin, J.-Z.; Xin, H. [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States); Nickoloff, B.J., E-mail: bnickol@lumc.edu [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States)

    2010-05-28

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

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

    LENUS (Irish Health Repository)

    O'Neill, Amanda J

    2011-10-07

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

  9. The impact of metformin and salinomycin on transforming growth factor β-induced epithelial-to-mesenchymal transition in non-small cell lung cancer cell lines

    Science.gov (United States)

    KOECK, STEFAN; AMANN, ARNO; HUBER, JULIA M.; GAMERITH, GABRIELE; HILBE, WOLFGANG; ZWIERZINA, HEINZ

    2016-01-01

    The epithelial-to-mesenchymal transition (EMT) is highly involved in the development of metastases. EMT transforms epithelial carcinoma cells into mesenchymal-like cells, characterized by increased cell migration and invasiveness. Transforming growth factor β (TGFβ) appears to be crucial in this process. Metformin and salinomycin have demonstrated an EMT inhibitory effect. The current experiments indicate that these substances specifically inhibit TGFβ-induced EMT in non-small cell lung cancer (NSCLC) cell lines. The NSCLC cell lines A549 and HCC4006 were stimulated with TGFβ for 48 h to induce EMT. Metformin or salinomycin was added simultaneously with TGFβ to inhibit TGFβ-induced EMT. Western blot analyses of E-cadherin and vimentin were performed to detect changes in EMT marker expression, and a wound healing assay was conducted to determine the potential effects on cell migration. The effects of the two drugs on cell viability were also investigated using MTS tetrazolium dye assays. The results revealed that cells undergoing EMT by application of TGFβ exhibited a downregulation of E-cadherin and an upregulation of vimentin protein expression on western blot analyses, and an increased capacity for cell migration. Simultaneous application of TGFβ and metformin specifically inhibited EMT and increased E-cadherin expression. At the higher dose tested, salinomycin also inhibited EMT, despite an increase in vimentin expression in the two cell lines. Furthermore, metformin and salinomycin, at the two concentrations tested, inhibited cell migration. These findings demonstrate that metformin and salinomycin are able to block EMT and inhibit EMT-induced cell migration. Thus, these two substances are novel EMT inhibiting drugs that have the potential to specifically control EMT and metastatic spread in NSCLC. PMID:27073581

  10. The impact of metformin and salinomycin on transforming growth factor β-induced epithelial-to-mesenchymal transition in non-small cell lung cancer cell lines.

    Science.gov (United States)

    Koeck, Stefan; Amann, Arno; Huber, Julia M; Gamerith, Gabriele; Hilbe, Wolfgang; Zwierzina, Heinz

    2016-04-01

    The epithelial-to-mesenchymal transition (EMT) is highly involved in the development of metastases. EMT transforms epithelial carcinoma cells into mesenchymal-like cells, characterized by increased cell migration and invasiveness. Transforming growth factor β (TGFβ) appears to be crucial in this process. Metformin and salinomycin have demonstrated an EMT inhibitory effect. The current experiments indicate that these substances specifically inhibit TGFβ-induced EMT in non-small cell lung cancer (NSCLC) cell lines. The NSCLC cell lines A549 and HCC4006 were stimulated with TGFβ for 48 h to induce EMT. Metformin or salinomycin was added simultaneously with TGFβ to inhibit TGFβ-induced EMT. Western blot analyses of E-cadherin and vimentin were performed to detect changes in EMT marker expression, and a wound healing assay was conducted to determine the potential effects on cell migration. The effects of the two drugs on cell viability were also investigated using MTS tetrazolium dye assays. The results revealed that cells undergoing EMT by application of TGFβ exhibited a downregulation of E-cadherin and an upregulation of vimentin protein expression on western blot analyses, and an increased capacity for cell migration. Simultaneous application of TGFβ and metformin specifically inhibited EMT and increased E-cadherin expression. At the higher dose tested, salinomycin also inhibited EMT, despite an increase in vimentin expression in the two cell lines. Furthermore, metformin and salinomycin, at the two concentrations tested, inhibited cell migration. These findings demonstrate that metformin and salinomycin are able to block EMT and inhibit EMT-induced cell migration. Thus, these two substances are novel EMT inhibiting drugs that have the potential to specifically control EMT and metastatic spread in NSCLC.

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

    Directory of Open Access Journals (Sweden)

    Somayeh Khosravi-Farsani

    2015-01-01

    Full Text Available Objective: The existence of female germ-line stem cells (FGSCs has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. Materials and Methods: In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH and stage-specific embryonic antigen-1 (SSEA1 markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3, alkaline phosphatase (AP activity test and immunocytochemistry. Results: Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 ± 0.49% (Mean ± SDV positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. Conclusion: We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries.

  12. Characterization of cancer stem-like cells in the side population cells of human gastric cancer cell line MKN-45

    Institute of Scientific and Technical Information of China (English)

    Hai-hong ZHANG; Ai-zhen CAI; Xue-ming WEI; Li DING; Feng-zhi LI; Ai-ming ZHENG; Da-jiang DAI

    2013-01-01

    Objective:Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells,including several gastric cancer cell lines.This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45.Methods:We used fluorescence activated cell sorting (FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45 (cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells.Results:This study found that the SP cells had higher clone formation efficiency than major population (MP) cells.Five stemness-related gene expression profiles,including OCT-4,SOX-2,NANOG,CD44,and adenosine triphosphate (ATP)-binding cassette transporters gene ABCG2,were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).Western blot was used to show the difference of protein expression between SP and MP cells.Both results show that there was significantly higher protein expression in SP cells than in MP cells.When inoculated into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice,SP cells show higher tumorigenesis tendency than MP cells.Conclusions:These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.

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

    Science.gov (United States)

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

    2008-11-01

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

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

    Science.gov (United States)

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

    2016-08-31

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

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

    Science.gov (United States)

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

    2017-01-01

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

  16. 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.

  17. Expression of the epidermal growth factor receptor in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Damstrup, L; Rygaard, K; Spang-Thomsen, M;

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... demonstrated that the cells bound between 3 and 52 fmol/mg protein with a KD ranging from 0.5 x 10(-10) to 2.7 x 10(-10) M. EGF binding to the receptor was confirmed by affinity-labeling EGF to the EGF receptor. The cross-linked complex had a M(r) of 170,000-180,000. Northern blotting showed the expression...... of EGF receptor mRNA in all 10 cell lines that were found to be EGF receptor-positive and in one cell line that was found to be EGF receptor-negative in the radioreceptor assay and affinity labeling. Our results provide, for the first time, evidence that a large proportion of a broad panel of small cell...

  18. Cell Motility and Invasiveness of Neurofibromin-Deficient Neural Crest Cells and Malignant Triton Tumor Lines

    Science.gov (United States)

    2005-06-01

    immunoblotting techniques to characterize signaling pathways activated by TGF-beta and PDGF-BB in MPNST -like sarcoma cell lines isolated from cisNfl+/-;p53...mouse model to include characterizations of genomic instability in the context of malignant transformation, and to test possible modifiers of MPNST ...growth and invasiveness. 15. SUBJECT TERMS neurofibromatosis type 1; neural crest cells; cell motility and Migration; PDGF; TGF-beta; MPNST

  19. Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week.

    Science.gov (United States)

    Nikodemova, Maria; Kimyon, Rebecca S; De, Ishani; Small, Alissa L; Collier, Lara S; Watters, Jyoti J

    2015-01-15

    During postnatal development, microglia, CNS resident innate immune cells, are essential for synaptic pruning, neuronal apoptosis and remodeling. During this period microglia undergo morphological and phenotypic transformations; however, little is known about how microglial number and density is regulated during postnatal CNS development. We found that after an initial increase during the first 14 postnatal days, microglial numbers in mouse brain began declining in the third postnatal week and were reduced by 50% by 6weeks of age; these "adult" levels were maintained until at least 9months of age. Microglial CD11b levels increased, whereas CD45 and ER-MP58 declined between P10 and adulthood, consistent with a maturing microglial phenotype. Our data indicate that both increased microglial apoptosis and a decreased proliferative capacity contribute to the developmental reduction in microglial numbers. We found no correlation between developmental reductions in microglial numbers and brain mRNA levels of Cd200, Cx3Cl1, M-Csf or Il-34. We tested the ability of M-Csf-overexpression, a key growth factor promoting microglial proliferation and survival, to prevent microglial loss in the third postnatal week. Mice overexpressing M-Csf in astrocytes had higher numbers of microglia at all ages tested. However, the developmental decline in microglial numbers still occurred, suggesting that chronically elevated M-CSF is unable to overcome the developmental decrease in microglial numbers. Whereas the identity of the factor(s) regulating microglial number and density during development remains to be determined, it is likely that microglia respond to a "maturation" signal since the reduction in microglial numbers coincides with CNS maturation.

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

    Directory of Open Access Journals (Sweden)

    Weyhe Dirk

    2010-10-01

    Full Text Available Abstract Background The anti-infective agent Taurolidine (TRD has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Methods Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3 were incubated with TRD (100 μM, 250 μM and 1000 μM. Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. Results TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3 as well as genes involved in the ER stress response (PPP1R15A, in ubiquitination (TRAF6 and mitochondrial apoptotic pathways (PMAIP1. Conclusions This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis.

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  2. Tooth regeneration from newly established cell lines from a molar tooth germ epithelium.

    Science.gov (United States)

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

    2007-04-13

    In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.

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

    Directory of Open Access Journals (Sweden)

    Tobias Hannes

    2015-01-01

    Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

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

    Directory of Open Access Journals (Sweden)

    Behra Martine

    2012-01-01

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

  5. Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Jie Du; Xiaoqing Gao; Li Deng; Nengbin Chang; Huailin Xiong; Yu Zheng

    2014-01-01

    Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro-tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su-pernatant were signiifcantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes-enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen-chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

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

    OpenAIRE

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

    1993-01-01

    The platinum drug chemosensitivity of five human cervical squamous cell carcinoma cell lines (HX/151, HX/155, HX/156, HX/160 and HX/171) derived from previously untreated patients has been determined. Compared to our data obtained previously using human ovarian carcinoma cell lines, all five lines were relatively resistant to cisplatin, carboplatin, iproplatin and tetraplatin. One of the lines (HX/156) was exceptionally sensitive to the novel platinum (IV) ammine/amine dicarboxylates JM216 [b...

  7. Expression of tumor antigens on primary ovarian cancer cells compared to established ovarian cancer cell lines

    Science.gov (United States)

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

    2016-01-01

    In order to select a suitable combination of cancer cell lines as an appropriate source of antigens for dendritic cell-based immunotherapy of ovarian cancer, we analyzed the expression level of 21 tumor associated antigens (BIRC5, CA125, CEA, DDX43, EPCAM, FOLR1, Her-2/neu, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MUC-1, NY-ESO-1, PRAME, p53, TPBG, TRT, WT1) in 4 established ovarian cancer cell lines and in primary tumor cells isolated from the high-grade serous epithelial ovarian cancer tissue. More than 90% of tumor samples expressed very high levels of CA125, FOLR1, EPCAM and MUC-1 and elevated levels of Her-2/neu, similarly to OVCAR-3 cell line. The combination of OV-90 and OVCAR-3 cell lines showed the highest overlap with patients' samples in the TAA expression profile. PMID:27323861

  8. Development of improved vaccine cell lines against rotavirus

    Science.gov (United States)

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

    2017-01-01

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

  9. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  10. Comprehensive characterization of genomic instability in pluripotent stem cells and their derived neuroprogenitor cell lines

    Directory of Open Access Journals (Sweden)

    Nestor Luis Lopez Corrales

    2012-12-01

    Full Text Available The genomic integrity of two human pluripotent stem cells and their derived neuroprogenitor cell lines was studied, applying a combination of high-resolution genetic methodologies. The usefulness of combining array-comparative genomic hybridization (aCGH and multiplex fluorescence in situ hybridization (M-FISH techniques should be delineated to exclude/detect a maximum of possible genomic structural aberrations. Interestingly, in parts different genomic imbalances at chromosomal and subchromosomal levels were detected in pluripotent stem cells and their derivatives. Some of the copy number variations were inherited from the original cell line, whereas other modifications were presumably acquired during the differentiation and manipulation procedures. These results underline the necessity to study both pluripotent stem cells and their differentiated progeny by as many approaches as possible in order to assess their genomic stability before using them in clinical therapies.

  11. Lipid analysis of eight human breast cancer cell lines with ToF-SIMS.

    Science.gov (United States)

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

    2015-06-28

    In this work, four triple negative (TN) cell lines, three ER+ and PR+ receptor positive (RP) cell lines, and one ER+, PR+, and HER2+ cell line were chemically distinguished from one another using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA). PCA scores separation was observed between the individual cell lines within a given classification (TN and RP) and there were distinctly different trends found in the fatty acid and lipid compositions of the two different classifications. These trends indicated that the RP cell lines separated out based on the carbon chain length of the lipids while the TN cell lines showed separation based on cholesterol-related peaks (in the positive ion data). Both cell types separated out by trends in fatty acid chain length and saturation in the negative ions. These chemical differences may be manifestations of unique metabolic processes within each of the different cell lines. Additionally, the HER2+ cell line was distinguished from three other RP cell types as having a unique distribution of fatty acids including anticorrelation to 18-carbon chain fatty acids. As these cell lines could not be grown in the same growth media, a combination of chemical fixation, rinsing, C60 (+) presputtering, and selection of cellular regions-of-interest is also presented as a successful method to acquire ToF-SIMS data from cell lines grown in different media.

  12. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines.

    Science.gov (United States)

    Varamini, P; Soltani, M; Ghaderi, A

    2009-01-01

    There are reports on the presence of various compounds exerting different biological activities in Ruta graveolens, a plant of Rutaceae family. The aim of the present study was to evaluate in vitro cytotoxicity of the total extract of R. graveolens against tumor cell lines of different origin. Aerial parts of the plant was extracted with 70% ethanol by sonication method and cytotoxic activity was examined on RAJI, RAMOS, RPMI8866, U937, Jurkat, MDA-MB-453, MCF-7, LNCap-FGC-10, 5637, HeLa, SK-OV-3, A549, Mehr-80 and also peripheral blood mononuclear cells (PBMC) by the use of WST-1 assay. Results were expressed as IC(50) values. R. graveolens extract showed high cytotoxic activity against RAJI and RAMOS, two Burkitt's lymphoma cell lines, with an IC(50) equal to 24.3 microg/ml and 35.2 microg/ml respectively and LNCap-FGC-10, a prostate adenocarcinoma cell line with an IC(50) equal to 27.6 microg/ml as well as Mehr-80, a newly established Large Cell Lung Carcinoma (IC(50)=46.2 microg/ml). No significant anti-proliferative activity was observed on other cell lines including MCF-7, MDA-MB-453, SK-OV-3, HeLa, 5637, JURKAT and RPMI8866. Adverse cytotoxic effect of R. graveolens was investigated against PBMCs and a significantly lower effect of this extract (IC(50)=104 microg/ml) was seen on normal cells compared with RAJI and RAMOS, two haematopoietic cell lines.

  13. Single-cell printing to form three-dimensional lines of olfactory ensheathing cells

    Energy Technology Data Exchange (ETDEWEB)

    Othon, Christina M; Ringeisen, Bradley R [Naval Research Laboratory/Code 6113, 4555 Overlook Ave. SW, Washington, DC 20375 (United States); Wu Xingjia; Anders, Juanita J [Department of Anatomy, Physiology and Genetics, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814 (United States)], E-mail: ringeisen@nrl.navy.mil

    2008-09-01

    Biological laser printing (BioLP(TM)) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes ({approx}{mu}Ls) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 {mu}m, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth.

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

    Directory of Open Access Journals (Sweden)

    Létourneau Isabelle J

    2012-08-01

    Full Text Available Abstract Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133 were derived from solid tumor (TOV and ascites (OV, at specific time points at diagnosis and relapse (R. Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133, or cisplatin/topotecan (2295. Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2, the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R and OV2295(R2 cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian

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

    Science.gov (United States)

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

    2016-06-01

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

  16. 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.

  17. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    Science.gov (United States)

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

    2015-01-01

    Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  18. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl].

    Science.gov (United States)

    Shimizu, T

    1997-11-01

    Solcoseryl is composed of extracts from calf blood, and is a drug known to activate tissue respiration. In the present study, I demonstrated the cell biological effects of Solcoseryl on a human small cell lung cancer cell line, PC-6, by analyzing cell morphology, cell growth, expression of neuronal differentiation markers, and the ras proto-oncogene product(ras p21). Exposure of PC-6 cells to Solcoseryl at the concentration of 200 microliters/ml induced (1) cell morphological changes, including neurodendrite-like projections from the cell surface, and (2) complete inhibition of cell growth, that was shown by the loss of Ki-67 expression. Solcoseryl also induced the expression of neurofilament protein and acetylcholinesterase, both of which are markers of neuronal differentiation. Moreover, it upregulated the expression of the ras proto-oncogene product, ras p21. Taken together, these data suggest that Solcoseryl is composed of component(s) which can induce neuronal differentiation of the human small cell lung cancer cell line, PC-6.

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

    Directory of Open Access Journals (Sweden)

    Hiraki,Shunkichi

    1979-02-01

    Full Text Available Human leukemic B-cell (BALL-1, T-cell (TALL-1 and null-cell (NALL-1 lines have been established from three patients with acute lymphoblastic leukemia (ALL. To study the heterotransplantability and in vivo growth characteristics, attempts were made to transplant these ALL cell lines into newborn Syrian hamsters treated with rabbit anti-hamster thymocyte serum. Intraperitoneal implantation of 1.8-3.5 x 10(7 cells gave rise to invasive tumors in all recipients after 15 to 41 days. In addition to a common in vivo feature of mesenteric and retroperitoneal tumors, BALL-1 line was characterized by infiltration of the skin, massive ascites and bone marrow invasion. TALL-1 cells infiltrated various organs including the lymph nodes, liver, gallbladder, spleen, bone marrow, central nervous system and eyes. NALL-1 line grew slowly, producing the least tumors, although there were distant metastases in the lungs. Tumor cells were detected in the blood of 2 of 3 BALL-1-bearing hamsters and in the blood of 4 of 5 TALL-1-bearing hamsters. Thus, these three ALL cell lines were found to exhibit a characteristic biological behavior in hamsters, which might be related to the different cell lineage.

  20. Chemical inhibition of autophagy: Examining its potential to increase the specific productivity of recombinant CHO cell lines.

    Science.gov (United States)

    Baek, Eric; Kim, Che Lin; Kim, Mi Gyeom; Lee, Jae Seong; Lee, Gyun Min

    2016-09-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc.

  1. Inhibition of N-linked glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma cell lines

    Directory of Open Access Journals (Sweden)

    Del Grosso Federica

    2011-12-01

    Full Text Available Abstract Background The Anaplastic Lymphoma Kinase (ALK is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t(2;5 translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL. Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB, a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression. Methods We employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L, UKF-NB3 (ALK mutation R1275Q and NB1 (ALK amplification. As a control, we used the NB cell lines LA1-5S and NB5 (no ALK expression, and the ALCL cell line SU-DHL1 (NPM-ALK. Results Tunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors (AKT, ERK1/2, STAT3 were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP suggested that apoptotic pathways may be

  2. Identification of a nuclear-localized nuclease from wheat cells undergoing programmed cell death that is able to trigger DNA fragmentation and apoptotic morphology on nuclei from human cells.

    Science.gov (United States)

    Domínguez, Fernando; Cejudo, Francisco J

    2006-08-01

    PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed.

  3. Use of mammalian target of rapamycin inhibitors after failure of tyrosine kinase inhibitors in patients with metastatic renal cell carcinoma undergoing hemodialysis: A single-center experience with four cases.

    Science.gov (United States)

    Omae, Kenji; Kondo, Tsunenori; Takagi, Toshio; Iizuka, Junpei; Kobayashi, Hirohito; Hashimoto, Yasunobu; Tanabe, Kazunari

    2016-07-01

    We retrospectively identified patients with end-stage renal disease undergoing hemodialysis treated with the mammalian target of rapamycin inhibitors as a second- and/or third-line targeted therapy after treatment failure with the tyrosine kinase inhibitors for metastatic renal cell carcinoma. Patient medical records were reviewed to evaluate the response to therapies and treatment-related toxicities. Four patients were identified. All patients had undergone nephrectomy, and one had received immunotherapy before targeted therapy. Two patients had clear cell histology, and the other two had papillary histology. All patients were classified into the intermediate risk group according to the Memorial Sloan-Kettering Cancer Center risk model. All patients were treated with everolimus as a second- or third-line therapy, and two patients were treated with temsirolimus as a second- or third-line therapy after treatment failure with sorafenib or sunitinib. The median duration of everolimus therapy was 6.7 months, whereas that of temsirolimus was 9.5 months. All patients had stable disease as the best response during each period of therapy. There were no severe adverse events. The use of mammalian target of rapamycin inhibitors in patients who previously failed to respond to tyrosine kinase inhibitors appears to be feasible in patients with end-stage renal disease requiring hemodialysis.

  4. Drug treatment of cancer cell lines: a way to select for cancer stem cells?

    Science.gov (United States)

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

    2011-03-04

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

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

    Directory of Open Access Journals (Sweden)

    Ilaria Chiodi

    2011-03-01

    Full Text Available Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A. Ivana; Mondello, Chiara, E-mail: mondello@igm.cnr.it [Institute of Molecular Genetics, CNR, via Abbiategrasso 207, 27100 Pavia (Italy)

    2011-03-04

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  7. Photodynamic therapy-induced programmed cell death in carcinoma cell lines

    Science.gov (United States)

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

    1993-06-01

    The mode of cell death following photodynamic therapy (PDT) was investigated from the perspective of programmed cell death (apoptosis). Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a), and rat mammary carcinoma (MTF7) were treated by PDT following sensitization with dihematoporphyrin ether (DHE). The response of these carcinoma cell lines to PDT was variable. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder pattern indicative of internucleosomal cleavage of DNA during apoptosis. MTF7 and PC3 responded to PDT by inducing apoptosis while H322a had no apoptotic response. The magnitude of the response and the PDT dosage required to induce the effect were different in PC3 and MTF7. MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal apoptosis at the LD50 but had a marked response at the LD85. Furthermore, the onset of apoptosis followed slower kinetics in PC3 (2 hr - 4 hr) than in MTF7 (cells were killed by PDT but failed to exhibit any apoptotic response. This study indicates that apoptosis may occur during PDT induced cell death, but this pathway is not universal for all cancer cell lines.

  8. Hypotension due to Chemotherapy in a Patient with Small Cell Lung Cancer and Lambert-Eaton Myasthenic Syndrome Undergoing Hemodialysis: A First Case Report

    Directory of Open Access Journals (Sweden)

    Taiji Kuwata

    2012-01-01

    Full Text Available We present the first case of small cell lung cancer with Lambert-Eaton myasthenic syndrome during hemodialysis (HD. A 72-year-old male patient receiving HD experienced progressive muscle weakness. He was diagnosed with small cell lung cancer with Lambert-Eaton myasthenic syndrome due to an increased serum level of anti-voltage-gated calcium channel antibody and aspiration cytology on endobronchial ultrasonography for the swelling of a subcarinal lymph node. He received chemotherapy consisting of carboplatin (300 mg/m2 and etoposide (50 mg/m2, to which he had a partial response. However, the second therapy course could not be administered because of the unexpected development of severe hematological adverse events, which also prevented him from undergoing further HD. This case indicates that caution should be taken when using chemotherapy for such patients because of hypotension due to chemotherapy, with which it is impossible to undergo HD.

  9. Dose Escalation of Total Marrow Irradiation With Concurrent Chemotherapy in Patients With Advanced Acute Leukemia Undergoing Allogeneic Hematopoietic Cell Transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Jeffrey Y.C., E-mail: jwong@coh.org [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States); Forman, Stephen; Somlo, George [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States); Rosenthal, Joseph [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States); Department of Pediatrics, City of Hope National Medical Center, Duarte, California (United States); Liu An; Schultheiss, Timothy; Radany, Eric [Department of Radiation Oncology, City of Hope National Medical Center, Duarte, California (United States); Palmer, Joycelynne [Department of Biostatistics, City of Hope National Medical Center, Duarte, California (United States); Stein, Anthony [Department of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center, Duarte, California (United States)

    2013-01-01

    Purpose: We have demonstrated that toxicities are acceptable with total marrow irradiation (TMI) at 16 Gy without chemotherapy or TMI at 12 Gy and the reduced intensity regimen of fludarabine/melphalan in patients undergoing hematopoietic cell transplantation (HCT). This article reports results of a study of TMI combined with higher intensity chemotherapy regimens in 2 phase I trials in patients with advanced acute myelogenous leukemia or acute lymphoblastic leukemia (AML/ALL) who would do poorly on standard intent-to-cure HCT regimens. Methods and Materials: Trial 1 consisted of TMI on Days -10 to -6, etoposide (VP16) on Day -5 (60 mg/kg), and cyclophosphamide (CY) on Day -3 (100 mg/kg). TMI dose was 12 (n=3 patients), 13.5 (n=3 patients), and 15 (n=6 patients) Gy at 1.5 Gy twice daily. Trial 2 consisted of busulfan (BU) on Days -12 to -8 (800 {mu}M min), TMI on Days -8 to -4, and VP16 on Day -3 (30 mg/kg). TMI dose was 12 (n=18) and 13.5 (n=2) Gy at 1.5 Gy twice daily. Results: Trial 1 had 12 patients with a median age of 33 years. Six patients had induction failures (IF), and 6 had first relapses (1RL), 9 with leukemia blast involvement of bone marrow ranging from 10%-98%, 5 with circulating blasts (24%-85%), and 2 with chloromas. No dose-limiting toxicities were observed. Eleven patients achieved complete remission at Day 30. With a median follow-up of 14.75 months, 5 patients remained in complete remission from 13.5-37.7 months. Trial 2 had 20 patients with a median age of 41 years. Thirteen patients had IF, and 5 had 1RL, 2 in second relapse, 19 with marrow blasts (3%-100%) and 13 with peripheral blasts (6%-63%). Grade 4 dose-limiting toxicities were seen at 13.5 Gy (stomatitis and hepatotoxicity). Stomatitis was the most frequent toxicity in both trials. Conclusions: TMI dose escalation to 15 Gy is possible when combined with CY/VP16 and is associated with acceptable toxicities and encouraging outcomes. TMI dose escalation is not possible with BU/VP16 due to

  10. Quercetin modulates Wnt signaling components in prostate cancer cell line by inhibiting cell viability, migration, and metastases.

    Science.gov (United States)

    Baruah, Meghna M; Khandwekar, Anand P; Sharma, Neeti

    2016-10-01

    Epithelial-mesenchymal transition (EMT) is a plastic transition in tumor progression during which cancer cells undergo dramatic changes acquiring highly invasive properties. Transforming growth factor-β (TGF-β) is an inducer of EMT in epithelial cells and is obligatory for acquiring invasive phenotype in carcinoma. TGF-β plays a vital role in metastasis and tumorigenesis in prostate cancer, and mutations in the components of Wnt signaling pathways are associated with various kinds of cancers including prostate cancer. The purpose of this study was to identify alterations in Wnt signaling pathway components involved during prostate cancer progression and to determine the effect of quercetin on TGF-β-induced EMT in prostate cancer (PC-3) cell line. The expression of epithelial and mesenchymal markers and the components of Wnt signaling pathway were evaluated by real-time polymerase chain reaction. It was observed that quercetin prevented TGF-β-induced expression of vimentin and N-cadherin and increased the expression of E-cadherin in PC-3 cells, thus preventing TGF-β-induced EMT. Furthermore, the relative expression of Twist, Snail, and Slug showed that quercetin significantly decreased TGF-β-induced expression of Twist, Snail, and Slug. In the present study, the expression of epithelial markers were found to be upregulated in naive state and downregulated in induced state whereas the mesenchymal markers were found to be downregulated in naive state and upregulated in induced state. Thus, our study concludes that quercetin may prevent prostate cancer metastasis by regulating the components of Wnt pathway.

  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.

    1987-03-01

    Plateau-phase mouse clonal bone marrow stromal cell lines D2XRII and C3H cl 11 produce decreasing levels of M-CSF (CSF-1), a specific macrophage progenitor cell humoral regulator, following X-irradiation in vitro. The decrease did not go below 40% of control levels, even after irradiation doses of 50,000 rad (500 Gy). In contrast, a distinct humoral regulator stimulating growth of GM-CSF/IL-3 factor-dependent (FD) hematopoietic progenitor cell lines was detected following radiation to doses above 2000 rad. This humoral factor was not detectable in conditioned medium from irradiated cells, weakly detected using factor-dependent target cell populations in agar overlay, and was prominently detected by liquid co-cultivation of factor-dependent cells with irradiated stromal cell cultures. Subclonal lines of FD cells, derived after co-cultivation revealed karyotypic abnormalities and induced myeloblastic tumors in syngeneic mice. Five-eight weeks co-cultivation was required for induction of factor independence and malignancy and was associated with dense cell to cell contact between FD cells and stromal cells demonstrated by light and electron microscopy. Increases in hematopoietic to stromal cell surface area, total number of adherent cells per flask, total non-adherent cell colonies per flask, and cumulative non-adherent cell production were observed after irradiation. The present data may prove very relevant to an understanding of the cell to cell interactions during X-irradiation-induced leukemia.

  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.

    2004-07-01

    It has been established that the treatment with Histamine (Hi) produces a significant growth inhibition of different cell lines derived from human neoplasia. In a model of Knockout mice completely depleted of endogenous Hi, it was observed a significant delay in bone marroe repopulation after whole body irradiation. These results are in agreement with the hypothesis that histamine has a role in the regulation of haematopoiesis as well as an inhibitory effect on apoptosis. The objective of this paper was to study the possible effect of Hi as protector of normal cells and radiosensitizer of malignant ones. To study the effect of Hi on small-intestine and bone marrow, thirty made mice were randomly separeted into two groups: Control irradiated (C), and irradiated receiving Histamine (HI-group). All animals received a single dose of 10 Gy on whole-body employing a ''137Cs source of 189 TB{sub q} (Dose rate: 7.7 Gy/min) calibrated with TLD 700 dosimeter. Hi-group recieved a daily se injection (0.1 mg/kg) starting 20 hs before irradiation. Mice were sacrificed 5 days after irradiation. Histopathological analysis indicated that intestinal mucosae of C group showed important injury, whist mucosae of Hi-treated mice showed mild mucosal atrophy with conservation of villous projections and absence of vascular congestive changes. In order to investigate the effect of Hi on radiosensitivity of transformed cells, MDA-MB-231 (human breast carcinoma cells) were irradiated in vitro with doses ranging from 0 to 10 Gy. Results of radiobiological parameters indicate a significant increase on radiosensitivity of malignant cells. Employing specific fluorescent dyes and flow cytometric analysis we determined that the intracellular levels of hydrogen peroxide (H{sub 2}O{sub 2}) are significant increased by Hi 10 {mu}M in control and also in irradiated MDA-MB-231 cells, while the levels of superoxide (SO{sub 2}) were not significantly modified by Hi-treatment. (Author) 9 refs.

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

    Directory of Open Access Journals (Sweden)

    Masoud Hashemi Arabi

    2014-05-01

    Four breast cancer cell lines, MCF-7 ، T47D ، MDA-MB231 and MDA-MB468 were purchased from National cell Bank of Iran based in Iran Pasture Institute and were cultured in high glucose DMEM supplemented with 10% FCS. Cells were stained with antiCD44-PE and antiCD24-FITC antibodies and Status of CD44 and CD24 as markers of breast cancer stem cells were evaluated using flow cytometer and fluorescent microscopy.Evaluation of CD44 and CD24 as markers of breast cancer stem cells showed that MDA-MB231 with 97±1.2% CD44+/CD24-/low cells is significantly different from the others that they were mainly CD44 and CD24 positive cells(p

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

    OpenAIRE

    D’Angelo, Rosemarie C.; Ouzounova, Maria; Davis, April; Choi, Daejin; Tchuenkam, Stevie M.; Kim, Gwangil; Luther, Tahra; Quraishi, Ahmed A.; Senbabaoglu, Yasin; Conley, Sarah J; Shawn G Clouthier; Hassan, Khaled A.; Wicha, Max S; Korkaya, Hasan

    2015-01-01

    Developmental pathways such as Notch play a pivotal role in tissue specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch+) or reduced activity (Notch-) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays we investigated the role of Notch ...

  15. In vitro evaluation of a new nitrosourea, TCNU, against human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Roed, H; Vindeløv, L L; Spang-Thomsen, M;

    1987-01-01

    The cytotoxic activity of a new nitrosourea, TCNU, was compared with that of BCNU in five human small cell lung cancer cell lines in vitro. TCNU was found to be equivalent or inferior to BCNU when compared on a microgram to microgram basis. If the potential of in vitro phase II trials for selecti...... of new drugs can be validated, it can be concluded that TCNU is not superior to other nitrosoureas for the treatment of SCCL....

  16. Comparative In Vitro Immune Stimulation Analysis of Primary Human B Cells and B Cell Lines

    Science.gov (United States)

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

    2016-01-01

    B cell specific immunomodulatory drugs still remain an unmet medical need. Utilisation of validated simplified in vitro models would allow readily obtaining new insights in the complexity of B cell regulation. For this purpose we investigated which human B lymphocyte stimulation assays may be ideally suited to investigate new B lymphocyte immunosuppressants. Primary polyclonal human B cells underwent in vitro stimulation and their proliferation, production of immunoglobulins (Igs) and of cytokines, and expression of cell surface molecules were analysed using various stimuli. ODN2006, a toll-like receptor 9 (TLR9) agonist, was the most potent general B cell stimulus. Subsequently, we investigated on which human B cell lines ODN2006 evoked the broadest immunostimulatory effects. The Namalwa cell line proved to be the most responsive upon TLR9 stimulation and hence may serve as a relevant, homogeneous, and stable B cell model in an in vitro phenotypic assay for the discovery of new targets and inhibitors of the B cell activation processes. As for the read-out for such screening assay, it is proposed that the expression of activation and costimulatory surface markers reliably reflects B lymphocyte activation. PMID:28116319

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polym...

  18. Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidop teran Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Yan-lei Wu; Lei Jiang; Yoshifumi Hashimoto; Robert R.Granados; Guo-xun Li

    2011-01-01

    Lepidopteran heat-tolerant(ht)cell lines have been obtained with sf-9,sf-21 and several Bombyx cells.They have a distinct karyotype,membrane lipid composition,morphology and growth kinetics from the parental cell lines.In this paper,we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility.Adaptation of cell lines sf-9,BTI-TN-5131-4(High5)and BTI-TN-MG1(MG 1)to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature.The process of adaption to a higher culture temperature was accomplished over a period of 2 months.The cell lines with the temperature adaption were designated as sf9-ht33,sf9-ht35,High5-ht33,High5-ht35,MG1-ht33,MG1-ht35.These cell lines have been subcultured over 70 passages.Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines.The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines.Cell shapes did not show obvious change,however,the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption.When the cell lines were infected with Autographa californica nuclear polyhedrosis virus(AcMNPV)at 28℃,33℃,35℃ and 37℃,production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.

  19. Dynamic JUNQ inclusion bodies are asymmetrically inherited in mammalian cell lines through the asymmetric partitioning of vimentin.

    Science.gov (United States)

    Ogrodnik, Mikołaj; Salmonowicz, Hanna; Brown, Rachel; Turkowska, Joanna; Średniawa, Władysław; Pattabiraman, Sundararaghavan; Amen, Triana; Abraham, Ayelet-chen; Eichler, Noam; Lyakhovetsky, Roman; Kaganovich, Daniel

    2014-06-01

    Aging is associated with the accumulation of several types of damage: in particular, damage to the proteome. Recent work points to a conserved replicative rejuvenation mechanism that works by preventing the inheritance of damaged and misfolded proteins by specific cells during division. Asymmetric inheritance of misfolded and aggregated proteins has been shown in bacteria and yeast, but relatively little evidence exists for a similar mechanism in mammalian cells. Here, we demonstrate, using long-term 4D imaging, that the vimentin intermediate filament establishes mitotic polarity in mammalian cell lines and mediates the asymmetric partitioning of damaged proteins. We show that mammalian JUNQ inclusion bodies containing soluble misfolded proteins are inherited asymmetrically, similarly to JUNQ quality-control inclusions observed in yeast. Mammalian IPOD-like inclusion bodies, meanwhile, are not always inherited by the same cell as the JUNQ. Our study suggests that the mammalian cytoskeleton and intermediate filaments provide the physical scaffold for asymmetric inheritance of dynamic quality-control JUNQ inclusions. Mammalian IPOD inclusions containing amyloidogenic proteins are not partitioned as effectively during mitosis as their counterparts in yeast. These findings provide a valuable mechanistic basis for studying the process of asymmetric inheritance in mammalian cells, including cells potentially undergoing polar divisions, such as differentiating stem cells and cancer cells.

  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

    2006-01-01

    Full Text Available Abstract Background Pluripotent mouse embryonic stem (ES cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321 or Stem Cell Factor (SCF. Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium. RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed.

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

    Science.gov (United States)

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

    2006-01-01

    Background Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF). Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium). RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed. PMID:16436212

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

    Science.gov (United States)

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

    2008-05-01

    Cell lines are very useful for clinical and basic research. The establishment of uterine malignant tumor cell lines with unusual histology is especially important. We describe the establishment and characterization of a new human endometrial stromal sarcoma cell line of the uterus. The cell line OMC-9 was established from a tumor mass in the uterine body of a 55-year-old woman. Characteristics of the cell line studied include morphology, chromosome analysis, heterotransplantation, tumor markers and chemosensitivity. This cell line has grown well for 196 months and has been subcultured more than 50 times. Monolayer cultured cells are polygonal in shape, appear to be spindle-shaped or multipolar and have a tendency to pile up without contact inhibition. The 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.

    Science.gov (United States)

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

    2014-01-01

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

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

    DEFF Research Database (Denmark)

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

    1988-01-01

    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

    2008-09-01

    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.

    Science.gov (United States)

    Martin, G R

    1981-12-01

    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: jaepiljeon@hanmail.net [Division of Brain Diseases, Center for Biomedical Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of)

    2013-11-15

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

  8. Evaluating Prestin's Changing Biophysical Attributes in Development Using a Tet-Induced Cell Line

    Science.gov (United States)

    Bian, Shumin; Koo, Bon W.; Kelleher, Stephen; Santos-Sacchi, Joseph; Navaratnam, Dhasakumar

    2011-11-01

    The motor protein prestin is a member of the SLC26 anion transporter family, and expressed in the lateral wall of OHCs. It is now widely recognized that prestin is required for mammalian cochlear amplification. Expression of prestin precedes the onset of hearing in mice and undergoes a functional maturation within the membrane coincident with the onset of hearing. We have developed several tetracycline-inducible prestin expressing cell lines that duplicate prestins functional maturation in vivo. Thus, following induction there is an initial stage of increase in the charge carried by an individual motor (z) that accompanies a phase of slow growth in charge density. A plateau in z follows and is accompanied by rapid increase in charge density. The latter strongly correlates with an increasing ratio between an apparently larger and smaller monomer of prestin, suggesting that the latter exerts a dominant negative effect on function. Through the experimental period there is a progressive shift in the voltage of peak capacitance, similar to that observed in developing OHCs. Finally, we observed a non-selective ionic current after induction, the size of which correlated with charge density.

  9. Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro.

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2010-11-15

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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...

  12. 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;

    2013-01-01

    /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....

  13. 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

    2008-01-01

    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.

  14. 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

    2010-05-04

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

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

    Directory of Open Access Journals (Sweden)

    Lu Yontao

    2010-06-01

    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

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

    DEFF Research Database (Denmark)

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines...... based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted...... 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...

  17. EXPRESSION OF THE O6-METHYLGUANINE-DNA METHYLTRANSFERASE GENE IN EIGHT HUMAN TUMOR CELL LINES

    Institute of Scientific and Technical Information of China (English)

    陈建敏; 章扬培; 吴英

    1994-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    David Hare

    2016-11-01

    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.

  19. CELLULAR BASIS FOR DIFFERENTIAL SENSITIVITY TO CISPLATIN IN HUMAN GERM-CELL TUMOR AND COLON-CARCINOMA CELL-LINES

    NARCIS (Netherlands)

    SARK, MWJ; TIMMERBOSSCHA, H; MEIJER, C; UGES, DRA; SLUITER, WJ; PETERS, WHM; MULDER, NH; DEVRIES, EGE

    1995-01-01

    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

  20. 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;

    2005-01-01

    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...

  1. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    Science.gov (United States)

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

    1987-11-01

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

  2. Experimental impact of aspirin exposure on rat intestinal bacteria, epithelial cells and cell line.

    Science.gov (United States)

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

    2010-10-01

    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.

  3. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology.

    Science.gov (United States)

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

    2016-10-14

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

  4. 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.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Meineke Viktor

    2011-05-01

    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

  7. 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

    2001-01-01

    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.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. 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.

  10. 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)

    1985-06-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-05-01

    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.

  12. Tumor suppressors status in cancer cell line Encyclopedia.

    Science.gov (United States)

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

    2013-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Holz David

    2008-01-01

    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.

    Science.gov (United States)

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

    2012-09-01

    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.

    Science.gov (United States)

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

    2013-11-01

    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. Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

    Science.gov (United States)

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

    2013-10-01

    This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P 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.

  17. 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.

    Science.gov (United States)

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

    2013-02-01

    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.

  18. Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5.

    Directory of Open Access Journals (Sweden)

    Yaíma L Lightfoot

    Full Text Available Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA depleted βLox5 cells, or βLox5 ρ(0 cells. βLox5 ρ(0 cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ(0 cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    J Tavakkol Afshari

    2010-07-01

    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.

  1. Check your cultures! A list of cross-contaminated or misidentified cell lines.

    Science.gov (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

    2010-07-01

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

  2. Whole-exome characterization of pancreatic neuroendocrine tumor cell lines BON-1 and QGP-1.

    Science.gov (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

    2015-04-01

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

  3. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.

    Science.gov (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

    2015-11-13

    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.

  4. 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

    2005-01-01

    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.

  5. FTIR characterization of animal lung cells: normal and precancerous modified e10 cell line

    Science.gov (United States)

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

    2012-06-01

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

  6. The culture of cancer cell lines as tumorspheres does not systematically result in cancer stem cell enrichment.

    Science.gov (United States)

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

    2014-01-01

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

  7. Lactobacillus in Preventing Infection in Patients Undergoing a Donor Stem Cell Transplant for Hematologic Cancer or Myelodysplastic Syndrome

    Science.gov (United States)

    2017-02-02

    Breast Cancer; Chronic Myeloproliferative Disorders; Leukemia; Lymphoma; Multiple Myeloma and Plasma Cell Neoplasm; Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Neoplasms; Neuroblastoma; Ovarian Cancer; Testicular Germ Cell Tumor

  8. Real-life comparison of severe vascular events and other non-hematological complications in patients with chronic myeloid leukemia undergoing second-line nilotinib or dasatinib treatment.

    Science.gov (United States)

    Gora-Tybor, Joanna; Medras, Ewa; Calbecka, Malgorzata; Kolkowska-Leśniak, Agnieszka; Ponikowska-Szyba, Edyta; Robak, Tadeusz; Jamroziak, Krzysztof

    2015-01-01

    We retrospectively analyzed the rates of significant non-hematological adverse events (AEs) in 105 patients with chronic myeloid leukemia (CML) treated with second-generation tyrosine kinase inhibitor (TKIs) dasatinib or nilotinib used as second-line therapy in Polish tertiary care centers. Our analysis revealed that in a "real life setting," nearly half of patients with CML on second-generation TKIs suffer from therapy complications. Grade 2-5 non-hematological AEs were observed in 40% of patients treated with nilotinib and in 42% treated with dasatinib (p=0.83). Severe vascular events including peripheral artery occlusive disease (PAOD) occurred in 11% of patients on nilotinib and 4% on dasatinib (p=0.16). Pleural effusion occurred more often in the dasatinib group (26%) than in the nilotinib group (2%) (p=0.003). Importantly, most AEs occurred late, after more than 1 year of treatment. Since AEs are most often the reason for poor therapy compliance, careful monitoring of tolerability is crucial for an optimal treatment response in CML.

  9. Dose Monitoring of Busulfan and Combination Chemotherapy in Hodgkin or Non-Hodgkin Lymphoma Undergoing Stem Cell Transplant

    Science.gov (United States)

    2015-08-12

    Adult Grade III Lymphomatoid Granulomatosis; Adult Nasal Type Extranodal NK/T-cell Lymphoma; Anaplastic Large Cell Lymphoma; Angioimmunoblastic T-cell Lymphoma; Childhood Burkitt Lymphoma; Childhood Diffuse Large Cell Lymphoma; Childhood Grade III Lymphomatoid Granulomatosis; Childhood Immunoblastic Large Cell Lymphoma; Childhood Nasal Type Extranodal NK/T-cell Lymphoma; Contiguous Stage II Adult Burkitt Lymphoma; Contiguous Stage II Adult Diffuse Large Cell Lymphoma; Contiguous Stage II Adult Diffuse Mixed Cell Lymphoma; Contiguous Stage II Adult Diffuse Small Cleaved Cell Lymphoma; Contiguous Stage II Adult Immunoblastic Large Cell Lymphoma; Contiguous Stage II Adult Lymphoblastic Lymphoma; Contiguous Stage II Grade 1 Follicular Lymphoma; Contiguous Stage II Grade 2 Follicular Lymphoma; Contiguous Stage II Grade 3 Follicular Lymphoma; Contiguous Stage II Mantle Cell Lymphoma; Contiguous Stage II Marginal Zone Lymphoma; Contiguous Stage II Small Lymphocytic Lymphoma; Cutaneous B-cell Non-Hodgkin Lymphoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Hepatosplenic T-cell Lymphoma; Intraocular Lymphoma; Nodal Marginal Zone B-cell Lymphoma; Noncontiguous Stage II Adult Burkitt Lymphoma; Noncontiguous Stage II Adult Diffuse Large Cell Lymphoma; Noncontiguous Stage II Adult Diffuse Mixed Cell Lymphoma; Noncontiguous Stage II Adult Diffuse Small Cleaved Cell Lymphoma; Noncontiguous Stage II Adult Immunoblastic Large Cell Lymphoma; Noncontiguous Stage II Adult Lymphoblastic Lymphoma; Noncontiguous Stage II Grade 1 Follicular Lymphoma; Noncontiguous Stage II Grade 2 Follicular Lymphoma; Noncontiguous Stage II Grade 3 Follicular Lymphoma; Noncontiguous Stage II Mantle Cell Lymphoma; Noncontiguous Stage II Marginal Zone Lymphoma; Noncontiguous Stage II Small Lymphocytic Lymphoma; Noncutaneous Extranodal Lymphoma; Peripheral T-cell Lymphoma; Recurrent Adult Burkitt Lymphoma; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult

  10. 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

    2013-01-01

    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

  11. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses.

    Science.gov (United States)

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

    1985-01-01

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

  12. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses*

    Science.gov (United States)

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

    1985-01-01

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

  13. Morphological and molecular characterization of an undifferentiated soft tissue sarcoma cell line and derivative clones.

    Science.gov (United States)

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

    1997-09-01

    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.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    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....

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

    Directory of Open Access Journals (Sweden)

    Oliveira Paula A

    2009-01-01

    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.

  16. 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

    2007-01-01

    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.

  17. Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells.

    Science.gov (United States)

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

    2011-02-01

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

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

    Directory of Open Access Journals (Sweden)

    Silvia A Fuertes Marraco

    2012-11-01

    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.

  19. Postendocytic sorting of constitutively internalized dopamine transporter in cell lines and dopaminergic neurons

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Bjørn-Yoshimoto, Walden Emil; Jørgensen, Trine Nygaard;

    2010-01-01

    The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cell...

  20. 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

    2006-01-01

    AIM: To investigate the relationship between the inhibited growth (cytotoxic activity) of berberine and apoptotic pathway with its molecular mechanism of action.METHODS: The in vitro cytotoxic techniques were complemented by cell cycle analysis and determination of sub-G1 for apoptosis in human gastric carcinoma SNU-5 cells. Percentage of viable cells, cell cycle, and sub-G1 group (apoptosis) were examined and determined by the flow cytometric methods. The associated proteins for cell cycle arrest and apoptosis were examined by Western blotting.RESULTS: For SNU-5 cell line, the IC (50) was found to be 48 μmol/L of berberine. In SNU-5 cells treated with 25-200 μmol/L berberine, G2/M cell cycle arrest was observed which was associated with a marked increment of the expression of p53, Wee1 and CDk1 proteins and decreased cyclin B. A concentration-dependent decrease of cells in G0/G1 phase and an increase in G2/M phase were detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved 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.

  1. TGF-β1/SMAD SIGNALING PATHWAY MEDIATES p53-DEPENDENT APOPTOSIS IN HEPATOMA CELL LINES

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    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.

  2. 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

    2011-01-01

    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

  3. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

    Science.gov (United States)

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

    2015-02-01

    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.

  4. Graft-Versus-Host Disease Prophylaxis in Treating Patients With Hematologic Malignancies Undergoing Unrelated Donor Peripheral Blood Stem Cell Transplant

    Science.gov (United States)

    2016-08-18

    Acute Lymphoblastic Leukemia; Acute Myeloid Leukemia; Aggressive Non-Hodgkin Lymphoma; Chronic Lymphocytic Leukemia; Diffuse Large B-Cell Lymphoma; Hematopoietic and Lymphoid Cell Neoplasm; Indolent Non-Hodgkin Lymphoma; Mantle Cell Lymphoma; Myelodysplastic Syndrome; Myeloproliferative Neoplasm; Prolymphocytic Leukemia; Recurrent Chronic Lymphocytic Leukemia; Recurrent Plasma Cell Myeloma; Refractory Chronic Lymphocytic Leukemia; Refractory Chronic Myelogenous Leukemia, BCR-ABL1 Positive; Refractory Hodgkin Lymphoma; Small Lymphocytic Lymphoma; T-Cell Chronic Lymphocytic Leukemia; Waldenstrom Macroglobulinemia

  5. The Tribolium castaneum cell line TcA: a new tool kit for cell biology.

    Science.gov (United States)

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

    2014-10-30

    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.

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

    Science.gov (United States)

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

    1989-01-01

    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.

  7. Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

    Science.gov (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

    2016-01-01

    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

  8. Biological activity assays of cell-free reassembled nuclei——Injecting cell-free reassembled nuclei into unfertilized eggs can induce the eggs to cleave and reconstitute asters,and the injected nuclei undergo cell cycle changes

    Institute of Scientific and Technical Information of China (English)

    张传茂; 曲健; 梁金华; 翟中和

    1995-01-01

    Nucleus may reassemble spontaneously in cell-free mixture of HeLa metaphase chromosomes,Xenopus egg extracts and ATP-regenerating system,and the nucleus shows some biological activities.It isfound that,after being injected into unfertilized mature eggs,the cell-free reassembled nuclei can cause theeggs to cleave and reconstitute asters in their cytoplasm,and the injected nuclei undergo changes in response tocell cycle regulators stored in the eggs,and that reinjecting cytostatic factors(CSF)into the eggs can stabilizethe eggs in mitotic phase,cause the nuclei disassembly and chromatin condensation to chromosomes.

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

    OpenAIRE

    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

    2011-01-01

    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 ...

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

    Science.gov (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.

    2011-01-01

    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

  11. Efficacy of oral cryotherapy on oral mucositis prevention in patients with hematological malignancies undergoing hematopoietic stem cell transplantation: a meta-analysis of randomized controlled trials.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Controversy exists regarding whether oral cryotherapy can prevent oral mucositis (OM in patients with hematological malignancies undergoing hematopoietic stem cell transplantation (HSCT. The aim of the present meta-analysis was to evaluate the efficacy of oral cryotherapy for OM prevention in patients with hematological malignancies undergoing HSCT.PubMed and the Cochrane Library were searched through October 2014. Randomized controlled trials (RCTs comparing the effect of oral cryotherapy with no treatment or with other interventions for OM in patients undergoing HSCT were included. The primary outcomes were the incidence, severity, and duration of OM. The secondary outcomes included length of analgesic use, total parenteral nutrition (TPN use, and length of hospital stay.Seven RCTs involving eight articles analyzing 458 patients were included. Oral cryotherapy significantly decreased the incidence of severe OM (RR = 0.52, 95% CI = 0.27 to 0.99 and OM severity (SMD = -2.07, 95% CI = -3.90 to -0.25. In addition, the duration of TPN use and the length of hospitalization were markedly reduced (SMD = -0.56, 95% CI = -0.92 to -0.19; SMD = -0.44, 95% CI = -0.76 to -0.13; respectively. However, the pooled results were uncertain for the duration of OM and analgesic use (SMD = -0.13, 95% CI = -0.41 to 0.15; SMD = -1.15, 95% CI = -2.57 to 0.27; respectively.Oral cryotherapy is a readily applicable and cost-effective prophylaxis for OM in patients undergoing HSCT.

  12. Efficacy of Oral Cryotherapy on Oral Mucositis Prevention in Patients with Hematological Malignancies Undergoing Hematopoietic Stem Cell Transplantation: A Meta-Analysis of Randomized Controlled Trials

    Science.gov (United States)

    Zhai, Ruiren; Zhao, Shasha; Luo, Lan; Li, Dandan; Zhao, Xiaoli; Wei, Huaping; Pang, Zhaoxia; Wang, Lili; Liu, Daihong; Wang, Quanshun; Gao, Chunji

    2015-01-01

    Objectives Controversy exists regarding whether oral cryotherapy can prevent oral mucositis (OM) in patients with hematological malignancies undergoing hematopoietic stem cell transplantation (HSCT). The aim of the present meta-analysis was to evaluate the efficacy of oral cryotherapy for OM prevention in patients with hematological malignancies undergoing HSCT. Methods PubMed and the Cochrane Library were searched through October 2014. Randomized controlled trials (RCTs) comparing the effect of oral cryotherapy with no treatment or with other interventions for OM in patients undergoing HSCT were included. The primary outcomes were the incidence, severity, and duration of OM. The secondary outcomes included length of analgesic use, total parenteral nutrition (TPN) use, and length of hospital stay. Results Seven RCTs involving eight articles analyzing 458 patients were included. Oral cryotherapy significantly decreased the incidence of severe OM (RR = 0.52, 95% CI = 0.27 to 0.99) and OM severity (SMD = -2.07, 95% CI = -3.90 to -0.25). In addition, the duration of TPN use and the length of hospitalization were markedly reduced (SMD = -0.56, 95% CI = -0.92 to -0.19; SMD = -0.44, 95% CI = -0.76 to -0.13; respectively). However, the pooled results were uncertain for the duration of OM and analgesic use (SMD = -0.13, 95% CI = -0.41 to 0.15; SMD = -1.15, 95% CI = -2.57 to 0.27; respectively). Conclusions Oral cryotherapy is a readily applicable and cost-effective prophylaxis for OM in patients undergoing HSCT. PMID:26024220

  13. 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: akikuri@hotmail.com [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)

    2015-08-07

    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.

  14. Cell and molecular biology of SAE, a cell line from the spiny dogfish shark, Squalus acanthias.

    Science.gov (United States)

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

    2007-02-01

    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.

  15. Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.

    Science.gov (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

    2012-08-10

    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.

  16. 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)

    2002-01-01

    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

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

    Institute of Scientific and Technical Information of China (English)

    潘耀柱

    2013-01-01

    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

  18. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Science.gov (United States)

    2010-01-01

    ... 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...

  19. 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

    1996-01-01

    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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    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...

  1. Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells.

    Science.gov (United States)

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

    2012-02-01

    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.

  2. Amygdalin induces apoptosis in human cervical cancer cell line HeLa cells.

    Science.gov (United States)

    Chen, Yu; Ma, Jinshu; Wang, Fang; Hu, Jie; Cui, Ai; Wei, Chengguo; Yang, Qing; Li, Fan

    2013-02-01

    Amygdalin, a naturally occurring substance, has been suggested to be efficacious as an anticancer substance. The effect of amygdalin on cervical cancer cells has never been studied. In this study, we found that the viability of human cervical cancer HeLa cell line was significantly inhibited by amygdalin. 4,6-Diamino-2-phenyl indole (DAPI) staining showed that amygdalin-treated HeLa cells developed typical apoptotic changes. The development of apoptosis in the amygdalin-treated HeLa cells were confirmed by double staining of amygdalin-treated HeLa cells with annexin V-FITC and propidium iodide (PI) along with increase in caspase-3 activity in these cells. Further studies indicated that antiapoptotic protein Bcl-2 was downregulated whereas proapoptotic Bax protein was upregulated in the amygdalin-treated HeLa cells implying involvement of the intrinsic pathway of apoptosis. In vivo, amygdalin administration inhibited the growth of HeLa cell xenografts through a mechanism of apoptosis. The results in the present study suggest that amygdalin may offer a new therapeutic option for patients with cervical cancer.

  3. Induction of cell death by ascorbic acid derivatives in human renal carcinoma and glioblastoma cell lines.

    Science.gov (United States)

    Makino, Y; Sakagami, H; Takeda, M

    1999-01-01

    Sodium-L-ascorbate, L-ascorbic acid, D-isoascorbic acid, sodium 5,6-benzylidene-L-ascorbate and sodium-6-beta-O-galactosyl-L-ascorbate, which produce ascorbyl radicals during the oxidative degradation, also induced cytotoxicity against cultured human renal carcinoma (TC-1) and glioblastoma multiform tumor (T98G) cell lines. On the other hand, L-ascorbic acid 2-phosphate magnesium and L-ascorbic acid 2-sulfate dipotassium salt, which do not produce the ascorbyl radical, were inactive. This suggests the possible role of the ascorbyl radical for cell death induction. T98G cells were more resistant to ascorbate analogs than TC-1 and HL-60 cells, possibly due to higher intracellular glutathione concentrations. Ascorbate treatment induced rapid elevation of both intracellular concentration of cAMP and Ca2+ in HL-60 cells, but not in TC-1 and T98G cells. However, the elevation of cAMP by theophyline and N,2-dibutyryl adenosine 3,5 cyclic monophosphate (dibutyryl cAMP) resulted in a decrease in the viable cell number. This suggests the possible role of cAMP for ascorbate-induced cell death.

  4. Apoptotic-like death occurs through a caspase-independent route in colon carcinoma cells undergoing mitotic catastrophe.

    Science.gov (United States)

    Llovera, Laia; Mansilla, Sylvia; Portugal, José

    2012-12-29

    We have examined the relationship between chemotherapy-induced mitotic catastrophe and cell death by apoptosis in both wild-type and p53(-/-) HCT116 human colon carcinoma cells treated with nanomolar concentrations of paclitaxel (PTX), a drug that acts on tubulin altering the normal development of mitosis. After treatment, HCT116 cells entered mitosis regardless of the presence of functional p53, which resulted in changes in the distribution of cells in the different phases of the cell cycle, and in cell death. In the presence of PTX, the percentage of polyploid cells observed was higher in p53-deficient cells, indicating that mitotic slippage was favored compared to wild-type cells, with the presence of large multinucleate cells. PTX caused mitotic catastrophe and about 50-60% cells that were entering an aberrant mitosis died through an apoptotic-like pathway characterized by the presence of phosphatidylserine in the outer cell membrane, which occurred in the absence of significant activation of caspases. Lack of p53 facilitated endoreduplication and polyploidy in PTX-treated cells, but cells were still killed with similar efficacy through the same apoptotic-like mechanism in the absence of caspase activity.

  5. Generation of KCL033 clinical grade human embryonic stem cell line

    OpenAIRE

    2016-01-01

    The KCL033 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility...

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

    Science.gov (United States)

    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; Clouthier, Shawn G; Hassan, Khaled A; Wicha, Max S; Korkaya, Hasan

    2015-03-01

    Developmental pathways such as Notch play a pivotal role in tissue-specific stem cell self-renewal as well as in tumor development. However, the role of Notch signaling in breast cancer stem cells (CSC) remains to be determined. We utilized a lentiviral Notch reporter system to identify a subset of cells with a higher Notch activity (Notch(+)) or reduced activity (Notch(-)) in multiple breast cancer cell lines. Using in vitro and mouse xenotransplantation assays, we investigated the role of the Notch pathway in breast CSC regulation. Breast cancer cells with increased Notch activity displayed increased sphere formation as well as expression of breast CSC markers. Interestingly Notch(+) cells displayed higher Notch4 expression in both basal and luminal breast cancer cell lines. Moreover, Notch(+) cells demonstrated tumor initiation capacity at serial dilutions in mouse xenografts, whereas Notch(-) cells failed to generate tumors. γ-Secretase inhibitor (GSI), a Notch blocker but not a chemotherapeutic agent, effectively targets these Notch(+) cells in vitro and in mouse xenografts. Furthermore, elevated Notch4 and Hey1 expression in primary patient samples correlated with poor patient survival. Our study revealed a molecular mechanism for the role of Notch-mediated regulation of breast CSCs and provided a compelling rationale for CSC-targeted therapeutics.

  7. Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

    Science.gov (United States)

    Weber, C.; Pohl, S.; Poertner, R.; Pino-Grace, Pablo; Freimark, D.; Wallrapp, C.; Geigle, P.; Czermak, P.

    Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

  8. Radiobiological modeling analysis of the optimal fraction scheme in patients with peripheral non-small cell lung cancer undergoing stereotactic body radiotherapy

    OpenAIRE

    Bao-Tian Huang; Jia-Yang Lu; Pei-Xian Lin; Jian-Zhou Chen; De-Rui Li; Chuang-Zhen Chen

    2015-01-01

    This study aimed to determine the optimal fraction scheme (FS) in patients with small peripheral non-small cell lung cancer (NSCLC) undergoing stereotactic body radiotherapy (SBRT) with the 4 × 12 Gy scheme as the reference. CT simulation data for sixteen patients diagnosed with primary NSCLC or metastatic tumor with a single peripheral lesion ≤3 cm were used in this study. Volumetric modulated arc therapy (VMAT) plans were designed based on ten different FS of 1 × 25 Gy, 1 × 30 Gy, 1 × 34 Gy...

  9. Early infections in patients undergoing high-dose treatment with stem cell support: a comparison of patients with non-Hodgkin lymphoma and multiple myeloma

    DEFF Research Database (Denmark)

    Gang, A O; Arpi, M.; Gang, U.J.O.;

    2010-01-01

    Background: Infections are life-threatening complications in patients undergoing high-dose chemotherapy with stem cell support (HDT). Knowledge of the infectious pathogens is essential to make a safe outpatient setting. Methods: We conducted a retrospective study of 208 patients treated with HDT...... related mortality was similar between the groups. Conclusion: The frequency of isolated pathogens, positive blood cultures, and the diversity of pathogens were higher in MM patients as compared to NHL patients. However, this did not translate into higher transplantation-related mortality, probably because...

  10. BCSH/BSBMT/UK clinical virology network guideline: diagnosis and management of common respiratory viral infections in patients undergoing treatment for haematological malignancies or stem cell transplantation.

    Science.gov (United States)

    Dignan, Fiona L; Clark, Andrew; Aitken, Celia; Gilleece, Maria; Jayakar, Vishal; Krishnamurthy, Pramila; Pagliuca, Antonio; Potter, Michael N; Shaw, Bronwen; Skinner, Roderick; Turner, Andrew; Wynn, Robert F; Coyle, Peter

    2016-05-01

    A joint working group established by the Haemato-oncology subgroup of the British Committee for Standards in Haematology, the British Society for Bone Marrow Transplantation and the UK Clinical Virology Network has reviewed the available literature and made recommendations for the diagnosis and management of respiratory viral infections in patients with haematological malignancies or those undergoing haematopoietic stem cell transplantation. This guideline includes recommendations for the diagnosis, prevention and treatment of respiratory viral infections in adults and children. The suggestions and recommendations are primarily intended for physicians practising in the United Kingdom.

  11. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, K.; Tezuka, H.; Sakoda, H.; Konno, M.; Nagata, K.; Uchiyama, T.; Uchino, H.; Mori, K.J.

    1989-02-01

    Stromal cell lines, designated MS-1, -2, -3, -4, -5, -6, and -7 were established by irradiating the adherent cells in long-term bone marrow cultures with 900-rad x-rays. Two of the cell lines, MS-1 and MS-5, have the capacity to support the growth of hemopoietic stem cells (spleen colony-forming cells and granulocyte-macrophage colony-forming cells) for greater than 2 months in vitro. These two cell lines were alkaline phosphatase-, peroxidase-, and factor VIII-negative and positive for periodic acid-Schiff and nonspecific esterase. Extracellular matrix proteins such as fibronectin, laminin, and collagen type I were produced by these two cell lines. Neither MS-1 cell- nor MS-5 cell-conditioned medium supported the growth of hemopoietic stem cells, and hemopoietic stem cells were found preferentially to be under and on MS-1 and MS-5 layers rather than in suspension. Close contact with the MS-1 cell layer or the MS-5 cell layer appears to be essential in maintaining hemopoiesis in vitro. Conditioned media from MS-1 cells and MS-5 cells stimulated granulocyte colony formation from murine bone marrow cells in semisolid culture.

  12. Development of PIN and Prostate Adenocarcinoma Cell Lines: A Model System for Multistage Tumor Progression

    Directory of Open Access Journals (Sweden)

    Colin R. Soares

    2002-01-01

    Full Text Available Existing prostate cancer cell lines have been derived from late stages of human prostate cancer. In this paper, we present two cell lines generated from prostatic intraepithelial neoplasia (PIN, the precursor lesion for prostate adenocarcinoma. Pr-111 and Pr-117 were established from PIN lesions that developed in the C3(1/Tag transgenic model of prostate cancer. Pr-111 and Pr-117 cells express simian virus 40 large T antigen (SV40 Tag and are immortalized in culture, distinguishing them from normal prostate cells. The growth rates of these two cell lines are quite different; with Pr-111 cells growing much more slowly (doubling time approximately 40 hours compared to Pr-117 cells (doubling time approximately 22 hours, and also show significantly different growth rates in different media. Both prostate cell lines express cytokeratin and androgen receptor (AR with Pr-111 cells demonstrating androgen-dependent growth and Pr-117 cells exhibiting androgen-responsive growth characteristics. Athymic nude mice injected with Pr-111 cells either do not develop tumors or develop tumors after a long latency period of 14 weeks. Pr-117 cells, however, develop tumors by 3 to 6 weeks, suggesting that Pr-117 cells represent a later stage of tumor progression. These two novel cell lines will be useful for studying early stages of prostate tumor development and androgen responsiveness.

  13. Propagation of Asian isolates of canine distemper virus (CDV in hamster cell lines

    Directory of Open Access Journals (Sweden)

    Yamaguchi Ryoji

    2009-10-01

    Full Text Available Abstract Backgrounds The aim of this study was to confirm the propagation of various canine distemper viruses (CDV in hamster cell lines of HmLu and BHK, since only a little is known about the possibility of propagation of CDV in rodent cells irrespective of their epidemiological importance. Methods The growth of CDV in hamster cell lines was monitored by titration using Vero.dogSLAMtag (Vero-DST cells that had been proven to be susceptible to almost all field isolates of CDV, with the preparations of cell-free and cell-associated virus from the cultures infected with recent Asian isolates of CDV (13 strains and by observing the development of cytopathic effect (CPE in infected cultures of hamster cell lines. Results Eleven of 13 strains grew in HmLu cells, and 12 of 13 strains grew in BHK cells with apparent CPE of cell fusion in the late stage of infection. Two strains and a strain of Asia 1 group could not grow in HmLu cells and BHK cells, respectively. Conclusion The present study demonstrates at the first time that hamster cell lines can propagate the majority of Asian field isolates of CDV. The usage of two hamster cell lines suggested to be useful to characterize the field isolates biologically.

  14. Increased sensitivity of an adriamycin-resistant human small cell lung carcinoma cell line to mitochondrial inhibitors

    NARCIS (Netherlands)

    de Jong, Steven; Holtrop, M; de Vries, H; de Vries, Liesbeth; Mulder, N H

    1992-01-01

    The energy metabolism of an atypical multidrug resistant human small cell lung carcinoma cell line (GLC4/ADR) was studied. The glycolytic rate was 30% reduced and the glucose-6-phosphate dehydrogenase activity 2-fold increased in GLC4/ADR compared to the parental sensitive line (GLC4). Although mito

  15. Cell line-specific accumulation of the baculovirus non-hr origin of DNA replication in infected insect cells

    NARCIS (Netherlands)

    Pijlman, G.P.; Vermeesch, A.M.G.; Vlak, J.M.

    2003-01-01

    Successive Viral passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in the S. exigua cell line Se301 leads to the rapid accumulation of the non-hr origin of DNA replication (ori) as large concatemers. Passage of SeMNPV in two other S. exigua cell lines, SeUCR1 and SeIZD2109, did

  16. The transcriptional diversity of 25 Drosophila cell lines

    Energy Technology Data Exchange (ETDEWEB)