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Sample records for human cell line

  1. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    OpenAIRE

    Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

    1988-01-01

    The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting f...

  2. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    Science.gov (United States)

    Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

    1988-09-01

    The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

  3. Fraction against Human Cancer Cell Lines

    African Journals Online (AJOL)

    fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and ... The morphology of the HepG2 cell nucleus was investigated by Hoechst 33342, ..... Gong F, Liang Y, Xie P, Chau F. Information theory.

  4. 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.

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

    NARCIS (Netherlands)

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

    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

  6. Peptidomic analysis of human cell lines

    Science.gov (United States)

    Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

    2011-01-01

    Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

  7. (Asteraceae) Fraction against Human Cancer Cell Lines

    African Journals Online (AJOL)

    Purpose: To investigate the anti-proliferative and apoptotic activity of crude and dichloromethane fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and L929). Methods: A. sieberi was extracted with methanol and further purification was carried out using liquidliquid extraction ...

  8. 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.

  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. Novel human multiple myeloma cell line UHKT-893

    Czech Academy of Sciences Publication Activity Database

    Uherková, L.; Vančurová, I.; Vyhlídalová, I.; Pleschnerová, M.; Špička, I.; Mihalová, R.; Březinová, J.; Hodný, Zdeněk; Čermáková, K.; Polanská, V.; Marinov, I.; Jedelský, P.L.; Kuželová, K.; Stöckbauer, P.

    2013-01-01

    Roč. 37, č. 3 (2013), s. 320-326 ISSN 0145-2126 Institutional support: RVO:68378050 Keywords : human myeloma cell line * human multiple myeloma * plasma cell * IL-6 dependence * immunoglobulin * free light chain Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.692, year: 2013

  11. Peroxisomal abnormalities in the immortalized human hepatocyte (IHH) cell line.

    Science.gov (United States)

    Klouwer, Femke C C; Koster, Janet; Ferdinandusse, Sacha; Waterham, Hans R

    2017-04-01

    The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

  12. Radiation response of haematopoietic cell lines of human origin

    International Nuclear Information System (INIS)

    Lehnert, S.; Rybka, W.B.; Suissa, S.; Giambattisto, D.

    1986-01-01

    Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radio-sensitivity of the cells varied widely, the D 0 s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivities. (author)

  13. Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: Utility and pitfalls

    Directory of Open Access Journals (Sweden)

    Ashley R.P. Hinson

    2013-07-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  15. Radiation sensitivity of human lung cancer cell lines

    International Nuclear Information System (INIS)

    Carmichael, J.; Degraff, W.G.; Gamson, J.; Russo, G.; Mitchell, J.B.; Gazdar, A.F.; Minna, J.D.; Levitt, M.L.

    1989-01-01

    X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens. (author)

  16. Bifenthrin activates homotypic aggregation in human T-cell lines.

    Science.gov (United States)

    Hoffman, Nataly; Tran, Van; Daniyan, Anthony; Ojugbele, Olutosin; Pryor, Stephen C; Bonventre, Josephine A; Flynn, Katherine; Weeks, Benjamin S

    2006-03-01

    Here, we addressed the concern that, despite the lack of overt toxicity, exposure to low levels of the common household pyrethroid pesticide, bifenthrin, could cause harm to the immune system. To do this, we measure the effect of bifenthrin on phytohemagglutinin (PHA) activation of homotypic aggregation in human T-cell lines. The human CD4+ H9, and Jurkat cell lines and the human promonocyte U937 cell line, were exposed to varying concentrations of bifenthrin. Cell viability was determined using the AlmarBlue Toxicity Assay. Concentrations of bifenthrin which did not reduce cell viability were determined and these concentrations were tested for the effect of bifenthrin on PHA-mediated homotypic aggregation. Blocking antibodies to ICAM and LFA-1 were used to disrupt aggregation and a nonspecific IgG was used as a control. Bifenthrin was found to be nontoxic at concentrations ranging from 10(-4) to 10(-13) M. Bifenthrin did not inhibit PHA induced cell aggregation in all cell lines tested. However, at 10(-4) M, bifenthrin to form aggregates stimulated homotypic aggregation in the H9 and Jurkat T-cell lines. The bifenthrin-induced aggregate formation, like that seen with PHA, was blocked by treating the cells with antibodies to either LFA-1 or ICAM. The results here show that bifenthrin activates T-cell function by stimulating ICAM/LFA-1 mediated homotypic aggregation. This data suggests that exposure to bifenthrin, even at "acceptable" limits, can increase the risk for and frequency of inflammatory responses and diseases such as asthma.

  17. 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.

  18. Apoptosis induction of epifriedelinol on human cervical cancer cell line

    African Journals Online (AJOL)

    Background: Present investigation evaluates the antitumor activity of epifriedelinol for the management of cervical cancer by inducing process of apoptosis. Methods: Human Cervical Cancer Cell Line, C33A and HeLa were selected for study and treated with epifriedelinol at a concentration of (50-1000 μg/ml). Cytotoxicity of ...

  19. 77 FR 5489 - Identification of Human Cell Lines Project

    Science.gov (United States)

    2012-02-03

    ... individual or species. With the advent of standardized, simple, and rapid methods for human cell line... project will undergo STR profiling, a DNA profiling method that examines/screens for STRs (DNA elements 2... distinct DNA profile and when the STR DNA fragment sizes are converted to numeric values, the DNA profiles...

  20. Fibronectin synthesized by a human hepatoma cell line

    International Nuclear Information System (INIS)

    Glasgow, J.E.; Colman, R.W.

    1984-01-01

    Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-[ 35 S]methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. It was shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis

  1. Human embryonic stem cell lines model experimental human cytomegalovirus latency.

    Science.gov (United States)

    Penkert, Rhiannon R; Kalejta, Robert F

    2013-05-28

    Herpesviruses are highly successful pathogens that persist for the lifetime of their hosts primarily because of their ability to establish and maintain latent infections from which the virus is capable of productively reactivating. Human cytomegalovirus (HCMV), a betaherpesvirus, establishes latency in CD34(+) hematopoietic progenitor cells during natural infections in the body. Experimental infection of CD34(+) cells ex vivo has demonstrated that expression of the viral gene products that drive productive infection is silenced by an intrinsic immune defense mediated by Daxx and histone deacetylases through heterochromatinization of the viral genome during the establishment of latency. Additional mechanistic details about the establishment, let alone maintenance and reactivation, of HCMV latency remain scarce. This is partly due to the technical challenges of CD34(+) cell culture, most notably, the difficulty in preventing spontaneous differentiation that drives reactivation and renders them permissive for productive infection. Here we demonstrate that HCMV can establish, maintain, and reactivate in vitro from experimental latency in cultures of human embryonic stem cells (ESCs), for which spurious differentiation can be prevented or controlled. Furthermore, we show that known molecular aspects of HCMV latency are faithfully recapitulated in these cells. In total, we present ESCs as a novel, tractable model for studies of HCMV latency.

  2. Effects of hypoxia on human cancer cell line chemosensitivity

    Science.gov (United States)

    2013-01-01

    Background Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia. Methods A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA). Results Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs. Conclusions A panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia. PMID:23829203

  3. 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.

  4. Effects of cholera toxin on human colon carcinoma cell lines.

    Science.gov (United States)

    Barkla, D H; Whitehead, R H; Hayward, I P

    1992-10-01

    This study reports on changes in morphology and membrane transport in 5 human colon carcinoma cell lines treated with cholera toxin (CT). Three of the cell lines that grew as monolayers (LIM 1215, LIM 1899, LIM 2099) and 1 that grew as floating clumps (LIM 2408) did not show morphological changes after CT treatment. However, cell line LIM 1863 that grows as floating "crypt-like" organoids showed rapid and distinctive changes in morphology and membrane transport after CT treatment. At 1 and 6 hrs after CT treatment, light and transmission electron microscopy revealed rapid dilatation of the central lumen of organoids and the appearance of 2 populations of apical vesicular inclusions. The first population was unusual in being non-membrane bound and limited by fuzzy filamentous material. The second population was membrane bound. Scanning electron microscopy at 1-6 hr after CT treatment showed swelling and loss of surface microvilli on some, but not all, cells. At 24 hr after CT treatment the majority of organoids showed evidence of fluid accumulation and small apical vesicles coalesced to form large single vacuoles that obliterated normal cell morphology. By 48 hr, continued swelling produced extreme attenuation of the plasma membrane with cells taking on an "endothelial cell-like" appearance. The response to CT was dose-dependent. Uptake studies using 86Rubidium and blocking studies using ouabain and amiloride indicated that CT is acting on the Na+/K+ ATPase membrane pump to cause the increased fluid uptake by LIM 1863 cells. This study is the first to report specific morphological changes in intestine-derived cells in response to CT.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Culture of human cell lines by a pathogen-inactivated human platelet lysate.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

    2016-08-01

    Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

  6. Derivation of novel genetically diverse human embryonic stem cell lines.

    Science.gov (United States)

    Stefanova, Valentina T; Grifo, James A; Hansis, Christoph

    2012-06-10

    Human embryonic stem cells (hESCs) have the potential to revolutionize many biomedical fields ranging from basic research to disease modeling, regenerative medicine, drug discovery, and toxicity testing. A multitude of hESC lines have been derived worldwide since the first 5 lines by Thomson et al. 13 years ago, but many of these are poorly characterized, unavailable, or do not represent desired traits, thus making them unsuitable for application purposes. In order to provide the scientific community with better options, we have derived 12 new hESC lines at New York University from discarded genetically normal and abnormal embryos using the latest techniques. We examined the genetic status of the NYUES lines in detail as well as their molecular and cellular features and DNA fingerprinting profile. Furthermore, we differentiated our hESCs into the tissues most affected by a specific condition or into clinically desired cell types. To our knowledge, a number of characteristics of our hESCs have not been previously reported, for example, mutation for alpha thalassemia X-linked mental retardation syndrome, linkage to conditions with a genetic component such as asthma or poor sperm morphology, and novel combinations of ethnic backgrounds. Importantly, all of our undifferentiated euploid female lines tested to date did not show X chromosome inactivation, believed to result in superior potency. We continue to derive new hESC lines and add them to the NIH registry and other registries. This should facilitate the use of our hESCs and lead to advancements for patient-benefitting applications.

  7. Clonogenic cell line survival of a human liver cancer cell line SMMC-7721 after carbon ion irradiation with different LET

    International Nuclear Information System (INIS)

    Lei Suwen; Su Xu; Wang Jifang; Li Wenjian

    2003-01-01

    Objective: To investigate the survival fraction of a human liver cancer cell line SMMC-7721 following irradiation with carbon ions with different LET. Methods: cells of the human liver cancer cell line SMMC-7721 were irradiated with carbon ions (LET=30 and 70 keV/μm). The survival fraction was determined with clonogenic assay after 9 days incubation in a 5% CO 2 incubator at 37 degree C. Results: When the survival fractions of 70 keV/μm were D s = 0.1 and D s=0.01 absorption dose were 2.94 and 5.88 Gy respectively, and those of 30 keV/μm were 4.00 and 8.00 Gy respectively. Conclusion: For the SMMC-7721 cell line, 70 keV/μm is more effective for cell killing than 30 keV/μm

  8. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Science.gov (United States)

    2011-03-24

    ... differentiate among cell lines, as described in Designation: ASN-0002 Authentication of Human Cell Lines... NIST (contact information above). III. Data OMB Control Number: None. Form Number: None. Type of Review...

  9. 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.

  10. Lining cells on normal human vertebral bone surfaces

    International Nuclear Information System (INIS)

    Henning, C.B.; Lloyd, E.L.

    1982-01-01

    Thoracic vertebrae from two individuals with no bone disease were studied with the electron microscope to determine cell morphology in relation to bone mineral. The work was undertaken to determine if cell morphology or spatial relationships between the bone lining cells and bone mineral could account for the relative infrequency of bone tumors which arise at this site following radium intake, when compared with other sites, such as the head of the femur. Cells lining the vertebral mineral were found to be generally rounded in appearance with varied numbers of cytoplasmic granules, and they appeared to have a high density per unit of surface area. These features contrasted with the single layer of flattened cells characteristic of the bone lining cells of the femur. A tentative discussion of the reasons for the relative infrequency of tumors in the vertebrae following radium acquisition is presented

  11. Variations in Humanized and Defined Culture Conditions Supporting Derivation of New Human Embryonic Stem Cell Lines

    DEFF Research Database (Denmark)

    Fletcher, Judy M; Ferrier, Patricia M; Gardner, John O

    2006-01-01

    matrix substrate of purified human laminin (Ln) with transitional reliance on mitotically inactivated human fibroblast (HDF) feeder cells. With this integrated system hESC lines were isolated using either HDF conditioned medium supplemented with a bovine-sourced serum replacement (bSRM), or a defined...

  12. Functional analysis of the human calcyclin gene promoter in a panel of human melanoma cell lines

    NARCIS (Netherlands)

    van Groningen, J. J.; Weterman, M. A.; Swart, G. W.; Bloemers, H. P.

    1995-01-01

    By comparing two subsequent human tumor stages we previously described calcyclin as a new potential melanoma associated neoplastic progression marker positively linked with metastasis. In this study the calcyclin expression levels in a representative panel of human melanoma cell lines were

  13. Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

    Science.gov (United States)

    Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

    2016-08-01

    Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

  14. Glycoproteins and sialyl transferase of human B lymphoblastoid cell lines

    International Nuclear Information System (INIS)

    Lui, S.W.L.; Ng, M.H.

    1980-01-01

    We used two radiolabeling methods to study glycoproteins on the surface of lymphoblastoid cells. One of the methods affects tritiation of residues which are oxidized with galactose oxidase and the other causes tritiation of neuraminic acid residues. This approach was shown to allow a better resolution of cell surface glycoproteins than if either method were used alone. Glycoproteins of B 1 - 19 cells which harbor the Epstein-Barr virus genomes were compared with those of its parental cell line, BJAB, which does not harbor the viral genomes. These studies did not reveal a unique viral protein. A 28,000 mol. wt. glycoprotein was found to be the most prominent neuraminic acidlabeled product of B 1 - 19 cells and also of the two other cell lines, Raji and Ly38, which harbor the EBV genomes. A similar molecular weight species from BJAB cells identified by galactose oxidase labeling might be deficient in neuraminic acid residues as it was poorly labeled by the periodate oxidation method. The neuraminic acid content and level of sialyl transferase of BJAB cells were found to be lower than those of the other cell lines studied. (auth.)

  15. Modelling cell population growth with applications to cancer therapy in human tumour cell lines.

    Science.gov (United States)

    Basse, Britta; Baguley, Bruce C; Marshall, Elaine S; Wake, Graeme C; Wall, David J N

    2004-01-01

    In this paper we present an overview of the work undertaken to model a population of cells and the effects of cancer therapy. We began with a theoretical one compartment size structured cell population model and investigated its asymptotic steady size distributions (SSDs) (On a cell growth model for plankton, MMB JIMA 21 (2004) 49). However these size distributions are not similar to the DNA (size) distributions obtained experimentally via the flow cytometric analysis of human tumour cell lines (data obtained from the Auckland Cancer Society Research Centre, New Zealand). In our one compartment model, size was a generic term, but in order to obtain realistic steady size distributions we chose size to be DNA content and devised a multi-compartment mathematical model for the cell division cycle where each compartment corresponds to a distinct phase of the cell cycle (J. Math. Biol. 47 (2003) 295). We then incorporated another compartment describing the possible induction of apoptosis (cell death) from mitosis phase (Modelling cell death in human tumour cell lines exposed to anticancer drug paclitaxel, J. Math. Biol. 2004, in press). This enabled us to compare our model to flow cytometric data of a melanoma cell line where the anticancer drug, paclitaxel, had been added. The model gives a dynamic picture of the effects of paclitaxel on the cell cycle. We hope to use the model to describe the effects of other cancer therapies on a number of different cell lines. Copyright 2004 Elsevier Ltd.

  16. Role of free radicals in an adriamycin-resistant human small cell lung cancer cell line

    NARCIS (Netherlands)

    Meijer, C.; Mulder, N H; Timmer-Bosscha, H; Zijlstra, J G; de Vries, E G

    1987-01-01

    In two Adriamycin (Adr) resistant sublines (GLC4-Adr1 and GLC4-Adr2) of a human small cell lung carcinoma cell line, GLC4, cross-resistance for radiation was found. GLC4-Adr1 has an acquired Adr resistance factor of 44 after culturing without Adr for 20 days and GLC4-Adr2, the same subline cultured

  17. Expression of caspase-3 gene in apoptotic HL-60 cell and different human tumor cell lines

    International Nuclear Information System (INIS)

    Li Xiaoming; Song Tianbao

    1999-01-01

    Objective: To research the expression of caspase-3 gene in the apoptotic and the control HL-60 cells and in the different human tumor cell lines. Methods: Caspase-3 mRNA in the control and γ-radiation-induced apoptotic HL-60 cells, and in the 6 types of human tumor cell lines, was analysed by Northern blot. Results: The caspase-3 gene transcript was more highly expressed in leukemia cells HL-60, CEM, K562 and neuroblastoma SH-SY5Y than in cervical adenocarcinoma HeLa and breast carcinoma MCF7, and more highly in the radiation-induced apoptotic HL-60 than in the control HL-60 cells. Conclusion: The high level of expression of caspase-3 may aid the efforts to understand the tumor cell sensitivity to radiation, apoptosis and its inherent ability to survive

  18. 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.

  19. Interleukin-2 production by human leukemia cell lines of pre-B cell origin

    International Nuclear Information System (INIS)

    Holan, V.; Minowada, J.

    1993-01-01

    Cells of 7 tested human leukemia cell lines of pre-B cell origin (as characterized by immunophenotyping and by the expression of cytoplasmic micro chains, but not by surface immunoglobulins) produced after stimulation with bacterial lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) a lymphokine activity which supported the growth of the interleukin-2 (IL-2)-dependent CTLL-2 cell line. Three pieces of evidence indicate that the secreted lymphokine was functionally and antigenically very similar, if not identical, to human IL-2: (1) The lymphokine supported the growth of murine IL-2-dependent CTLL-2 cells, which did not respond to human lymphokines other than IL-2, but it did not stimulate the growth of murine IL-3-dependent FDC-P2 cells, (2) the biological activity of the lymphokine was was inhibited by monoclonal antibody (mAb) anti-human-IL-2, and (3) the proliferation of IL-2-dependent cells in the presence of the active materials was completely inhibited by the inclusion of the anti-mouse-IL-2 receptor (IL-2R) mAb. Since leukemia cells of immature B-cell origin also synthesize IL-2R, the human pre-B cell leukemias could represent another type of hematological malignancy where the autocrine processes of IL-2 production and utilization are involved in the expansion of the disease. (author)

  20. Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.

    Directory of Open Access Journals (Sweden)

    Ryo Kurita

    Full Text Available Transfusion of red blood cells (RBCs is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

  1. Generation of hiPSTZ16 (ISMMSi003-A cell line from normal human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    Marion Dejosez

    2018-01-01

    Full Text Available Human foreskin fibroblasts from a commercial source were reprogrammed into induced pluripotent stem cells to establish a clonal stem cell line, hiPSTZ16 (ISMMSi003-A. These cells show a normal karyotype and full differentiation potential in teratoma assays. The described cells provide a useful resource in combination with other iPS cell lines generated from normal human foreskin fibroblasts to study source- and reprogramming method-independent effects in downstream applications.

  2. Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongxia; Cui, Ruina [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Guo, Xuejiang [State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029 (China); Hu, Jiayue [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Dai, Jiayin, E-mail: daijy@ioz.ac.cn [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

    2016-08-05

    Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

  3. Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

    International Nuclear Information System (INIS)

    Zhang, Hongxia; Cui, Ruina; Guo, Xuejiang; Hu, Jiayue; Dai, Jiayin

    2016-01-01

    Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

  4. Caffeine markedly sensitizes human mesothelioma cell lines to pemetrexed

    Science.gov (United States)

    Min, Sang Hee; Goldman, I. David; Zhao, Rongbao

    2013-01-01

    Pemetrexed is a new generation antifolate approved for the treatment of mesothelioma and non-small cell lung cancer. Caffeine is known to augment radiation or chemotherapeutic drug-induced cell killing. The current study addresses the impact of caffeine on the activity of pemetrexed in mesothelioma cell lines. Caffeine enhanced pemetrexed activity in all four mesothelioma cell lines tested (H2052, H2373, H28 and MSTO-211H). Caffeine sensitized H2052 cells in a dose- and schedule-dependent manner, and was associated with a markedly decreased clonogenic survival. Caffeine sensitization occurred only in cells subjected to pulse, but not continuous, exposure to pemetrexed. Similar pemetrexed sensitization was also observed with the clinically better tolerated caffeine analog, theobromine. Pemetrexed sensitization by caffeine was associated with an increase in pemetrexed-induced phosphorylation of ataxia-telangiectasia-mutated (ATM) and Chk1. These data indicate that caffeine and its analog, theobromine, may be a useful approach to enhance pemetrexed-based chemotherapy. PMID:17594092

  5. Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a non-integration system

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Uhm

    2017-05-01

    Full Text Available We generated human induced pluripotent stem cells (hiPSCs from dermal fibroblasts using a Sendai virus (SeV-based gene delivery method. The generated hiPSC line, KSCBi002-A, has a normal karyotype (46,XY. The pluripotency and differentiation capacity were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from the National Stem Cell Bank, Korea National Institute of Health.

  6. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues.

    Science.gov (United States)

    Barallon, Rita; Bauer, Steven R; Butler, John; Capes-Davis, Amanda; Dirks, Wilhelm G; Elmore, Eugene; Furtado, Manohar; Kline, Margaret C; Kohara, Arihiro; Los, Georgyi V; MacLeod, Roderick A F; Masters, John R W; Nardone, Mark; Nardone, Roland M; Nims, Raymond W; Price, Paul J; Reid, Yvonne A; Shewale, Jaiprakash; Sykes, Gregory; Steuer, Anton F; Storts, Douglas R; Thomson, Jim; Taraporewala, Zenobia; Alston-Roberts, Christine; Kerrigan, Liz

    2010-10-01

    Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.

  7. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

    Science.gov (United States)

    Barallon, Rita; Bauer, Steven R.; Butler, John; Capes-Davis, Amanda; Dirks, Wilhelm G.; Furtado, Manohar; Kline, Margaret C.; Kohara, Arihiro; Los, Georgyi V.; MacLeod, Roderick A. F.; Masters, John R. W.; Nardone, Mark; Nardone, Roland M.; Nims, Raymond W.; Price, Paul J.; Reid, Yvonne A.; Shewale, Jaiprakash; Sykes, Gregory; Steuer, Anton F.; Storts, Douglas R.; Thomson, Jim; Taraporewala, Zenobia; Alston-Roberts, Christine; Kerrigan, Liz

    2010-01-01

    Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues. PMID:20614197

  8. Effect of New Water-Soluble Dendritic Phthalocyanines on Human Colorectal and Liver Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Ebru YABAŞ

    2017-08-01

    Full Text Available Human hepatocellular carcinoma (HepG2 cells and colorectal adenocarcinoma (DLD-1 cells were treated with the synthesized water soluble phthalocyanine derivatives to understand the effect of the compounds both on colorectal and liver cancer cells. The compounds inhibited cell proliferation and displayed cytotoxic effect on these cancer cell lines however; the effect of the compounds on healthy control fibroblast cell line was comparatively lower. The compounds can be employed for cancer treatment as anticancer agents.

  9. 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 selection...... of new drugs can be validated, it can be concluded that TCNU is not superior to other nitrosoureas for the treatment of SCCL....

  10. 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...... antibodies. There was a threshold level, below which the receptor could not be blocked. In addition, illumination caused the cells to upregulate the cyclin-dependent kinase inhibitor p21WAF1, irrespective of the p53 status. Since the EGF receptor is often overexpressed in cancers and other proliferative skin...... disorders, it might be possible to significantly reduce the proliferative potential of these cells making them good targets for laser-pulsed UV light treatment....

  11. Radiosensitization of human prostate cell line LNCAP by [6]- gingerol

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Josias Paulino Leal; Bellini, Maria Helena [Instituto de Pesquisas Energéticas e Nucleares (IPEN/CNEN-SP), São Paulo, SP (Brazil)

    2017-07-01

    Full text: Introduction: Prostate cancer is the second most prevalent malignancy and second leading cause of cancer-related deaths among men in the world. Several different diagnostic and therapeutic approaches have been developed in order to decrease the death rates. A number of experimental and clinical studies have showed antiproliferative, pro-apoptotic, anti-metastatic and anti-angiogenic effects of several phytochemicals. [6]-Gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3- decanone), the major pungent principle of ginger, has anti-oxidant, anti-inflammation and antitumor promoting activities. Aim: The purpose of this study was to evaluate the radiosensitizing activity of [6]-Gingerol in the human prostate cancer cells. Methods: The viability was assessed (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) tetrazolium (MTS) assay. The prostate human cells (LNCAP) (2,5×103 cells/well) were seeded into 96-well plates, after 24 hr they were treated with 150 and 300μg/mL of [6]-Gingerol or vehicle alone (0.1% DMSO) in serum containing media. After incubation, MTS solution was added to the plate at a final concentration of 0.5 mg/mL. The cells were incubated for 2 hr in dark at 37. The resulting MTS-products were determined by measuring the absorbance at 490 nm with ELISA reader. In the clonogenic cell survival assay, the cells were divided into two groups: A) control, B) treated with [6]-Gingerol, C) irradiated control and D) treated with [6]-Gingerol and irradiated. The cells were irradiated by 60Co source in the range from 0 to 15 Gy, using the GammaCell 220 - Irradiation Unit of Canadian-Atomic Energy Commision Ltd. (CTR-IPEN). After 10-14 days of culture in normoxia conditions, cell colonies were fixed and stained with methanol 20% and crystal violet 0.5% and counted. Multiple comparisons were assessed by One-way ANOVA followed by Bonferroni´s tests with GraphPad Prism version 6.0 software. p< 0.05 was considered statistically

  12. Radiosensitization of human prostate cell line LNCAP by [6]- gingerol

    International Nuclear Information System (INIS)

    Silva, Josias Paulino Leal; Bellini, Maria Helena

    2017-01-01

    Full text: Introduction: Prostate cancer is the second most prevalent malignancy and second leading cause of cancer-related deaths among men in the world. Several different diagnostic and therapeutic approaches have been developed in order to decrease the death rates. A number of experimental and clinical studies have showed antiproliferative, pro-apoptotic, anti-metastatic and anti-angiogenic effects of several phytochemicals. [6]-Gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3- decanone), the major pungent principle of ginger, has anti-oxidant, anti-inflammation and antitumor promoting activities. Aim: The purpose of this study was to evaluate the radiosensitizing activity of [6]-Gingerol in the human prostate cancer cells. Methods: The viability was assessed (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) tetrazolium (MTS) assay. The prostate human cells (LNCAP) (2,5×103 cells/well) were seeded into 96-well plates, after 24 hr they were treated with 150 and 300μg/mL of [6]-Gingerol or vehicle alone (0.1% DMSO) in serum containing media. After incubation, MTS solution was added to the plate at a final concentration of 0.5 mg/mL. The cells were incubated for 2 hr in dark at 37. The resulting MTS-products were determined by measuring the absorbance at 490 nm with ELISA reader. In the clonogenic cell survival assay, the cells were divided into two groups: A) control, B) treated with [6]-Gingerol, C) irradiated control and D) treated with [6]-Gingerol and irradiated. The cells were irradiated by 60Co source in the range from 0 to 15 Gy, using the GammaCell 220 - Irradiation Unit of Canadian-Atomic Energy Commision Ltd. (CTR-IPEN). After 10-14 days of culture in normoxia conditions, cell colonies were fixed and stained with methanol 20% and crystal violet 0.5% and counted. Multiple comparisons were assessed by One-way ANOVA followed by Bonferroni´s tests with GraphPad Prism version 6.0 software. p< 0.05 was considered statistically

  13. Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

    International Nuclear Information System (INIS)

    Yang, Qiwei; Tian, Yufeng; Ostler, Kelly R; Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Godley, Lucy A; Cohn, Susan L

    2010-01-01

    Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells alters the DNA methylation

  14. 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...... for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies....

  15. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    Science.gov (United States)

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  16. Biobanking human embryonic stem cell lines: policy, ethics and efficiency.

    Science.gov (United States)

    Holm, Søren

    2015-12-01

    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 discussions relating to their establishment or maintenance. It is argued (1) that 'ethical arguments' are often more important in the establishment phase and 'efficiency arguments' more important in the maintenance phase, and (2) that arguments relating to the interests of embryo and gamete donors 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 ignored in the policy making process.

  17. Response of the MG-63 human osteosarcoma cell line grown as multicellular spheroids to neutron irradiation

    International Nuclear Information System (INIS)

    Kubota, Nobuo; Kakehi, Masae; Matsubara, Shou; Koike, Sachiko; Ando, Koichi.

    1993-01-01

    Multicellular tumor spheroids are composed of the mixed populations of cells with regard to cell proliferation, nutrition, oxygenation and radiosensitivity. Human osteogenic sarcoma is generally considered clinically radioresistant. However, the in vitro cell survival curves for human osteogenic sarcoma cell lines do not differ from those of other tumor cell lines. In this study, the responses of human osteogenic sarcoma cell line to gamma ray and neutrons were investigated by using spheroid system. The spheroids of the osteogenic sarcoma cell line are considered to be a good in vitro model of radioresistant tumors. The purpose of this study is to measure the response of the spheroids to fast neutron irradiation. MG-63 human osteogenic sarcoma cell line was used for this study. The cell line was cultured in alpha-MEM with supplement. Cell survival was estimated after the trypsinization of spheroids 24 hours after irradiation. The method of measuring spheroid cure is explained. The mean number of surviving cells per spheroid can be obtained from the mean clonogenic number and cell survival curve. The cell survival of MG-63 spheroids exposed to gamma ray and neutrons and the dose effect curves for spheroid cure after irradiation are shown. (K.I.)

  18. 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

  19. Radiosensitivity evaluation of Human tumor cell lines by single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Zhang Yipei; Cao Jia; Wang Yan; Du Liqing; Li Jin; Wang Qin; Fan Feiyue; Liu Qiang

    2011-01-01

    Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using single cell gel electrophoresis (SCGE). Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction (SF) and DNA damage were detected by MTT assay, nested PCR technique and comet assay respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4 and 8 Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. SCGE: The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusion: The multi-utilization of many biological parameter is hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

  20. Radiobiological studies with a series of human cell lines of varying glutathione content

    International Nuclear Information System (INIS)

    Astor, M.B.

    1984-01-01

    Radiation responses of a series of four human fibroblast lines obtained from a family affected with 5-oxoprolinuria were determined. Cell suspensions were irradiated under hypoxic conditions and the oxygen enhancement ratio was determined for each cell line. Results are compared with previous studies

  1. 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.

  2. Derivation of Huntington Disease affected Genea046 human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea046 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying HTT gene CAG expansion of 45 repeats, indicative of Huntington Disease. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XX by CGH and STR analysis demonstrated a female Allele pattern. The hESC line had pluripotent cell morphology, 85% of cells expressed Nanog, 92% Oct4, 75% Tra1–60 and 99% SSEA4 and demonstrated Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and visible contamination.

  3. Radiosensitization of C225 on human non-small cell lung cancer cell line H-520

    International Nuclear Information System (INIS)

    Zhang Yingdong; Wang Junjie; Liu Feng; Zhao Yong

    2008-01-01

    Objective: To investigate the efficacy of C225 (cetuximab), a chimeric human-mouse anti-epithelial growth factor receptor monoclonal antibody, combined with 60 Co gamma irradiation against human non-small cell lung cancer cell line H-520. Methods: H-520 cells were treated either with different dose of 60 Co irradiation (1,2,4,6,8 and 10 Gy)alone or together with C225 (100 nmol/L). Colony forming capacity was determined to create the survival curve 10 days after the treatment. Cells in different groups were harvested 72 hours after irradiation for apoptosis analysis or 48 hours after irradiation for cell cycle analysis by flow cytometry assay. Results: The clone number in combinational treatment group was less than that in irradiation only group, which suggested that the cell survival rate in the combinational treatment group was significantly decreased comparing with irradiation only group (F=6.36, P O + G 1 phases for C225 treatment, in G 2 + M phases for 60 Co irradiation, and in both G 0 + G 1 and G 2 + M phases for C225 in combination with 60 Co irradiation. Conclusions: C225 has radiosensitizing effects on H-520 cells, which may through the enhancement of 60 Co irradiation-induced cell death and cell cycle arrest. This study provides a supportive evidence for clinical treatment in non-small cell lung cancer. (authors)

  4. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    International Nuclear Information System (INIS)

    Youakim, A.; Herscovics, A.

    1985-01-01

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2- 3 H]mannose or L-[5,6- 3 H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2- 3 H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2- 3 H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6- 3 H]glucosamine and L-[1- 14 C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3 H-labeled N-acetylglucosamine and N-acetylgalactosamine

  5. [The characters and specific features of new human embryonic stem cells lines].

    Science.gov (United States)

    Krylova, T A; Kol'tsova, A M; Zenin, V V; Gordeeva, O F; Musorina, A S; Goriachaia, T S; Shlykova, S A; Kamenetskaia, Iu K; Pinaev, G P; Polianskaia, G G

    2009-01-01

    Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

  6. In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Paula M. Kustiawan

    2014-07-01

    Conclusions: Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s.

  7. BETULINIC ACID WAS MORE CYTOTOXIC TOWARDS THE HUMAN BREAST CANCER CELL LINE MDA-MB-231 THAN THE HUMAN PROMYELOCYTIC LEUKAEMIA CELL LINE HL-60

    Directory of Open Access Journals (Sweden)

    LATIFAH SAIFUL YAZAN

    2009-01-01

    Full Text Available Betulinic acid (BA is a pentacyclic triterpene found in several botanical sources that has been shown to cause apoptosis in a number of cell lines. This study was undertaken to determine the in vitro cytotoxic properties of BA towards the human mammary carcinoma cell line MDA-MB-231 and the human promyelocytic leukaemia cell line HL-60 and the mode of the induced cell death. The cytotoxicity and mode of cell death of BA were determined using the MTT assay and DNAfragmentation analysis, respectively. In our study, the compound was found to be cytotoxic to MDA-MB-231 and HL-60 cells with IC50 values of 58 μg/mL and 134 μg/mL, respectively. Cells treated with high concentrations of BA exhibited features characteristic of apoptosis such as blebbing, shrinking and a number of small cytoplasm body masses when viewed under an inverted light microscope after 24 h. The incidence of apoptosis in MDA-MB-231 was further confirmed bythe DNA fragmentation analysis, with the formation of DNA fragments of oligonucleosomal size (180-200 base pairs, giving a ladder-like pattern on agarose gel electrophoresis. BA was more cytotoxic towards MDA-MB-231 than HL-60 cells, and induced apoptosis in MDA-MB-231 cells.

  8. Generation of iPSC line epiHUVEC from human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Peggy Matz

    2015-11-01

    Full Text Available Human umbilical vein endothelial cells (HUVECs were used to generate the iPSC line epiHUVEC employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHUVEC and the human embryonic stem cell line — H1 have a Pearson correlation of 0.899.

  9. Generation of iPSC lines from primary human chorionic villi cells

    Directory of Open Access Journals (Sweden)

    Björn Lichtner

    2015-11-01

    Full Text Available Primary human chorionic villi (CV cells were used to generate the iPSC line by retroviral transduction of the four Yamanaka-factors OCT4, SOX2, KLF4 and c-MYC. Pluripotency was confirmed both in vivo and in vitro. The transcriptomes of the CV-derived iPSC lines and the human embryonic stem cell lines—H1 and H9 have a Pearson correlation of 0.929 and 0.943 respectively.

  10. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

  11. A stable murine-based RD114 retroviral packaging line efficiently transduces human hematopoietic cells.

    Science.gov (United States)

    Ward, Maureen; Sattler, Rose; Grossman, I Robert; Bell, Anthony J; Skerrett, Donna; Baxi, Laxmi; Bank, Arthur

    2003-11-01

    Several barriers exist to high-efficiency transfer of therapeutic genes into human hematopoietic stem cells (HSCs) using complex oncoretroviral vectors. Human clinical trials to date have used Moloney leukemia virus-based amphotropic and gibbon ape leukemia virus-based envelopes in stable retroviral packaging lines. However, retroviruses pseudotyped with these envelopes have low titers due to the inability to concentrate viral supernatants efficiently by centrifugation without damaging the virus and low transduction efficiencies because of low-level expression of viral target receptors on human HSC. The RD114 envelope from the feline endogenous virus has been shown to transduce human CD34+ cells using transient packaging systems and to be concentrated to high titers by centrifugation. Stable packaging systems have potential advantages over transient systems because greater and more reproducible viral productions can be attained. We have, therefore, constructed and tested a stable RD114-expressing packaging line capable of high-level transduction of human CD34+ cells. Viral particles from this cell line were concentrated up to 100-fold (up to 10(7) viral particles/ml) by ultracentrifugation. Human hematopoietic progenitors from cord blood and sickle cell CD34+ cells were efficiently transduced with a Neo(R)-containing vector after a single exposure to concentrated RD114-pseudotyped virus produced from this cell line. Up to 78% of progenitors from transduced cord blood CD34+ cells and 51% of progenitors from sickle cell CD34+ cells expressed the NeoR gene. We also show transfer of a human beta-globin gene into progenitor cells from CD34+ cells from sickle cell patients with this new RD114 stable packaging system. The results indicate that this packaging line may eventually be useful in human clinical trials of globin gene therapy.

  12. Derivation and characterization of the NIH registry human stem cell line NYSCF100 line under defined feeder-free conditions

    Directory of Open Access Journals (Sweden)

    Ana Sevilla

    2018-05-01

    Full Text Available The human embryonic stem cell line NYSCFe001-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe001-A line, registered as NYSCF100 on the NIH registry, presents normal karyotype, is mycoplasma free, expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro.

  13. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.

    Science.gov (United States)

    Alley, M C; Scudiero, D A; Monks, A; Hursey, M L; Czerwinski, M J; Fine, D L; Abbott, B J; Mayo, J G; Shoemaker, R H; Boyd, M R

    1988-02-01

    For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

  14. Modulatory effects of quercetin on proliferation and differentiation of the human colorectal cell line Caco-2

    NARCIS (Netherlands)

    Dihal, A.A.; Woutersen, R.A.; Ommen, B.v.; Rietjens, I.M.C.M.; Stierum, R.H.

    2006-01-01

    The effect of the dietary flavonoid quercetin was investigated on proliferation and differentiation of the human colon cancer cell line Caco-2. Confluent Caco-2 monolayers exposed to quercetin showed a biphasic effect on cell proliferation and a decrease in cell differentiation (0.001

  15. Single-dose and fractionated irradiation of four human lung cancer cell lines in vitro

    International Nuclear Information System (INIS)

    Brodin, O.; Lennartsson, L.; Nilsson, S.

    1991-01-01

    Four established human lung cancer cell lines were exposed to single-dose irradiation. The survival curves of 2 small cell lung carcinomas (SCLC) were characterized by a limited capacity for repair with small and moderate shoulders with extrapolation numbers (n) of 1.05 and 1.60 respectively. Two non-small cell lung carcinoma (NSCLC) cell lines, one squamous cell (SQCLC) and one large cell (LCLC) had large shoulders with n-values of 73 and 15 respectively. The radiosensitivity when measured as D 0 did not, however, differ as much from cell line to cell line, with values from 1.22 to 1.65. The surviving fraction after 2 Gy (SF2) was 0.24 and 0.42 respectively in the SCLC cell lines and 0.90 and 0.88 respectively in the NSCLC cell lines. Fractionated irradiation delivered according to 3 different schedules was also investigated. All the schedules delivered a total dose of 10 Gy in 5 days and were applied in 1, 2 and 5 Gy dose fractions respectively. Survival followed the pattern found after single-dose irradiation; it was lowest in the SCLC cell line with the lowest SF and highest in the two NSCLC cell lines. In the SCLC cell lines all schedules were approximately equally efficient. In the LCLC and in the SQCLC cell lines, the 5 Gy schedule killed more cells than the 1 and 2 Gy schedules. The results indicate that the size of the shoulder of the survival curve is essential when choosing the most tumoricidal fractionation schedule. (orig.)

  16. Dihydrochalcone Compounds Isolated from Crabapple Leaves Showed Anticancer Effects on Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Qin

    2015-11-01

    Full Text Available Seven dihydrochalcone compounds were isolated from the leaves of Malus crabapples, cv. “Radiant”, and their chemical structures were elucidated by UV, IR, ESI-MS, 1H-NMR and 13C-NMR analyses. These compounds, which include trilobatin (A1, phloretin (A2, 3-hydroxyphloretin (A3, phloretin rutinoside (A4, phlorizin (A5, 6′′-O-coumaroyl-4′-O-glucopyranosylphloretin (A6, and 3′′′-methoxy-6′′-O-feruloy-4′-O-glucopyranosyl-phloretin (A7, all belong to the phloretin class and its derivatives. Compounds A6 and A7 are two new rare dihydrochalcone compounds. The results of a MTT cancer cell growth inhibition assay demonstrated that phloretin and these derivatives showed significant positive anticancer activities against several human cancer cell lines, including the A549 human lung cancer cell line, Bel 7402 liver cancer cell line, HepG2 human ileocecal cancer cell line, and HT-29 human colon cancer cell line. A7 had significant effects on all cancer cell lines, suggesting potential applications for phloretin and its derivatives. Adding a methoxyl group to phloretin dramatically increases phloretin’s anticancer activity.

  17. Expression and function of β-adrenergic receptors in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    Maeki, T.; Andersson, L.C.; Kontula, K.K.

    1992-01-01

    We investigated the expression and functional characteristics of β-adrenoceptors in a panel of 10 phenotypically different human hematopoietic cell lines. A binding assay with [ 125 I]iodocyanopindolol as the ligand revealed that cell lines of myelomonocytic or histiocytic derivation (HL-60, ML-2, RC-2A, U-937) expressed high numbers of β-adrenoceptors. An intermediate density of receptors was found in a non-T, non-B cell leukemia line (Nall-1), whereas T-cell (JM, CCRF-CEM), B-cell (Raji) or erythroleukemic cell lines (K-562, HEL) displayed minimala or undetectable binding of the radioligand. Isoprenaline-stimulated cAMP production by the cells correlated to their extent of β-adrenoceptor expression. Southern blot hybridization analysis of genomic DNA from the cell lines with a 32 P-labelled β 2 -adrenoceptor cDNA probe revealed no evidence for major rearrangement or amplification of the receptor gene. Incubation with isoprenaline in vitro suppressed the proliferation of the receptor-rich RC-2A cells but did not affect the growth rate of the receptor-deficient K-562 cells. Treatment with propranolol slightly enhanced the proliferation of the RC-2A cells but did not markedly alter the growth rate of two other cell lines, regardless of their β-adrenoceptor status. These findings indicate a regulatory influence by the sympathoadrenergic system on selected cells of the myelomonocytic lineage. (au)

  18. [Establishment and characterization of a cell line derived from human ovarian mucinous cystadenocarcinoma].

    Science.gov (United States)

    Wan, Q; Xu, D; Li, Z

    2001-07-01

    To establish a cell line of human ovarian cancer, and study its characterization. The cell line was established by the cultivation of subsides walls, and kept by freezing. The morphology was observed by microscope and electromicroscope. The authors studied its growth and propagation, the agglutination test of phytohemagglutinin (PHA), the chromosome analysis, heterotransplanting, immuno-histochemistry staining, the analysis of hormone, the pollution examination and the test of sensitivity to virus etc. A new human ovarian carcinoma cell line, designated ovarian mucinous cystadenocarcinoma 685 (OMC685), was established from mucinous cystadenocarcinoma. This cell line had subcultured to 91 generations, and some had been frozen for 8 years and revived, still grew well. This cell line possessed the feature of glandular epithelium cancer cell. The cells grew exuberantly, and the agglutinating test of PHA was positive. Karyotype was subtriploid with distortion. Heterotransplantations, alcian blue periobic acid-schiff (AbPAS), mucicarmine, alcian blue stainings, estradiol (E2) and progesterone were all positive. Without being polluted, it was sensitive to polivirus-I, adenovirus 7 and measles virus. OMC685 is a distinct human ovarian tumous cell line.

  19. Derivation of Two New Human Embryonic Stem Cell Lines from Nonviable Human Embryos

    Directory of Open Access Journals (Sweden)

    Svetlana Gavrilov

    2011-01-01

    Full Text Available We report the derivation and characterization of two new human embryonic stem cells (hESC lines (CU1 and CU2 from embryos with an irreversible loss of integrated organismic function. In addition, we analyzed retrospective data of morphological progression from embryonic day (ED 5 to ED6 for 2480 embryos not suitable for clinical use to assess grading criteria indicative of loss of viability on ED5. Our analysis indicated that a large proportion of in vitro fertilization (IVF embryos not suitable for clinical use could be used for hESC derivation. Based on these combined findings, we propose that criteria commonly used in IVF clinics to determine optimal embryos for uterine transfer can be employed to predict the potential for hESC derivation from poor quality embryos without the destruction of vital human embryos.

  20. [Effects of icotinib hydrochloride on the proliferation and apoptosis of human lung cancer cell lines].

    Science.gov (United States)

    Ma, Li; Han, Xiao-hong; Wang, Shuai; Wang, Jian-fei; Shi, Yuan-kai

    2012-09-25

    To explore the effects of icotinib on the proliferation and apoptosis of various lung cancer cell lines. Human lung cancer cell lines HCC827, H1650, H1975, A549 and human epidermal cancer cell line A431 were treated in vitro with icotinib or gefitinib at a concentration gradient of 0 - 40 µmol/L. Their proliferation effects were analyzed by the thiazolyl blue (MTT) assay and the apoptotic effects detected by flow cytometer. The downstream signaling proteins were detected by Western blot. The median inhibitory concentrations (IC(50)) of icotinib for A431 and HCC827 cell lines were (0.04 ± 0.02) and (0.15 ± 0.06) µmol/L respectively. No significant differences existed between the inhibitions of gefitinib and icotinib on A431, HCC827, H1650, H1975 and A549 cell lines (all P > 0.05). Compared with H1650, H1975 and A549 cell lines, icotinib significantly inhibited A431 (P = 0.009, 0.005 and 0.000) and HCC827 (P = 0.001, 0.001 and 0.000) cell lines. And it lowered the expressions of p-AKT, p-ERK and survivin protein expression through the inhibited activity of p-EGFR protein. Icotinib can arrest the proliferation of lung adenocarcinoma cells with EGFR mutation or over-expression by inhibiting the signal pathways of AKT-ERK and survivin.

  1. Derivation and characterization of the NYSCFe003-A human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Ana Sevilla

    2017-12-01

    Full Text Available The human embryonic stem cell line NYSCFe003-A was derived from a day 5 to day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe003-A line expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro. The line presents normal karyotype and is mycoplasma free.

  2. Cytotoxic activity of kenaf (Hibiscus cannabinus L.) seed extract and oil against human cancer cell lines

    Science.gov (United States)

    Wong, Yu Hua; Tan, Wai Yan; Tan, Chin Ping; Long, Kamariah; Nyam, Kar Lin

    2014-01-01

    Objective To examine the cytotoxic properties of both the kenaf (Hibiscus cannabinus L.) seed extract and kenaf seed oil on human cervical cancer, human breast cancer, human colon cancer and human lung cancer cell lines. Methods The in vitro cytotoxic activity of the kenaf (Hibiscus cannabinus L.) seed extract and kenaf seed oil on human cancer cell lines was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and sulforhodamine B assays. Cell morphological changes were observed by using an inverted light microscope. Results The kenaf seed extract (KSE) exhibited a lower IC50 than kenaf seed oil (KSO) in all of the cancer cell lines. Morphological alterations in the cell lines after KSE and KSO treatment were observed. KSE and KSO possessed effective cytotoxic activities against all the cell lines been selected. Conclusions KSE and KSO could be potential sources of natural anti-cancer agents. Further investigations on using kenaf seeds for anti-proliferative properties are warranted. PMID:25183141

  3. Presence of dopamine D-2 receptors in human tumoral cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Sokoloff, P.; Riou, J.F.; Martres, M.P.; Schwartz, J.C. (Centre Paul Broca, Paris (France))

    1989-07-31

    ({sup 125}I) Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, ({sup 125}I) iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.

  4. 78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

    Science.gov (United States)

    2013-04-29

    ...; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH... Embryonic Stem Cell Line to be Approved for Use in NIH-Funded Research, 0925-0601, Expiration Date 04/30... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be...

  5. Effect of recombinant adenovirus encoding human p53 tumor suppressor gene combined with radiation therapy on human lymphoma cells lines

    International Nuclear Information System (INIS)

    Yu Zeyang; Fan Wo; Li Dongqing; Zhu Ran; Wan Jianmei; Wang Yongqing; Wu Jinchang

    2008-01-01

    This paper analyzes the inhibitory effect and radiation sensitization of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on human lymphoma cell lines. Human lymphoma cell lines were treated with rAd-p53, radiation therapy and combined treatment, respectively. The cell growth inhibition was assessed by MTF. The cell cycle and apoptosis were detected by flow cytometry, and the p53 protein expression was detected by Western blotting. The results showed that extrinsic p53 gene have expressed to some degree, but not at high level. The role of inhibition and radiation sensitivity of rAd-p53 was not significant to human lymphoma cell lines. (authors)

  6. Estrogenic effects of fusarielins in human breast cancer cell lines

    DEFF Research Database (Denmark)

    Søndergaard, Teis; Klitgaard, Louise Graabæk; Purup, Stig

    2012-01-01

    without the estrogen receptor-α and MCF-10a cells without estrogen receptors were not stimulated by fusarielins. Furthermore, the stimulation was prevented in MCF-7 cells when fusarielins were incubated in the presence of the estrogen receptor antagonist fulvestrant. These observations suggest...

  7. Derivation of the human embryonic stem cell line RCe006-A (RC-2

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe006-A (RC-2 was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12. Microsatellite DNA marker identity and HLA and blood group typing data are available.

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

    OpenAIRE

    Zhang Ping; Zhang Zhiyuan; Zhou Xiaojian; Qiu Weiliu; Chen Fangan; Chen Wantao

    2006-01-01

    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-differe...

  9. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Mu-Yun [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Shen, Yuh-Chiang [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); National Research Institute of Chinese Medicine, Taipei, Taiwan (China); Lu, Chien-Hsing [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Obstetrics and Gynecology, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yang, Shu-Yi [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Ho, Tsing-Fen [Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China); Peng, Yu-Ta [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Chang, Chia-Che, E-mail: chia_che@dragon.nchu.edu.tw [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

    2012-12-15

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified

  10. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    International Nuclear Information System (INIS)

    Pan, Mu-Yun; Shen, Yuh-Chiang; Lu, Chien-Hsing; Yang, Shu-Yi; Ho, Tsing-Fen; Peng, Yu-Ta; Chang, Chia-Che

    2012-01-01

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ► Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ► Prodigiosin is herein identified as an

  11. A COUP-TFII Human Embryonic Stem Cell Reporter Line to Identify and Select Atrial Cardiomyocytes

    NARCIS (Netherlands)

    Schwach, Verena; Verkerk, Arie O.; Mol, Mervyn; Monshouwer-Kloots, Jantine J.; Devalla, Harsha D.; Orlova, Valeria V.; Anastassiadis, Konstantinos; Mummery, Christine L.; Davis, Richard P.; Passier, Robert

    2017-01-01

    Reporter cell lines have already proven valuable in identifying, tracking, and purifying cardiac subtypes and progenitors during differentiation of human pluripotent stem cells (hPSCs). We previously showed that chick ovalbumin upstream promoter transcription factor II (COUP-TFII) is highly enriched

  12. The toxicity of silver and silica nanoparticles in comparable human and mouse cell lines

    DEFF Research Database (Denmark)

    Foldbjerg, Rasmus; Beer, Christiane; Sutherland, Duncan S

    The toxicity of silica (SiO2) and PVP-coated silver (Ag) nanoparticles (NPs) was investigated in two pairs of human or mouse cell lines originating from lung epithelium (A549 and ASB-XIV) and macrophages (THP-1 and J744A.1). Both NPs were characterized in H2O and cell media and demonstrated to be...

  13. Comparative pharmacology of a new recombinant FSH expressed by a human cell line

    DEFF Research Database (Denmark)

    Koechling, Wolfgang; Plaksin, Daniel; Croston, Glenn E.

    2017-01-01

    Recombinant FSH proteins are important therapeutic agents for the treatment of infertility, including follitropin alfa expressed in Chinese Hamster Ovary (CHO) cells and, more recently, follitropin delta expressed in the human cell line PER.C6. These recombinant FSH proteins have distinct glycosy...

  14. Hypoxia-induced resistance to doxorubicin and methotrexate in human melanoma cell lines in vitro.

    Science.gov (United States)

    Sanna, K; Rofstad, E K

    1994-07-15

    Rodent cell lines can develop resistance to doxorubicin and methotrexate during hypoxic stress. This has so far not been observed in human tumor cell lines. The purpose of our communication is to show that doxorubicin and methotrexate resistance can also develop in human melanoma cells during exposure to hypoxia. Four cell lines (BEX-c, COX-c, SAX-c, WIX-c) have been studied. Cells were exposed to hypoxia (O2 concentration WIX-c. BEX-c and SAX-c were sensitive to methotrexate without hypoxia pre-treatment, whereas COX-c and WIX-c were resistant initially. Hypoxia-induced drug resistance was present immediately after reoxygenation and tended to decrease with time but remained statistically significant even 42 hr after reoxygenation.

  15. A human beta cell line with drug inducible excision of immortalizing transgenes

    Science.gov (United States)

    Benazra, Marion; Lecomte, Marie-José; Colace, Claire; Müller, Andreas; Machado, Cécile; Pechberty, Severine; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Solimena, Michele; Scharfmann, Raphaël; Czernichow, Paul; Ravassard, Philippe

    2015-01-01

    Objectives Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen. Methods We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion. Results We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 μg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture. Conclusions EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture. PMID:26909308

  16. Antiproliferative Effects of Selected Chemotherapeutics in Human Ovarian Cancer Cell Line A2780

    Directory of Open Access Journals (Sweden)

    Kateřina Caltová

    2012-01-01

    Full Text Available The aim of our study was to determine the effect of selected cytostatics on a human ovarian cancer cell line A2780 as a model system for ovarian cancer treatment. This cell line is considered cisplatin-sensitive. Panel of tested cytostatics included cisplatin, paclitaxel, carboplatin, gemcitabine, topotecan and etoposide. These cytostatics have a different mechanism of action. To evaluate cytotoxic potential of the tested compounds, the methods measuring various toxicological endpoints were employed including morphological studies, MTT assay, dynamic monitoring of cell proliferation with xCELLigence, cell cycle analysis, caspase 3 activity and expression of proteins involved in cell cycle regulation and cell death. The A270 cell line showed different sensitivity towards the selected cytostatics, the highest cytotoxic effect was associated with paclitaxel and topotecan.

  17. Effects of arsenite on cell cycle progression in a human bladder cancer cell line

    International Nuclear Information System (INIS)

    Hernandez-Zavala, A.; Cordova, E.; Razo, L.M. del; Cebrian, M.E.; Garrido, E.

    2005-01-01

    Bladder cancer is one of the most important diseases associated with arsenic (As) exposure in view of its high prevalence and mortality rate. Experimental studies have shown that As exposure induces cell proliferation in the bladder of sodium arsenite (iAsIII) subchronically treated mice. However, there is little available information on its effects on the cell cycle of bladder cells. Thus, our purpose was to evaluate the effects of iAsIII on cell cycle progression and the response of p53 and p21 on the human-derived epithelial bladder cell line HT1197. iAsIII treatment (1-10 μM) for 24 h induced a dose-dependent increase in the proportion of cells in S-phase, which reached 65% at the highest dose. A progressive reduction in cell proliferation was also observed. BrdU was incorporated to cellular DNA in an interrupted form, suggesting an incomplete DNA synthesis. The time-course of iAsIII effects (10 μM) showed an increase in p53 protein content and a transient increase in p21 protein levels accompanying the changes in S-phase. These effects were correlated with iAs concentrations inside the cells, which were not able to metabolize inorganic arsenic. Our findings suggest that p21 was not able to block CDK2-cyclin E complex activity and was therefore unable to arrest cells in G1 allowing their progression into the S-phase. Further studies are needed to ascertain the mechanisms underlying the effects of iAsIII on the G1 to S phase transition in bladder cells

  18. Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines

    Directory of Open Access Journals (Sweden)

    Ding-Kun Kang

    Full Text Available Abstract Background: Local anesthetics (LAs are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Methods: Melanoma cell lines, A375 and Hs294T, were exposed to 1 h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Results: Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p < 0.001, concentration- (p < 0.001, and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72 h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10% ± 2% and 14% ± 2% in A375 and Hs294T, respectively after 72 h of exposure. Ropivacaine 0.75% after 72 h reduced viable cells to 15% ± 3% and 25% ± 3% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to the combination was 10% ± 2% and 18% ± 2% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to melphalan was 8% ± 1% and 12% ± 2%, in A375 and Hs294T, respectively. Conclusion: LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8.

  19. Expression pattern of matrix metalloproteinases in human gynecological cancer cell lines

    International Nuclear Information System (INIS)

    Schröpfer, Andrea; Kammerer, Ulrike; Kapp, Michaela; Dietl, Johannes; Feix, Sonja; Anacker, Jelena

    2010-01-01

    Matrix metalloproteinases (MMPs) are involved in the degradation of protein components of the extracellular matrix and thus play an important role in tumor invasion and metastasis. Their expression is related to the progression of gynecological cancers (e.g. endometrial, cervical or ovarian carcinoma). In this study we investigated the expression pattern of the 23 MMPs, currently known in humans, in different gynecological cancer cell lines. In total, cell lines from three endometrium carcinomas (Ishikawa, HEC-1-A, AN3 CA), three cervical carcinomas (HeLa, Caski, SiHa), three chorioncarcinomas (JEG, JAR, BeWo), two ovarian cancers (BG-1, OAW-42) and one teratocarcinoma (PA-1) were examined. The expression of MMPs was analyzed by RT-PCR, Western blot and gelatin zymography. We demonstrated that the cell lines examined can constitutively express a wide variety of MMPs on mRNA and protein level. While MMP-2, -11, -14 and -24 were widely expressed, no expression was seen for MMP-12, -16, -20, -25, -26, -27 in any of the cell lines. A broad range of 16 MMPs could be found in the PA1 cells and thus this cell line could be used as a positive control for general MMP experiments. While the three cervical cancer cell lines expressed 10-14 different MMPs, the median expression in endometrial and choriocarcinoma cells was 7 different enzymes. The two investigated ovarian cancer cell lines showed a distinctive difference in the number of expressed MMPs (2 vs. 10). Ishikawa, Caski, OAW-42 and BeWo cell lines could be the best choice for all future experiments on MMP regulation and their role in endometrial, cervical, ovarian or choriocarcinoma development, whereas the teratocarcinoma cell line PA1 could be used as a positive control for general MMP experiments

  20. Early death, late death and repair factor in three human tumour cell lines

    International Nuclear Information System (INIS)

    Courdi, A.; Gioanni, J.; Mari, D.; Chauvel, P.

    1997-01-01

    The in vivo colony method used to generate survival curves following exposure to ionizing irradiation allows to score large clones, representing surviving cells, and small colonies, representing late reproductive death. By subtraction, early-dying cells can be estimated. In the three human tumour cell lines examined, we have observed that early cell death is a major mode of action of irradiation. The contribution of early cell death to total mortality increases as the dose increases. Moreover, repair due to dose-splitting and delayed plating in densely-inhibited cells is not observed in early-dying cells. (authors)

  1. Monitoring cell line identity in collections of human induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Raquel Sarafian

    2018-04-01

    Full Text Available The ability to reprogram somatic cells into induced pluripotent stem cells (hiPSCs has led to the generation of large collections of cell lines from thousands of individuals with specific phenotypes, many of which will be shared among different research groups as invaluable tools for biomedical research. As hiPSC-based research involves extensive culture of many cell lines, the issue periodic cell line identification is particularly important to ensure that cell line identity remains accurate. Here we analyzed the different commercially available genotyping methods considering ease of in-house genotyping, cost and informativeness, and applied one of them in our workflow for hiPSC generation. We show that the chosen STR method was able to establish a unique DNA profile for each of the 35 individuals/hiPSC lines at the examined sites, as well as identify two discrepancies resulting from inadvertently exchanged samples. Our results highlight the importance of hiPSC line genotyping by an in-house method that allows periodic cell line identification and demonstrate that STR is a useful approach to supplement less frequent karyotyping and epigenetic evaluations. Keywords: Induced pluripotent stem cells, Genotyping, Cell line identification, Short tandem repeats, Quality control

  2. Cytogenetic Evolution of Human Ovarian Cell Lines Associated with Chemoresistance and Loss of Tumorigenicity

    Directory of Open Access Journals (Sweden)

    Stéphanie Struski

    2003-01-01

    Full Text Available In order to identify genomic changes associated with a resistant phenotype acquisition, we used comparative genomic hybridization (CGH to compare a human ovarian cell line, Igrov1, and four derived subcell lines, resistant to vincristine and presenting a reversion of malignant properties. Multicolor FISH (Multiplex‐FISH and Spectral Karyotype and conventional FISH are also used to elucidate the karyotype of parental cell line. The drug‐resistant subcell lines displayed many chromosomal abnormalities suggesting the implication of different pathways leading to a multidrug resistance phenotype. However, these cell lines shared two common rearrangements: an unbalanced translocation der(8t(8;13(p22;q? and a deletion of the 11p. These chromosomal imbalances could reflected the acquisition of the chemoresistance (der(8 or the loss of tumorigenicity properties (del(11p. Colour figure can be viewed on http://www.esacp.org/acp/2003/25‐3/struski.htm.

  3. Degradation of endothelial basement membrane by human breast cancer cell lines

    International Nuclear Information System (INIS)

    Yee, C.; Shiu, R.P.

    1986-01-01

    During metastasis, it is believed that tumor cells destroy the basement membrane (BM) of blood vessels in order to disseminate through the circulatory system. By radioactively labeling the extracellular matrix produced by primary endothelial cells in vitro, the ability of human breast cancer cells to degrade BM components was studied. We found that T-47D, a human breast cancer line, was able to degrade significant amounts of [35S]methionine-labeled and [3H]proline-labeled BM, but not 35SO4-labeled BM. Six other tumor cell lines of human breast origin were assayed in the same manner and were found to degrade BM to varying degrees. Several non-tumor cell lines tested showed relatively little degrading activity. The use of serum-free medium greatly enhanced degradation of the BM by tumor cells, suggesting a role for naturally occurring enzyme inhibitors in the serum. Direct cell contact with the BM was required for BM degradation, suggesting that the active enzymes are cell associated. The addition of hormones implicated in the etiology of breast cancer did not significantly alter the ability of T-47D cells to degrade the BM. The use of this assay affords future studies on the mechanism of invasion and metastasis of human breast cancer

  4. In vitro radiosensitivity of six human cell lines. A comparative study with different statistical models

    International Nuclear Information System (INIS)

    Fertil, B.; Deschavanne, P.J.; Lachet, B.; Malaise, E.P.

    1980-01-01

    The intrinsic radiosensitivity of human cell lines (five tumor and one nontransformed fibroblastic) was studied in vitro. The survival curves were fitted by the single-hit multitarget, the two-hit multitarget, the single-hit multitarget with initial slope, and the quadratic models. The accuracy of the experimental results permitted evaluation of the various fittings. Both a statistical test (comparison of variances left unexplained by the four models) and a biological consideration (check for independence of the fitted parameters vis-a-vis the portion of the survival curve in question) were carried out. The quadratic model came out best with each of them. It described the low-dose effects satisfactorily, revealing a single-hit lethal component. This finding and the fact that the six survival curves displayed a continuous curvature ruled out the adoption of the target models as well as the widely used linear regression. As calculated by the quadratic model, the parameters of the six cell lines lead to the following conclusions: (a) the intrinsic radiosensitivity varies greatly among the different cell lines; (b) the interpretation of the fibroblast survival curve is not basically different from that of the tumor cell lines; and (c) the radiosensitivity of these human cell lines is comparable to that of other mammalian cell lines

  5. Reliable generation of induced pluripotent stem cells from human lymphoblastoid cell lines.

    Science.gov (United States)

    Barrett, Robert; Ornelas, Loren; Yeager, Nicole; Mandefro, Berhan; Sahabian, Anais; Lenaeus, Lindsay; Targan, Stephan R; Svendsen, Clive N; Sareen, Dhruv

    2014-12-01

    Patient-specific induced pluripotent stem cells (iPSCs) hold great promise for many applications, including disease modeling to elucidate mechanisms involved in disease pathogenesis, drug screening, and ultimately regenerative medicine therapies. A frequently used starting source of cells for reprogramming has been dermal fibroblasts isolated from skin biopsies. However, numerous repositories containing lymphoblastoid cell lines (LCLs) generated from a wide array of patients also exist in abundance. To date, this rich bioresource has been severely underused for iPSC generation. We first attempted to create iPSCs from LCLs using two existing methods but were unsuccessful. Here we report a new and more reliable method for LCL reprogramming using episomal plasmids expressing pluripotency factors and p53 shRNA in combination with small molecules. The LCL-derived iPSCs (LCL-iPSCs) exhibited identical characteristics to fibroblast-derived iPSCs (fib-iPSCs), wherein they retained their genotype, exhibited a normal pluripotency profile, and readily differentiated into all three germ-layer cell types. As expected, they also maintained rearrangement of the heavy chain immunoglobulin locus. Importantly, we also show efficient iPSC generation from LCLs of patients with spinal muscular atrophy and inflammatory bowel disease. These LCL-iPSCs retained the disease mutation and could differentiate into neurons, spinal motor neurons, and intestinal organoids, all of which were virtually indistinguishable from differentiated cells derived from fib-iPSCs. This method for reliably deriving iPSCs from patient LCLs paves the way for using invaluable worldwide LCL repositories to generate new human iPSC lines, thus providing an enormous bioresource for disease modeling, drug discovery, and regenerative medicine applications. ©AlphaMed Press.

  6. Derivation of NEM2 affected human embryonic stem cell line Genea079

    Directory of Open Access Journals (Sweden)

    Biljana Dumevska

    2016-03-01

    Full Text Available The Genea079 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, carrying compound heterozygous mutations in the NEB gene, exon 55 deletion & c.15110dupA, indicative of Nemaline Myopathy Type 2 (NEM2. Following ICM outgrowth on inactivated human feeders, karyotype was confirmed as 46, XY and STR analysis demonstrated a male Allele pattern. The hESC line had pluripotent cell morphology, 86% of cells expressed Nanog, 95% Oct4, 54% Tra1-60 and 98% SSEA4 and gave a PluriTest Pluripotency score of 30.25, Novelty of 1.21. The cell line was negative for Mycoplasma and visible contamination.

  7. Overexpression of c-Jun contributes to sorafenib resistance in human hepatoma cell lines.

    Directory of Open Access Journals (Sweden)

    Yuki Haga

    Full Text Available Despite recent advances in treatment strategies, it is still difficult to cure patients with hepatocellular carcinoma (HCC. Sorafenib is the only approved multiple kinase inhibitor for systemic chemotherapy in patients with advanced HCC. The majority of advanced HCC patients are resistant to sorafenib. The mechanisms of sorafenib resistance are still unknown.The expression of molecules involved in the mitogen-activated protein kinase (MAPK signaling pathway in human hepatoma cell lines was examined in the presence or absence of sorafenib. Apoptosis of human hepatoma cells treated with sorafenib was investigated, and the expression of Jun proto-oncogene (c-Jun was measured.The expression and phosphorylation of c-Jun were enhanced in human hepatoma cell lines after treatment with sorafenib. Inhibiting c-Jun enhanced sorafenib-induced apoptosis. The overexpression of c-Jun impaired sorafenib-induced apoptosis. The expression of osteopontin, one of the established AP-1 target genes, was enhanced after treatment with sorafenib in human hepatoma cell lines.The protein c-Jun plays a role in sorafenib resistance in human hepatoma cell lines. The modulation and phosphorylation of c-Jun could be a new therapeutic option for enhancing responsiveness to sorafenib. Modulating c-Jun may be useful for certain HCC patients with sorafenib resistance.

  8. Expression of cDNAs in human Natural Killer cell lines by retroviral transduction.

    Science.gov (United States)

    Miah, S M Shahjahan; Campbell, Kerry S

    2010-01-01

    Human NK-like cell lines are difficult to transfect using standard mammalian expression vectors and conventional transfection protocols, but they are susceptible to retroviral transduction as a means to introduce cDNAs. Our laboratory has exploited this technique to study a number of receptors in human NK cell lines. The method utilizes a bicistronic retroviral vector that co-expresses either drug resistance or enhanced green fluorescent protein (EGFP) in parallel with the gene of interest. After a single infection with recombinant retrovirus, transduced NK cells can be sorted for expression of EGFP or the transduced cell surface marker. Alternatively, cells expressing the transduced cDNAs can be selected for by treatment with neomycin, puromycin, or hygromycin. Using this method, the sorted/selected cells uniformly express the gene of interest and the expression is stable for many weeks of culture.

  9. Derivation of the human embryonic stem cell line RCe014-A (RC-10

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe014-A (RC-10 was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  10. Derivation of the human embryonic stem cell line RCe010-A (RC-6

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe010-A (RC-6 was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.

  11. Derivation of the human embryonic stem cell line RCe012-A (RC-8

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-03-01

    Full Text Available The human embryonic stem cell line RCe012-A (RC-8 was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.

  12. Potentially lethal damage repair in cell lines of radioresistant human tumours and normal skin fibroblasts

    International Nuclear Information System (INIS)

    Marchese, M.J.; Minarik, L.; Hall, E.J.; Zaider, M.

    1985-01-01

    Radiation cell survival data were obtained in vitro for three cell lines isolated from human tumours traditionally considered to be radioresistant-two melanomas and one osteosarcoma-as well as from a diploid skin fibroblast cell line. One melanoma cell line was much more radioresistant than the other, while the osteosarcoma and fibroblast cell lines were more radiosensitive than either. For cells growing exponentially, little potentially lethal damage repair (PLDR) could be demonstrated by comparing survival data for cells in which subculture was delayed by 6 h with those sub-cultured immediately after treatment. For the malignant cells in plateau phase, which in these cells might be better termed 'slowed growth phase', since an appreciable fraction of the cells are still cycling, a small amount of PLDR was observed, but not as much as reported by other investigators in the literature. The normal fibroblasts, which achieved a truer plateau phase in terms of noncycling cells, showed a significantly larger amount of PLDR than the tumour cells. (author)

  13. Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

    Science.gov (United States)

    Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars

    2006-02-17

    Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

  14. Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

    OpenAIRE

    Bodel, P; Ralph, P; Wenc, K; Long, J C

    1980-01-01

    Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous ...

  15. Downregulation of telomerase activity in human promyelocytic cell line using RNA interference.

    Science.gov (United States)

    Miri-Moghaddam, E; Deezagi, A; Soheili, Z S

    2009-12-01

    Telomerase is a ribonucleoprotein complex. It consists of two main components, human telomerase reverse transcriptase (hTERT) and human telomerase RNA. High telomerase activity is present in most malignant cells, but it is barely detectable in majority of somatic cells. The direct correlation between telomerase reactivation and carcinogens has made hTERT a key target for anticancer therapeutic studies. In this study, for the first time, we evaluated the ability of the new generation of short interfering RNA (siRNA) to regulate telomerase activity in the human promyelocytic leukemia cell line (HL-60). Transient transfection cell line by hTERT siRNAs resulted in statistically significant suppression of hTERT messenger RNAs which were detected by quantitative real-time polymerase chain reaction, while the expressed hTERT protein levels were measured by flow cytometry. The results of telomeric repeat amplification protocol showed that telomerase activity was significantly reduced upon transfection of the HL-60 cell line with hTERT siRNAs. The results of this study showed that telomerase activity and cell proliferation were efficiently inhibited in the hTERT siRNA-treated leukemic cell line.

  16. 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......, FcepsilonRIalpha protein stability and the mRNA level of FcepsilonRIalpha-, beta- and the truncated beta-chain (beta(T)), and thereby determine the utility of these cell lines in investigations of the FcepsilonRI biology....

  17. Effects of phthalates on the human corneal endothelial cell line B4G12

    DEFF Research Database (Denmark)

    Krüger, Tanja; Cao, Yi; Kjærgaard, Søren K.

    2012-01-01

    Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2...... toxicity was observed for DBP and BBP. Upon DBP exposure at nontoxic concentrations, a significant increased gene expression and cytokine cell secretion were observed for interleukin-1ß (IL-1ß) and IL-8, and also an increased IL-6 secretion was observed. In conclusion, the human corneal endothelial cell...

  18. Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines

    International Nuclear Information System (INIS)

    Offerdahl, Danielle K.; Dorward, David W.; Hansen, Bryan T.; Bloom, Marshall E.

    2017-01-01

    The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60–100 nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20–30 nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. - Highlights: •First electron tomography of Zika virus cytoarchitecture. •Comparison of Zika virus infection in human neuroblastoma and mosquito cells. •Ultrastructure of Zika virus infection in human neuroblastoma and mosquito cells.

  19. Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Offerdahl, Danielle K. [Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT (United States); Dorward, David W.; Hansen, Bryan T. [Microscopy Unit, Research Technology Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT (United States); Bloom, Marshall E., E-mail: mbloom@nih.gov [Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT (United States)

    2017-01-15

    The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60–100 nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20–30 nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. - Highlights: •First electron tomography of Zika virus cytoarchitecture. •Comparison of Zika virus infection in human neuroblastoma and mosquito cells. •Ultrastructure of Zika virus infection in human neuroblastoma and mosquito cells.

  20. Effect of radiation combined with hyperthermia on human prostatic carcinoma cell lines in culture

    International Nuclear Information System (INIS)

    Kaver, I.; Ware, J.L.; Wilson, J.D.; Koontz, W.W. Jr.

    1991-01-01

    The effect of radiation combined with heat on three human prostatic carcinoma cell lines growing in vitro was investigated. Cells were exposed to different radiation doses followed by heat treatment at 43 degrees C for one hour. Heat treatment, given ten minutes after radiation, significantly enhanced the radiation response of all the cell lines studied. The combined effect of radiation and heat produced greater cytotoxicity than predicted from the additive effects of the two individual treatment modalities alone. These results indicate that a combined treatment regimen of radiation plus hyperthermia (43 degrees, 1 hr) might be an important tool in maintaining a better local control of prostatic cancer

  1. Basal HIF-1a expression levels are not predictive for radiosensitivity of human cancer cell lines

    International Nuclear Information System (INIS)

    Schilling, D.; Multhoff, G.; Helmholtz Center Munich, CCG - Innate Immunity in Tumor Biology, Munich; Bayer, C.; Emmerich, K.; Molls, M.; Vaupel, P.; Huber, R.M.

    2012-01-01

    High levels of hypoxia inducible factor (HIF)-1a in tumors are reported to be associated with tumor progression and resistance to therapy. To examine the impact of HIF-1a on radioresistance under normoxia, the sensitivity towards irradiation was measured in human tumor cell lines that differ significantly in their basal HIF-1a levels. HIF-1a levels were quantified in lysates of H1339, EPLC-272H, A549, SAS, XF354, FaDu, BHY, and CX- tumor cell lines by ELISA. Protein levels of HIF-1a, HIF-2a, carbonic anhydrase IX (CA IX), and GAPDH were assessed by Western blot analysis. Knock-down experiments were performed using HIF-1a siRNA. Clonogenic survival after irradiation was determined by the colony forming assay. According to their basal HIF-1a status, the tumor cell lines were divided into low (SAS, XF354, FaDu, A549, CX-), intermediate (EPLC-272H, BHY), and high (H1339) HIF-1a expressors. The functionality of the high basal HIF-1a expression in H1339 cells was proven by reduced CA IX expression after knocking-down HIF-1a. Linear regression analysis revealed no correlation between basal HIF-1a levels and the survival fraction at either 2 or 4 Gy in all tumor cell lines investigated. Our data suggest that basal HIF-1a levels in human tumor cell lines do not predict their radiosensitivity under normoxia. (orig.)

  2. No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

    Science.gov (United States)

    Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

    2010-01-01

    Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

  3. Synthesis of Chromonylthiazolidines and Their Cytotoxicity to Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Hoang Le Tuan Anh

    2015-01-01

    Full Text Available Nine new chromonylthiazolidine derivatives were successfully semi-synthesized from paeonol. All of the compounds, including starting materials, the intermediate compound and products, were evaluated for their cytotoxic effects toward eight human cancer cell lines. The synthesized chromonylthiazolidines displayed weak cytotoxic effects against the tested cancer cell lines, but selective cytotoxic effects were observed. Compounds 3a and 3b showed the most selective cytotoxic effects against human epidermoid carcinoma (IC50 44.1 ± 3.6 μg/mL and breast cancer (IC50 32.8 ± 1.4 μg/mL cell lines, respectively. The results suggest that chromoylthiazolidines are potential low-cost, and selective anticancer agents.

  4. Selenium-regulated hierarchy of human selenoproteome in cancerous and immortalized cells lines.

    Science.gov (United States)

    Touat-Hamici, Zahia; Bulteau, Anne-Laure; Bianga, Juliusz; Jean-Jacques, Hélène; Szpunar, Joanna; Lobinski, Ryszard; Chavatte, Laurent

    2018-04-13

    Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy. We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76 Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts. We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation. We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line. We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Effect of cisplatin on the clinically relevant radiosensitivity of human cervical carcinoma cell lines

    International Nuclear Information System (INIS)

    Britten, Richard A.; Evans, Andrew J.; Allalunis-Turner, M. Joan; Pearcey, Robert G.

    1996-01-01

    Purpose: To evaluate the effect of clinically relevant levels of cisplatin on the radiosensitivity of human cervical tumor cells, and to estimate what changes in local control rates might be expected to accrue from the concomitant use of cisplatin during fractionated radiotherapy. Methods and Materials: The effects of concomitant cisplatin (1 μg/ml, a typical intratumor concentration) on the clinically relevant radiosensitivity, i.e., surviving fraction after 2 G (SF 2 ) values, was determined in 19 cloned human cervical tumor cell lines. These early passage cell lines had SF 2 values ranging from 0.26 to 0.87. Results: The concomitant administration of cisplatin reduced the clinically relevant radiosensitivity in the majority (11 out of 19) of the human tumor cell lines investigated. In only 4 out of 19 was any radiosensitization observed, and in 4 out of 19 cell lines there was no significant change in radiosensitivity. However, the sum of the independent cell killing by radiation and cisplatin, was approximately twofold higher than after radiation alone. There was no apparent dependence of the cisplatin-induced changes in SF 2 values upon the level of cell killing by cisplatin. However, there is a suggestion that concomitant cisplatin administration may have a differential effect in inherently radiosensitive and resistant human tumor cell lines. Conclusions: Our data suggest that concomitant cisplatin/radiotherapy regimens may result in a higher level of local tumor control, but primarily through additive toxicity and not through radiosensitization. Future improvements in local tumor control may, thus, be derived by increasing the total dose of cisplatin

  6. Generation and properties of a new human ventral mesencephalic neural stem cell line

    DEFF Research Database (Denmark)

    Villa, Ana; Liste, Isabel; Courtois, Elise T

    2009-01-01

    . Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal......Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro...... derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing....

  7. Response of sensitive human ataxia and resistant T-1 cell lines to accelerated heavy ions

    International Nuclear Information System (INIS)

    Tobias, C.A.; Blakely, E.A.; Chang, P.Y.; Lommel, L.; Roots, R.

    1983-07-01

    The radiation dose responses of fibroblast from a patient with Ataxia telangiectasis (AT-2SF) and an established line of human T-1 cells were studied. Nearly monoenergetic accelerated neon and argon ions were used at the Berkeley Bevalac with various residual range values. The LET of the particles varied from 30 keV/μm to over 1000 keV/μm. All Ataxia survival curves were exponential functions of the dose. Their radiosensitivity reached peak values at 100 to 200 keV/μm. Human T-1 cells have effective sublethal damage repair as has been evidenced by split dose experiments, and they are much more resistant to low LET than to high LET radiation. The repair-misrepair model has been used to interpret these results. We have obtained mathematical expressions that describe the cross sections and inactivation coefficients for both human cell lines as a function of the LET and the type of particle used. The results suggest either that high-LET particles induce a greater number of radiolesions per track or that heavy-ions at high LET induce lesions that kill cells more effectively and that are different from those produced at low LET. We assume that the lesions induced in T-1 and Ataxia cells are qualitatively similar and that each cell line attempts to repair these lesions. The result in most irradiated Ataxia cells, however, is either lethal misrepair or incomplete repair leading to cell death. 63 references, 10 figures, 1 table

  8. 3-Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines.

    Science.gov (United States)

    Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Kwak, Byung Kook; Loffroy, Romaric; Vali, Mustafa

    2010-03-01

    Autophagy, a cellular response to stress, plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). Recently, we reported that the pyruvate analog 3-bromopyruvate (3-BrPA) promoted tumor cell death by targeting GAPDH. In continuance, we investigated the intracellular response of two human HCC cell lines (Hep3B and SK-Hep1) that differ in their status of key apoptotic regulators, p53 and Fas. 3-BrPA treatment induced endoplasmic reticulum (ER) stress, translation inhibition and apoptosis based on Western blot and qPCR, pulse labeling, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and active caspase-3 in both the cell lines. However, electron microscopy revealed that 3-BrPA treated SK-Hep1 cells underwent classical apoptotic cell death while Hep3B cells initially responded with the protective autophagy that failed to prevent eventual apoptosis. 3-BrPA treatment promotes apoptosis in human HCC cell lines, irrespective of the intracellular response.

  9. Preliminary study of steep pulse irreversible electroporation technology in human large cell lung cancer cell lines L9981

    Directory of Open Access Journals (Sweden)

    Song Zuoqing

    2013-01-01

    Full Text Available Our aim was to validate the effectiveness of steep pulse irreversible electroporation technology in human large cell lung cancer cells and to screen the optimal treatment of parameters for human large cell lung cancer cells. Three different sets of steep pulse therapy parameters were applied on the lung cancer cell line L9981. The cell line L9981 inhibition rate and proliferation capacity were detected by Vi-Cell vitality analysis and MTT. Steep pulsed irreversible electroporation technology for large cell lung cancer L9981 presents killing effects with various therapy parameters. The optimal treatment parameters are at a voltage amplitude of 2000V/cm, pulse width of 100μs, pulse frequency of 1 Hz, pulse number 10. With this group of parameters, steep pulse could have the best tumor cell-killing effects.

  10. Abnormal number cell division of human thyroid anaplastic carcinoma cell line, SW 1736

    Directory of Open Access Journals (Sweden)

    Keiichi Ikeda

    2015-12-01

    Full Text Available Cell division, during which a mother cell usually divides into two daughter cells during one cell cycle, is the most important physiological event of cell biology. We observed one-to-four cell division during imaging of live SW1736 human thyroid anaplastic carcinoma cells transfected with a plasmid expressing the hybrid protein of green fluorescent protein and histone 2B (plasmid eGFP-H2B. Analysis of the images revealed a mother cell divided into four daughter cells. And one of the abnormally divided daughter cells subsequently formed a dinucleate cell.

  11. Responses of human normal osteoblast cells and osteoblast-like cell line, MG-63 cells, to pulse electromagnetic field (PEMF

    Directory of Open Access Journals (Sweden)

    Suttatip Kamolmatyakul

    2008-01-01

    Full Text Available The objective of this in vitro study is to investigate the effect of pulsed electromagnetic field (PEMF on cellular proliferation and osteocalcin production of osteoblast-like cell line, MG-63 cells, and human normal osteoblast cells (NHOC obtained from surgical bone specimens. The cells were placed in 24-well culture plates in the amount of 3x104 cell/wells with 2 ml αMEM media supplemented with 10% FBS. The experimental plates were placed between a pair of Helmoltz coils powered by a pulse generator (PEMF, 50 Hz, 1.5 mV/cm in the upper compartment of a dual incubator (Forma. The control plates were placed in the lower compartment of the incubator without Helmotz coils. After three days, the cell proliferation was measured by the method modified from Mossman (J. Immunol Methods 1983; 65: 55-63. Other sets of plates were used for osteocalcin production assessment. Media from these sets were collected after 6 days and assessed for osteocalcin production using ELISA kits. The data were analyzed using a one-way analysis of variance (ANOVA. The results showed that MG-63 cells from the experimental group proliferated significantly more than those from the control group (20% increase, p<0.05. No significant difference in osteocalcin production was detected between the two groups. On the other hand, NHOC from the experimental group produced larger amount of osteocalcin (25% increase, p<0.05 and proliferated significantly more than those from the control group (100% increase, p<0.05. In conclusion, PEMF effect on osteoblasts might depend on their cell type of origin. For osteoblast-like cell line, MG-63 cells, PEMF increased proliferation rate but not osteocalcin production of the cells. However, PEMF stimulation effect on human normal osteoblast cells was most likely associated with enhancement of both osteocalcin production and cell proliferation.

  12. Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.

    Science.gov (United States)

    Emadi Baygi, Modjtaba; Soheili, Zahra Soheila; Schmitz, Ingo; Sameie, Shahram; Schulz, Wolfgang A

    2010-12-01

    The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.

  13. Generation and properties of a new human ventral mesencephalic neural stem cell line

    Energy Technology Data Exchange (ETDEWEB)

    Villa, Ana; Liste, Isabel; Courtois, Elise T.; Seiz, Emma G.; Ramos, Milagros [Center of Molecular Biology ' Severo Ochoa' , Autonomous University of Madrid-C.S.I.C., Campus Cantoblanco 28049-Madrid (Spain); Meyer, Morten [Department of Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Winslowparken 21,st, DK-500, Odense C (Denmark); Juliusson, Bengt; Kusk, Philip [NsGene A/S, Ballerup (Denmark); Martinez-Serrano, Alberto, E-mail: amserrano@cbm.uam.es [Center of Molecular Biology ' Severo Ochoa' , Autonomous University of Madrid-C.S.I.C., Campus Cantoblanco 28049-Madrid (Spain)

    2009-07-01

    Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal of growth factors, proliferation and expression of v-myc were dramatically reduced and the cells differentiated into astrocytes, oligodendrocytes and neurons. hVM1 cells yield a large number of dopaminergic neurons (about 12% of total cells are TH{sup +}) after differentiation, which also produce dopamine. In addition to proneural genes (NGN2, MASH1), differentiated cells show expression of several genuine mesencephalic dopaminergic markers such as: LMX1A, LMX1B, GIRK2, ADH2, NURR1, PITX3, VMAT2 and DAT, indicating that they retain their regional identity. Our data indicate that this cell line and its clonal derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing.

  14. Low-risk susceptibility alleles in 40 human breast cancer cell lines

    International Nuclear Information System (INIS)

    Riaz, Muhammad; Elstrodt, Fons; Hollestelle, Antoinette; Dehghan, Abbas; Klijn, Jan GM; Schutte, Mieke

    2009-01-01

    Low-risk breast cancer susceptibility alleles or SNPs confer only modest breast cancer risks ranging from just over 1.0 to1.3 fold. Yet, they are common among most populations and therefore are involved in the development of essentially all breast cancers. The mechanism by which the low-risk SNPs confer breast cancer risks is currently unclear. The breast cancer association consortium BCAC has hypothesized that the low-risk SNPs modulate expression levels of nearby located genes. Genotypes of five low-risk SNPs were determined for 40 human breast cancer cell lines, by direct sequencing of PCR-amplified genomic templates. We have analyzed expression of the four genes that are located nearby the low-risk SNPs, by using real-time RT-PCR and Human Exon microarrays. The SNP genotypes and additional phenotypic data on the breast cancer cell lines are presented. We did not detect any effect of the SNP genotypes on expression levels of the nearby-located genes MAP3K1, FGFR2, TNRC9 and LSP1. The SNP genotypes provide a base line for functional studies in a well-characterized cohort of 40 human breast cancer cell lines. Our expression analyses suggest that a putative disease mechanism through gene expression modulation is not operative in breast cancer cell lines

  15. Transcriptome variations among human embryonic stem cell lines are associated with their differentiation propensity.

    Directory of Open Access Journals (Sweden)

    Changbin Sun

    Full Text Available Human embryonic stem cells (hESCs have the potential to form any cell type in the body, making them attractive cell sources in drug screening, regenerative medicine, disease and developmental processes modeling. However, not all hESC lines have the equal potency to generate desired cell types in vitro. Significant variations have been observed for the differentiation efficiency of various human ESC lines. The precise underpinning molecular mechanisms are still unclear. In this work, we compared transcriptome variations of four hESC lines H7, HUES1, HUES8 and HUES9. We found that hESC lines have different gene expression profiles, and these differentially expressed genes (DEGs are significantly enriched in developmental processes, such as ectodermal, mesodermal and endodermal development. The enrichment difference between hESC lines was consistent with its lineage bias. Among these DEGs, some pluripotency factors and genes involved in signaling transduction showed great variations as well. The pleiotropic functions of these genes in controlling hESC identity and early lineage specification, implicated that different hESC lines may utilize distinct balance mechanisms to maintain pluripotent state. When the balance is broken in a certain environment, gene expression variation between them could impact on their different lineage specification behavior.

  16. Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Vethakanraj, Helen Shiphrah; Babu, Thabraz Ahmed; Sudarsanan, Ganesh Babu; Duraisamy, Prabhu Kumar; Ashok Kumar, Sekar, E-mail: sekarashok@gmail.com

    2015-08-28

    The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observed in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER{sup +} and ER{sup −} breast cancer cell lines. - Highlights: • Acid Ceramidase inhibitor, Ceranib 2 induced apoptosis in Breast cancer cell lines (MCF 7 and MDA MB 231 cell lines). • Apoptosis is mediated by DNA fragmentation and cell cycle arrest. • Ceranib 2 upregulated the expression of pro-apoptotic genes and down regulated anti-apoptotic gene expression. • More potent compared to the standard drug Tamoxifen.

  17. Targeting ceramide metabolic pathway induces apoptosis in human breast cancer cell lines

    International Nuclear Information System (INIS)

    Vethakanraj, Helen Shiphrah; Babu, Thabraz Ahmed; Sudarsanan, Ganesh Babu; Duraisamy, Prabhu Kumar; Ashok Kumar, Sekar

    2015-01-01

    The sphingolipid ceramide is a pro apoptotic molecule of ceramide metabolic pathway and is hydrolyzed to proliferative metabolite, sphingosine 1 phosphate by the action of acid ceramidase. Being upregulated in the tumors of breast, acid ceramidase acts as a potential target for breast cancer therapy. We aimed at targeting this enzyme with a small molecule acid ceramidase inhibitor, Ceranib 2 in human breast cancer cell lines MCF 7 and MDA MB 231. Ceranib 2 effectively inhibited the growth of both the cell lines in dose and time dependant manner. Morphological apoptotic hallmarks such as chromatin condensation, fragmented chromatin were observed in AO/EtBr staining. Moreover, ladder pattern of fragmented DNA observed in DNA gel electrophoresis proved the apoptotic activity of Ceranib 2 in breast cancer cell lines. The apoptotic events were associated with significant increase in the expression of pro-apoptotic genes (Bad, Bax and Bid) and down regulation of anti-apoptotic gene (Bcl 2). Interestingly, increase in sub G1 population of cell cycle phase analysis and elevated Annexin V positive cells after Ceranib 2 treatment substantiated its apoptotic activity in MCF 7 and MDA MB 231 cell lines. Thus, we report Ceranib 2 as a potent therapeutic agent against both ER + and ER − breast cancer cell lines. - Highlights: • Acid Ceramidase inhibitor, Ceranib 2 induced apoptosis in Breast cancer cell lines (MCF 7 and MDA MB 231 cell lines). • Apoptosis is mediated by DNA fragmentation and cell cycle arrest. • Ceranib 2 upregulated the expression of pro-apoptotic genes and down regulated anti-apoptotic gene expression. • More potent compared to the standard drug Tamoxifen

  18. Irradiation effect on the apoptosis induction in the human cancer cell lines and the gingival fibroblast

    International Nuclear Information System (INIS)

    Park, Mu Soon; Lee, Sam Sun; Choi, Soon Chul; Park, Tae Won; You, Dong Soo

    1998-01-01

    The radiation-induced apoptosis was studied for two human cancer cell lines (KB cells, RPMI 2650 cells) and the human gingival fibroblast cell line (HGF-1 cells). The single irradiation of 2, 10, 20 Gy was done with 241.5 cGy/min dose rate using the 137 Cs MK cell irradiator. The cell were stained with propidium iodide and examined under the fluoro-microscope and assayed with the flow cytometry a day after irradiation. Also, the LDH assay was done to determine the amount of necrotic cells. The obtained results were as follows : 1. On the fluoro-microscope, many fragmented nuclei were detected in the KB, RPMI 2650, and HGF-1 cells after irradiation. 2. On the DNA content histogram obtained from the flow cytometry, the percentages of the pre-G1 peak of the control and 2, 10 and 20 Gy irradiation group were 4.5, 55.0, 52.3, and 66.6% on KB cells, 2.7, 3.3, 31.8, and 32.6% on RPMI 2650 cells and 2.8, 21.8, 30.4, and 40.2% on HGF-1 cells respectively. 3. The number of G1-stage cells was abruptly decreased after 2 Gy irradiation on KB cells and 10 Gy irradiation on RPMI 2650 cells, But there was a slight decrease without regard to irradiation dose on HGF-1 cells. 4. There was no significantly different absorbance in extracellular LDH assay along the experimental cell lines

  19. Experimental investigation of the cytotoxicity of medium-borne signals in human prostate cancer cell line

    International Nuclear Information System (INIS)

    Sjostedt, Svetlana; Bezak, Eva

    2012-01-01

    Introduction. Evidence exists that exposure of non-irradiated cells to Irradiated Cell Conditioned Medium (ICCM) can cause effects similar to those resulting from direct radiation damage. This study attempts to validate the stochastic model, relating absorbed dose to the emission and processing of cell death signals by non-irradiated cells, in vitro in PC3 human prostate cancer cell line. Methods. The recipient cell survival was measured after exposure of cells to ICMM derived from donor cells: a) exposed to radiation doses from 2 Gy to 8 Gy and b) of concentrations varying from 2 x 10 2 to 6 x 10 6 irradiated with 2 Gy. Results. Exposure to ICCM, irradiated with doses between 2-8 Gy, resulted in a significant (p 2 cells was significantly higher (p < 0.5) compared to the rest of donor cell concentrations, indicating that the toxicity of ICCM depends on the cellular concentration of donor cells. Non-linear regression data fitting provided reasonable agreement with the microdosimetric model for the induction of cell killing through medium-borne signals. Conclusion. For the given cell line and given experimental conditions, significant decreases in cell survival were observed in non-irradiated cells exposed to ICCM derived from donor cells of various concentrations and irradiated with different doses

  20. 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......Tumour cell adhesion, detachment and aggregation seem to play an important part in tumour invasion and metastasis, and numerous cell adhesion molecules are expressed by tumour cells. Several families of cell-cell adhesion molecules have been described, of which two groups are particularly well...... 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....

  1. Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

    Science.gov (United States)

    Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

    1996-05-01

    The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

  2. Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

    OpenAIRE

    Feng, Yi; Tian, Zhong-Min; Wan, Ming-Xi; Zheng, Zhao-Bin

    2007-01-01

    AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.

  3. 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

    Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped...... 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...

  4. Radiation-induced apoptosis and cell cycle checkpoints in human colorectal tumour cell lines

    International Nuclear Information System (INIS)

    Playle, L.C.

    2001-03-01

    The p53 tumour suppressor gene is mutated in 75% of colorectal carcinomas and is critical for DNA damage-induced G1 cell cycle arrest. Data presented in this thesis demonstrate that after treatment with Ionizing Radiation (IR), colorectal tumour cell lines with mutant p53 are unable to arrest at G1 and undergo cell cycle arrest at G2. The staurosporine derivative, UCN-01, was shown to abrogate the IR-induced G2 checkpoint in colorectal tumour cell lines. Furthermore, in some cell lines, abrogation of the G2 checkpoint was associated with radiosensitisation. Data presented in this study demonstrate that 2 out of 5 cell lines with mutant p53 were sensitised to IR by UCN-01. In order to determine whether radiosensitisation correlated with lack of functional p53, transfected derivatives of an adenoma-derived cell line were studied, in which endogenous wild type p53 was disrupted by expression of a dominant negative p53 mutant protein (and with a vector control). In both these cell lines UCN-01 abrogated the G2 arrest however this was not associated with radiosensitisation, indicating that radiosensitisation is a cell type-specific phenomenon. Although 2 colorectal carcinoma cell lines, with mutant p53, were sensitised to IR by UCN-01, the mechanisms of p53-independent IR-induced apoptosis in the colon are essentially unknown. The mitogen-activated protein kinase (MAPK) pathways (that is the JNK, p38 and ERK pathways) have been implicated in apoptosis in a range of cell systems and in IR-induced apoptosis in some cell types. Data presented in this study show that, although the MAPKs can be activated by the known activator anisomycin, there is no evidence of a role for MAPKs in IR-induced apoptosis in colorectal tumour cell lines, regardless of p53 status. In summary, some colorectal tumour cell lines with mutant p53 can be sensitised to IR-induced cell death by G2 checkpoint abrogation and this may be an important treatment strategy, however mechanisms of IR-induced p53

  5. Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells.

    Science.gov (United States)

    Farley, J R; Stilt-Coffing, B

    2001-01-01

    Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P sALP activity (P sALP activity (P sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.

  6. [Anti-tumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line in vitro].

    Science.gov (United States)

    Li, Maolan; Lu, Wei; Zhang, Fei; Ding, Qichen; Wu, Xiangsong; Tan, Zhujun; Wu, Wenguang; Weng, Hao; Wang, Xuefeng; Shi, Weibin; Dong, Ping; Gu, Jun; Liu, Yingbin

    2014-11-04

    To explore the antitumor effects of DDP-PLLA-CNTs on human cholangiocarcinoma cell line. DDP-PLLA-CNTs were prepared with the method of ultrasound emulsification. The morphology of DDP-PLLA-CNTs was determined by scanning electron microscope (SEM). And its drug loading and drug release curve in vitro was detected by UV-Vis-NIR spectrophotometer. CCK8 was used to test the cytotoxic effects of DDP-PLLA-CNTs at different concentrations on QBC939 cell proliferation.Flow cytometry was employed to measure the changes of apoptotic rate. With excellent controlled-release characteristic of in vitro drug release, DDP-PLLA-CNTs inhibited the proliferation and significantly increased the apoptotic rate of QBC939 cell line. DDP-PLLA-CNTs have drug sustained-release characteristics and can significantly inhibit the proliferation of QBC939 cell line.

  7. Assessment of cytotoxicity of Portulaca oleracea Linn. against human colon adenocarcinoma and vero cell line

    Science.gov (United States)

    Mali, Prashant Y.

    2015-01-01

    Background: Portulaca oleracea Linn. (Portulacaceae) is commonly known as purslane in English. In traditional system it is used to cure diarrhea, dysentery, leprosy, ulcers, asthma, and piles, reduce small tumors and inflammations. Aim: To assess cytotoxic potential of chloroform extract of P. oleracea whole plant against human colon adenocarcinoma (HCT-15) and normal (Vero) cell line. Materials and Methods: Characterization of chloroform extract of P. oleracea by Fourier transform infrared (FTIR) spectroscopy was performed. Cytotoxicity (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was used for assessment of cytotoxic potential of chloroform extract of P. oleracea. The concentrations of 1000–0.05 μg/ml were used in the experiment. Doxorubicin was considered as standard reference drug. Results: FTIR spectrum showed the peak at 1019.52 and 1396.21 center. The 50% cell growth inhibition (IC50) of chloroform extract of P. oleracea and doxorubicin was 1132.02 μg/ml and 460.13 μg/ml against human colon adenocarcinoma and 767.60 μg/ml and 2392.71 μg/ml against Vero cell line, respectively. Conclusion: Chloroform extract of P. oleracea whole plant was less efficient or does not have cytotoxic activity against human colon adenocarcinoma cell line. It was not safe to normal Vero cell line. But, there is a need to isolate, identify, and confirm the phytoconstituents present in extract by sophisticated analytical techniques. PMID:27833374

  8. Production of Multiple Growth Factors by a Newly Established Human Thyroid Carcinoma Cell Line

    Science.gov (United States)

    Yoshida, Yataro; Ohashi, Kensaku; Sano, Emiko; Kobayashi, Hisataka; Endo, Keigo; Naruto, Masanobu; Nakamura, Toru

    1992-01-01

    A multiple growth factor‐producing tumor cell line (NIM‐1) was newly established from a patient with thyroid cancer and remarkable neutrophilia. NIM‐1 cells also caused severe neutrophilia in nude mice bearing tumors. NIM‐1‐conditioned medium (NIM‐1CM) contained activities that supported not only granulocyte, macrophage and eosinophil colony formation of human bone marrow cells but also the growth of colony‐stimulating factor (CSF)‐dependent cell lines, NFS60‐KX and TF‐1. Northern blot hybridization analysis revealed the constitutive expression of granulocyte‐CSF (G‐CSF), granulocyte/macrophage‐CSF (GM‐CSF) and interleukin(IL)‐6 mRNAs in NIM‐1 cells. Enzyme‐linked immunosorbent assays (ELISA) using NIM‐1CM also confirmed the production of IL‐la and a small amount of IL‐1β besides G‐CSF, GM‐CSF and IL‐6 in NIM‐1 cells. In addition, unexpected production of IL‐11 in NIM‐1 cells was detected by northern blot hybridization analysis and by bioassay using an IL‐11‐dependent cell line. Therefore, NIM‐1 cell line is shown to produce multiple cytokines including potentially megakaryopoietic growth factors such as GM‐CSF, IL‐6 and IL‐11. PMID:1372885

  9. FLAX OIL FROM TRANSGENIC LINUM USITATISSIMUM SELECTIVELY INHIBITS IN VITRO PROLIFERATION OF HUMAN CANCER CELL LINES.

    Science.gov (United States)

    Gebarowski, Tomasz; Gebczak, Katarzyna; Wiatrak, Benita; Kulma, Anna; Pelc, Katarzyna; Czuj, Tadeusz; Szopa, Jan; Gasiorowski, Kazimierz

    2017-03-01

    Emulsions made of oils from transgenic flaxseeds significantly decreased in vitro proliferation of six tested human cancer cell lines in 48-h cultures, as assessed with the standard sulforhodamine assay. However, the emulsions also increased proliferation rate of normal human dermal fibroblasts and, to a lower extend, of keratinocytes. Both inhibition of in vitro proliferation of human cancer cell lines and stimulation of proliferation of normal dermal fibroblasts and keratinocytes were especially strong with the emulsion type B and with emulsion type M. Oils from seeds of transgenic flax type B and M should be considered as valuable adjunct to standard cytostatic therapy of human cancers and also could be applied to improve the treatment of skin lesions in wound healing.

  10. Spontaneous lung metastasis formation of human Merkel cell carcinoma cell lines transplanted into scid mice.

    Science.gov (United States)

    Knips, Jill; Czech-Sioli, Manja; Spohn, Michael; Heiland, Max; Moll, Ingrid; Grundhoff, Adam; Schumacher, Udo; Fischer, Nicole

    2017-07-01

    Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation. © 2017 UICC.

  11. Effect of NCAM-transfection on growth and invasion of a human cancer cell line

    DEFF Research Database (Denmark)

    Edvardsen, K; Bock, E; Jirus, S

    1997-01-01

    of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth......-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.......A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial...

  12. Effects of hematin and Moessbauer gamma-radiation on human osteosarcoma cell lines

    International Nuclear Information System (INIS)

    Ortalli, I.; Pedrazzi, G.; Fano, V.; Ma, W.; Cai, S.; Giuliani, N.; Passeri, M.

    1996-01-01

    In the present work it is reported an investigation on cultures of the human osteosarcoma cell line 'MG-63' and the human osteoblastic cell line 'HOBIT'. It is examined the combined effects of hematin and 14,4keV gamma-radiation from a Moessbauer source. Preliminary results seem to confirm the trend already observed for the bone marrow system. Different degrees of growth inhibition were observed when hematin alone and hematin plus gamma-rays were administered to the cultures. It was previously found that hematin has no toxic effects on normal cells up to a concentration of 10 -4 M, but the same concentration produces various degrees of inhibition on cultures of tumor cells. While no significant effect could be attributed to irradiation alone, hematin plus irradiations show a larger inhibition than that expected for pure additive effects

  13. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

    OpenAIRE

    Barallon, Rita; Bauer, Steven R.; Butler, John; Capes-Davis, Amanda; Dirks, Wilhelm G.; Elmore, Eugene; Furtado, Manohar; Kline, Margaret C.; Kohara, Arihiro; Los, Georgyi V.; MacLeod, Roderick A. F.; Masters, John R. W.; Nardone, Mark; Nardone, Roland M.; Nims, Raymond W.

    2010-01-01

    Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer...

  14. Adaptive response to ionizing radiation induced by low doses of gamma rays in human cell lines

    International Nuclear Information System (INIS)

    Seong, Jinsil; Chang, Ok Suh; Gwi, Eon Kim

    1995-01-01

    Purpose: The aim of this study was to investigate whether the adaptive response could be induced in human lymphoblastoid cell lines and human tumor cell lines. The time necessary for the expression of the adaptive response was also investigated. Materials and Methods: Three lymphoblastoid cell lines from ataxia telangiectasia (AT) homozygote (GM 1526), AT heterozygote (GM 3382), and normal individual (3402p) and two hepatoma cell lines, Hep G2 and Hep 3B, were used in this study. Experiments were carried out by delivering 0.01 Gy followed by 0.5 Gy of gamma radiation to the exponentially growing cells. The time necessary for the expression of the adaptive response was determined by varying the time interval between the two doses from 1 to 72 h. In some experiments, 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, was added immediately after the 0.5 Gy exposure. The cultures were fixed 30 min (for the G 2 chromatid) and 6 h (for the S chromatid) after the 0.5 Gy exposure. Metaphase chromosome assay was carried out to score chromatid breaks as an end point. Results: A prior exposure to 0.01 Gy of gamma rays significantly reduced the number of chromatid breaks induced by subsequent higher doses (0.5 Gy) in all the tested cell lines. The magnitude of the adaptive response was similar among the cell lines despite their different radiosensitivities. In the G 2 chromatids, the adaptive response was observed both at short-time intervals, as early as 1 h, and at long-time intervals. In the S chromatids, however, the adaptive response was shown only at long-time intervals. When 3-aminobenzamide was added after the 0.5 Gy, the adaptive responses were abolished in all the experimental groups. Conclusion: The adaptive response was observed in human lymphoblastoid cell lines and hepatoma cell lines. The magnitude of the adaptive response did not seem to be related to the radiosensitivity of the cells. The elimination of the adaptive response with 3

  15. Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

    Science.gov (United States)

    Bodel, P; Ralph, P; Wenc, K; Long, J C

    1980-02-01

    Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

  16. Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line.

    Science.gov (United States)

    Shipley, Mackenzie M; Mangold, Colleen A; Szpara, Moriah L

    2016-02-17

    Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

  17. Derivation of the clinical grade human embryonic stem cell line RCe016-A (RC-12

    Directory of Open Access Journals (Sweden)

    P.A. De Sousa

    2016-05-01

    Full Text Available The human embryonic stem cell line RCe016-A (RC-12 was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12 shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15, which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.

  18. O6-alkylguanine-DNA-alkyltransferase activity and nitrosourea sensitivity in human cancer cell lines.

    OpenAIRE

    Walker, M. C.; Masters, J. R.; Margison, G. P.

    1992-01-01

    The DNA repair enzyme, O6-alkylguanine-DNA-alkyltransferase (ATase), is thought to be the principal mechanism controlling resistance to nitrosoureas and related alkylating agents. We compared the sensitivities of five human testis and five bladder tumour cell lines to two nitrosoureas (N-nitroso-N-methylurea (MNU) and mitozolomide) with cellular levels of ATase. Enzyme levels ranged from 3 to 206 fmol mg-1 protein (0.1 x 10(4) to 5.1 x 10(4) molecules/cell) in the testis lines and from 11 to ...

  19. Sensitivity to ionizing radiation and chemotherapeutic agents in gemcitabine-resistant human tumor cell lines

    International Nuclear Information System (INIS)

    Bree, Chris van; Kreder, Natasja Castro; Loves, Willem J.P.; Franken, Nicolaas A.P.; Peters, Godefridus J.; Haveman, Jaap

    2002-01-01

    Purpose: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells. Methods and Materials: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel, 5-fluorouracil (5-FU), methotrexate (MTX), cytarabine (ara-C), and dFdC was measured by a proliferation assay. Radiosensitivity and radioenhancement by dFdC of this cell panel and the human ovarian carcinoma cell line A2780 and its dFdC-resistant variant AG6000 were determined by clonogenic assay. Bivariate flowcytometry was performed to study cell cycle changes. Results: In the SWg, a complete deoxycytidine kinase (dCK) deficiency was found on mRNA and protein level. This was accompanied by a 10-fold decrease in dCK activity which resulted in the >1000-fold resistance to dFdC. Sensitivity to other anti-tumor drugs was not altered, except for ara-C (>100-fold resistance). Radiosensitivity was not altered in the dFdC-resistant cell lines SWg and AG6000. High concentrations (50-100 μM dFdC) induced radioenhancement in the dFdC-resistant cell lines similar to the radioenhancement obtained at lower concentrations (10 nM dFdC) in the parental lines. An early S-phase arrest was found in all cell lines after dFdC treatment where radioenhancement was achieved. Conclusions: In the dFdC-resistant lung tumor cell line SWg, the deficiency in dCK is related to the resistance to dFdC and ara-C. No cross-resistance was observed to other anti-tumor drugs used for the treatment in lung cancer. Sensitivity to ionizing radiation was not altered in two different dFdC-resistant cell lines. Resistance to dFdC does not eliminate the ability of dFdC to sensitize cells to radiation

  20. Establishment and characterization of a human uterine endometrial undifferentiated carcinoma cell line, TMG-L.

    Science.gov (United States)

    Hasegawa, Kiyoshi; Suzuki, Machiko; Ishikawa, Kunimi; Yasue, Akira; Kato, Rina; Nakamura, Azumi; Kuroki, Jun; Udagawa, Yasuhiro

    2003-03-01

    A new cell line of human uterine endometrial undifferentiated carcinoma, designated as TMG-L, was established from the metastatic lymph node of 56-year-old patient TMG-L cells have been cultured with Ham's F-12 medium supplemented with 10% FCS and grew as a loosely adherent monolayer with polygonal or spindle-shaped cells exhibiting poor cell-cell contact and piled up against each other, showing a tendency to grow as floating cells. The doubling time of this cell line was about 48 hours, and chromosomal analysis revealed aneuploidy at passage 25. The cells formed tumors in SCID mouse, the histology of which was similar to that of undifferentiated carcinoma component of primary tumor. TMG-L cells showed the loss of expression and membranous localization of either E-cadherin or alpha-catenin, implied corresponding loss of their adhesive function. And this dysfunction implicated the biological aggressive behavior of uterine endometrial undifferentiated carcinoma. This cell line appears to provide a useful system for studying uterine undifferentiated carcinoma in vivo and in vitro.

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

    International Nuclear Information System (INIS)

    Zhang, Ping; Zhang, Zhiyuan; Zhou, Xiaojian; Qiu, Weiliu; Chen, Fangan; Chen, Wantao

    2006-01-01

    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. 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. 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. 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 and further intervention in cisplatin resistance

  2. 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

  3. 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

    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...

  4. LINE-1 couples EMT programming with acquisition of oncogenic phenotypes in human bronchial epithelial cells.

    Science.gov (United States)

    Reyes-Reyes, Elsa M; Aispuro, Ivan; Tavera-Garcia, Marco A; Field, Matthew; Moore, Sara; Ramos, Irma; Ramos, Kenneth S

    2017-11-28

    Although several lines of evidence have established the central role of epithelial-to-mesenchymal-transition (EMT) in malignant progression of non-small cell lung cancers (NSCLCs), the molecular events connecting EMT to malignancy remain poorly understood. This study presents evidence that Long Interspersed Nuclear Element-1 (LINE-1) retrotransposon couples EMT programming with malignancy in human bronchial epithelial cells (BEAS-2B). This conclusion is supported by studies showing that: 1) activation of EMT programming by TGF-β1 increases LINE-1 mRNAs and protein; 2) the lung carcinogen benzo(a)pyrene coregulates TGF-β1 and LINE-1 mRNAs, with LINE-1 positioned downstream of TGF-β1 signaling; and, 3) forced expression of LINE-1 in BEAS-2B cells recapitulates EMT programming and induces malignant phenotypes and tumorigenesis in vivo . These findings identify a TGFβ1-LINE-1 axis as a critical effector pathway that can be targeted for the development of precision therapies during malignant progression of intractable NSCLCs.

  5. Comparison of thermoradiosensitization in two human melanoma cell lines and one fibroblast cell line by concurrent mild hyperthermia and low-dose-rate irradiation

    International Nuclear Information System (INIS)

    Raaphorst, G.P.; Bussey, A.; Heller, D.P.; Ng, C.E.

    1994-01-01

    Two human melanoma cell lines, one radioresistant (Sk-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41 degrees C). None of the cell lines expressed chronic thermotolerance during heating at 39-41 degrees C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40 degrees C but HT-144 cells were more sensitive at 41 degrees C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD. 24 refs., 5 figs., 1 tab

  6. Glycoengineering of Human Cell Lines Using Zinc Finger Nuclease Gene Targeting

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Bennett, Eric Paul; Clausen, Henrik

    2013-01-01

    Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome...... with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using zinc finger nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated...... in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc finger nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells....

  7. Adiponectin and Its Receptors Are Differentially Expressed in Human Tissues and Cell Lines of Distinct Origin

    Directory of Open Access Journals (Sweden)

    Simon Jasinski-Bergner

    2017-12-01

    Full Text Available Background: Adiponectin is secreted by adipose tissue and exerts high abundance and an anti-inflammatory potential. However, only little information exists about the expression profiles of adiponectin and its recently identified receptor CDH13 in non-tumorous human tissues and their association to clinical parameters. Methods: The expression levels of adiponectin and CDH13 were analyzed in heart, liver, kidney, spleen, skin, blood vessels, peripheral nerve and bone marrow of 21 human body donors, in 12 human cell lines, and in purified immune effector cell populations of healthy blood donors by immunohistochemistry, Western-blot, and semi-quantitative PCR. The obtained results were then correlated to clinical parameters, including age, sex and known diseases like cardiovascular and renal diseases. Results: Adiponectin expression in renal corpuscles was significantly higher in humans with known renal diseases. A coordinated expression of adiponectin and CDH13 was observed in the myocard. High levels of adiponectin could be detected in the bone marrow, in certain lymphoid tumor cell lines and in purified immune effector cell populations of healthy donors, in particular in cytotoxic T cells. Conclusion: For the first time, the expression profiles of adiponectin and CDH13 are analyzed in many human tissues in correlation to each other and to clinical parameters.

  8. 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

    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......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...

  9. Effects of gamma radiation on the OM431 human ocular melanoma cell line

    International Nuclear Information System (INIS)

    Logani, S.; Cho, A.S.; Su, L.D.; Withers, H.R.; McBride, W.H.; Hall, M.O.; Lee, D.A.; Milani, J.K.; Straatsma, B.R.

    1995-01-01

    In order to determine the dose responsiveness to radiation of ocular melanoma, we conducted an in vitro dose-response study on a monolayer cell culture using a clonogenic assay. The effects on cell survival were determined relative to unirradiated controls. A human epithelioid ocular melanoma cell line, OM431, was maintained in tissue culture and serial dilutions of viable cells were plated in flasks, allowed to settle and attach for 48 h, and subsequently irradiated with 1-10 Gy in single fractions. After 2 weeks, the number of reproducing clones (forming colonies with greater than 32 cells or five generations) were counted. The surviving fractions of cells were plotted on a cell survival curve using the linear quadratic model. The survival curve showed a large initial shoulder followed by an exponential decline in growth. Our data suggest that the OM431 ocular melanoma cell line responds to irradiation in a manner similar to other melanoma cell lines and is relatively radioresistent especially at lower doses. (author)

  10. DNA fragmentation and apoptosis induced by safranal in human prostate cancer cell line

    Directory of Open Access Journals (Sweden)

    Saeed Samarghandian

    2013-01-01

    Full Text Available Objectives: Apoptosis, an important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus (Saffron in different cancer types. However, limited effort has been made to correlate these effects to the active ingredients of saffron. The present study was designed to elucidate cytotoxic and apoptosis induction by safranal, the major coloring compound in saffron, in a human prostate cancer cell line (PC-3. Materials and Methods: PC-3 and human fetal lung fibroblast (MRC-5 cells were cultured and exposed to safranal (5, 10, 15, and 20 μg/ml. The 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay was performed to assess cytotoxicity. DNA fragmentation was assessed by gel electrophoresis. Cells were incubated with different concentrations of safranal, and cell morphologic changes and apoptosis were determined by the normal inverted microscope, Annexin V, and propidium iodide, followed by flow cytometric analysis, respectively. Results: MTT assay revealed a remarkable and concentration-dependent cytotoxic effect of safranal on PC-3 cells in comparison with non-malignant cell line. The morphologic alterations of the cells confirmed the MTT results. The IC 50 values against PC-3 cells were found to be 13.0 ΁ 0.07 and 6.4 ΁ 0.09 μg/ml at 48 and 72 h, respectively. Safranal induced an early and late apoptosis in the flow cytometry histogram of treated cells, indicating apoptosis is involved in this toxicity. DNA analysis revealed typical ladders as early as 48 and 72 h after treatment, indicative of apoptosis. Conclusions: Our preclinical study demonstrated a prostate cancer cell line to be highly sensitive to safranal-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of safranal action are not clearly understood, it appears to have potential as a therapeutic agent.

  11. Spontaneous transformation of human granulosa cell tumours into an aggressive phenotype: a metastasis model cell line

    International Nuclear Information System (INIS)

    Imai, Misa; Muraki, Miho; Takamatsu, Kiyoshi; Saito, Hidekazu; Seiki, Motoharu; Takahashi, Yuji

    2008-01-01

    Granulosa cell tumours (GCTs) are frequently seen in menopausal women and are relatively indolent. Although the physiological properties of normal granulosa cells have been studied extensively, little is known about the molecular mechanism of GCT progression. Here, we characterise the unique behavioural properties of a granulosa tumour cell line, KGN cells, for the molecular analysis of GCT progression. Population doubling was carried out to examine the proliferation capacity of KGN cells. Moreover, the invasive capacity of these cells was determined using the in vitro invasion assay. The expression level of tumour markers in KGN cells at different passages was then determined by Western blot analysis. Finally, the growth and metastasis of KGN cells injected subcutaneously (s.c.) into nude mice was observed 3 months after injection. During in vitro culture, the advanced passage KGN cells grew 2-fold faster than the early passage cells, as determined by the population doubling assay. Moreover, we found that the advanced passage cells were 2-fold more invasive than the early passage cells. The expression pattern of tumour markers, such as p53, osteopontin, BAX and BAG-1, supported the notion that with passage, KGN cells became more aggressive. Strikingly, KGN cells at both early and advanced passages metastasized to the bowel when injected s.c. into nude mice. In addition, more tumour nodules were formed when the advanced passage cells were implanted. KGN cells cultured in vitro acquire an aggressive phenotype, which was confirmed by the analysis of cellular activities and the expression of biomarkers. Interestingly, KGN cells injected s.c. are metastatic with nodule formation occurring mostly in the bowel. Thus, this cell line is a good model for analysing GCT progression and the mechanism of metastasis in vivo

  12. pSv3neo transfection and radiosensitivity of human cancer cell lines

    International Nuclear Information System (INIS)

    Parris, C.N.; Masters, J.R.W.; Green, M.H.L.

    1990-01-01

    Immortalisation of human fibroblasts by transfection with a plasmid, pSV3neo, results in an increase in their radioresistance. The change in radiosensitivity may either be a consequence of transformation or due to expression of the SV40 T-antigen in pSV3neo. To investigate these two possibilities, we transfected pSV3neo into cells already transformed and immortalised. The radiosensitivies of three human bladder cancer cell lines were unaltered in clones expressing T-antigen, indicating that the changes observed in fibroblasts probably are a consequence of transformation, and not the presence of SV40 T-antigen. (author)

  13. The human hepatocyte cell lines IHH and HepaRG: models to study glucose, lipid and lipoprotein metabolism.

    Science.gov (United States)

    Samanez, Carolina Huaman; Caron, Sandrine; Briand, Olivier; Dehondt, Hélène; Duplan, Isabelle; Kuipers, Folkert; Hennuyer, Nathalie; Clavey, Véronique; Staels, Bart

    2012-07-01

    Metabolic diseases reach epidemic proportions. A better knowledge of the associated alterations in the metabolic pathways in the liver is necessary. These studies need in vitro human cell models. Several human hepatoma models are used, but the response of many metabolic pathways to physiological stimuli is often lost. Here, we characterize two human hepatocyte cell lines, IHH and HepaRG, by analysing the expression and regulation of genes involved in glucose and lipid metabolism. Our results show that the glycolysis pathway is activated by glucose and insulin in both lines. Gluconeogenesis gene expression is induced by forskolin in IHH cells and inhibited by insulin in both cell lines. The lipogenic pathway is regulated by insulin in IHH cells. Finally, both cell lines secrete apolipoprotein B-containing lipoproteins, an effect promoted by increasing glucose concentrations. These two human cell lines are thus interesting models to study the regulation of glucose and lipid metabolism.

  14. Generation of human cortical neurons from a new immortal fetal neural stem cell line

    International Nuclear Information System (INIS)

    Cacci, E.; Villa, A.; Parmar, M.; Cavallaro, M.; Mandahl, N.; Lindvall, O.; Martinez-Serrano, A.; Kokaia, Z.

    2007-01-01

    Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology

  15. Carboxylated nanodiamonds are neither cytotoxic nor genotoxic on liver, kidney, intestine and lung human cell lines.

    Science.gov (United States)

    Paget, V; Sergent, J A; Grall, R; Altmeyer-Morel, S; Girard, H A; Petit, T; Gesset, C; Mermoux, M; Bergonzo, P; Arnault, J C; Chevillard, S

    2014-08-01

    Although nanodiamonds (NDs) appear as one of the most promising nanocarbon materials available so far for biomedical applications, their risk for human health remains unknown. Our work was aimed at defining the cytotoxicity and genotoxicity of two sets of commercial carboxylated NDs with diameters below 20 and 100 nm, on six human cell lines chosen as representative of potential target organs: HepG2 and Hep3B (liver), Caki-1 and Hek-293 (kidney), HT29 (intestine) and A549 (lung). Cytotoxicity of NDs was assessed by measuring cell impedance (xCELLigence® system) and cell survival/death by flow cytometry while genotoxicity was assessed by γ-H2Ax foci detection, which is considered the most sensitive technique for studying DNA double-strand breaks. To validate and check the sensitivity of the techniques, aminated polystyrene nanobeads were used as positive control in all assays. Cell incorporation of NDs was also studied by flow cytometry and luminescent N-V center photoluminescence (confirmed by Raman microscopy), to ensure that nanoparticles entered the cells. Overall, we show that NDs effectively entered the cells but NDs do not induce any significant cytotoxic or genotoxic effects on the six cell lines up to an exposure dose of 250 µg/mL. Taken together these results strongly support the huge potential of NDs for human nanomedicine but also their potential as negative control in nanotoxicology studies.

  16. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    International Nuclear Information System (INIS)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

    1991-01-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

  17. Functional activity of human sodium/iodide symporter in tumor cell lines

    International Nuclear Information System (INIS)

    Petrich, T.; Knapp, W.H.; Poetter, E.

    2003-01-01

    Aim: The sodium/iodide symporter (NIS) actively transports iodide into thyrocytes. Thus, NIS represents a key protein for diagnosis and radioiodine therapy of differentiated thyroid cancer. Additionally, in the future the NIS gene may be used for cancer gene therapy of non-thyroid-derived malignancies. In this study we evaluated the functionality of NIS with respect to iodide uptake in a panel of tumor cell lines and compared this to gene transfer efficiency. Methods: A human NIS-containing expression vector and reporter-gene vectors encoding and beta;-Galactosidase- or EGFP were used for transient transfection of 13 tumor cell lines. Following transfection measurements of NIS-mediated radioiodide uptake using Na 125 I and of transfection efficiency were performed. The latter included β;-Galactosidase activity measurements using a commercial kit and observation by fluorescence microscopy for EGFP expression. Results: In contrast to respective parental cells, most NIS-transfected cell lines displayed high, perchlorate-sensitive radioiodide uptake. Differences in radioiodide uptake between cell lines apparently corresponded to transfection efficiencies, as judged from reporter-gene assays. Conclusion: With respect to iodide uptake we provide evidence that NIS is functional in different cellular context. As iodide uptake capacity appears to be well correlated to gene transfer efficiency, cell type-specific actions on NIS (e. g. post-translational modification such as glycosylation) are not inhibitory to NIS function. Our data support the promising role of NIS in cancer gene therapy strategies. (orig.)

  18. 78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

    Science.gov (United States)

    2013-02-28

    ... Comment Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research... Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research. OMB No. 0925-0601-- Expiration Date... and Use of Information Collection: The form is used by applicants to request that human embryonic stem...

  19. Postirradiation expression of lethal mutations in an immortalized human keratinocyte cell line

    International Nuclear Information System (INIS)

    O'Reilly, S.; Mothersill, C.; Seymour, C.B.

    1994-01-01

    The quantification of the extent of delayed cell death and the rate and pattern of its occurrence in relation to the cell division cycle is important in radiotherapy and also in radiation transformation studies related to protection and dose limits. Here the numbers of lethal mutations occurring over 45 population doublings (clonal expansion to about 10 13 cells per cell originally surviving irradiation) was measured in an HPV 16 immortalized human keratinocyte cell lines used for transformation studies. The results showed that when postirradiation (dose range 1-6 Gy) growth curves were constructed, the difference in slopes could be accounted for entirely by correcting for the non-clonogenic fraction in the cell count, excluding a longer cell generation time as an explanation. When the cell loss was examined over the entire growth period of 6 weeks (about 45 doublings of the cell population), it was found to be dose dependent for the first two passages, but then to become more independent of dose. The results allow a time/cell generation dependent factor to be derived for the cell line and used in survival curve equations where effects of radiation are being measured at times distant from the original exposure. (author)

  20. Differential repair of platinum-DNA adducts in human bladder and testicular tumor continuous cell lines

    International Nuclear Information System (INIS)

    Bedford, P.; Fichtinger-Schepman, A.M.; Shellard, S.A.; Walker, M.C.; Masters, J.R.; Hill, B.T.

    1988-01-01

    The formation and removal of four platinum-DNA adducts were immunochemically quantitated in cultured cells derived from a human bladder carcinoma cell line (RT112) and from two lines derived from germ cell tumors of the testis (833K and SUSA), following exposure in vitro to 16.7 microM (5 micrograms/ml) cisplatin. RT112 cells were least sensitive to the drug and were proficient in the repair of all four adducts, whereas SUSA cells, which were 5-fold more sensitive, were deficient in the repair of DNA-DNA intrastrand cross-links in the sequences pApG and pGpG. Despite expressing a similar sensitivity to SUSA cells, 833K cells were proficient in the repair of all four adducts, although less so than the RT112 bladder tumor cells. In addition, SUSA cells were unable to repair DNA-DNA interstrand cross-links whereas 50-85% of these lesions were removed in RT112 and 833K cells 24 h following drug exposure. It is possible that the inability of SuSa cells to repair platinated DNA may account for their hypersensitivity to cisplatin

  1. Cytokeratin characterization of human prostatic carcinoma and its derived cell lines.

    Science.gov (United States)

    Nagle, R B; Ahmann, F R; McDaniel, K M; Paquin, M L; Clark, V A; Celniker, A

    1987-01-01

    Two murine monoclonal anti-cytokeratin antibodies with defined specificity were shown to distinguish between basal cells and luminal cells in human prostate tissue. Forty-one biopsies or transurethral resection specimens were characterized using these two antibodies. In cases of benign prostatic hyperplasia, focal loss of the basal cell layer was noted in areas of glandular proliferation. Ten cases of adenocarcinoma of the prostate, varying in Gleason's histological grade from 2 to 4, were also studied. In each case the carcinoma was shown to represent the luminal cell phenotype with no evidence of involvement of the basal cell phenotype. An analysis of three established metastatic prostatic carcinoma cell lines (DU-145, PC-3, and LNCaP) using two-dimensional electrophoresis showed that the cytokeratin complement of each cell line was slightly different but retained the phenotype of the luminal cell. It was concluded that during both hyperplasia and neoplastic transformation of the prostate, the luminal cell phenotype is primarily involved and that the basal cell phenotype does not appear to contribute to either intraluminal proliferation or invasive cell populations.

  2. In vitro cytotoxicity of alpha conjugates for human pancreatic cancer cell lines

    International Nuclear Information System (INIS)

    Qu, C.; Li, Y.; Rizvi, M.A.; Allen, B.; Samra, J.; Smith, R.

    2003-01-01

    Targeted Alpha therapy (TAT) can inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The aim of this study is to demonstrate the cytotoxicity of different alpha conjugates in vitro to human metastatic pancreatic cancer cell lines (CAPAN-1, CFPAN-1 and PANC-1). We are labeling the C595 and J591 (non-specific controls) monoclonal antibodies (Mabs) with 213 Bi were performed according to the standard methods in our laboratory. 213 Bi-C595 is specifically cytotoxic to CAPAN-1, CFPAN-1 and PANC-1cell lines in a concentration-dependent fashion. While non-specific alpha conjugates only killed very small fractions of pancreatic cancer cells. These alpha conjugates might be useful agents for the treatment of micro-metastases in pancreatic cancer patients with over-expression of the targeted receptors

  3. The F309S mutation increases factor VIII secretion in human cell line

    Directory of Open Access Journals (Sweden)

    Daianne Maciely Carvalho Fantacini

    2016-06-01

    Full Text Available ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

  4. Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a modified RNA system

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Uhm

    2017-10-01

    Full Text Available We generated human induced pluripotent stem cells (KSCBi002-B and KSCBi002-B-1 from the dermal fibroblasts of a donor using a modified RNA-based gene delivery method. According to GTG-banding analysis, the generated KSCBi002-B line has a cytogenetic abnormality (46,XY, t(1;4(q21;q25 that is distinct from that of the donor, whereas KSCBi002-B-1 has a normal karyotype (46,XY. These cell lines can be useful as a model for characterizing the hiPSCs generated by a non-viral and non-integrative system, or as a chromosomal balanced translocation model. These two cell lines are registered and available from the National Stem Cell Bank, Korea National Institute of Health.

  5. Derivation and characterization of human embryonic stem cell lines from the Chinese population

    Institute of Scientific and Technical Information of China (English)

    Zhao Wu; Huimin Dai; Lei Qian; Qing Tian; Lei Xiao; Xiaojun Tan; Hui Li; Lingjun Rao; Lixiazi He; Lei Bao; Jing Liao; Chun Cui; Zhenyu Zuo; Qiao Li

    2011-01-01

    Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.

  6. Demonstration of a novel HIV-1 restriction phenotype from a human T cell line.

    Directory of Open Access Journals (Sweden)

    Yanxing Han

    2008-07-01

    Full Text Available Although retroviruses may invade host cells, a productive infection can be established only after the virus counteracts inhibition from different types of host restriction factors. Fv1, APOBEC3G/F, TRIM5alpha, ZAP, and CD317 inhibit the replication of different retroviruses by interfering with viral uncoating, reverse transcription, nuclear import, RNA stability, and release. In humans, although APOBEC3G/3F and CD317 block HIV-1 replication, their antiviral activities are neutralized by viral proteins Vif and Vpu. So far, no human gene has been found to effectively block wild type HIV-1 replication under natural condition. Thus, identification of such a gene product would be of great medical importance for the development of HIV therapies.In this study, we discovered a new type of host restriction against the wild type HIV-1 from a CD4/CXCR4 double-positive human T cell line. We identified a CEM-derived cell line (CEM.NKR that is highly resistant to productive HIV-1 infection. Viral production was reduced by at least 1000-fold when compared to the other permissive human T cell lines such as H9, A3.01, and CEM-T4. Importantly, this resistance was evident at extremely high multiplicity of infection. Further analyses demonstrated that HIV-1 could finish the first round of replication in CEM.NKR cells, but the released virions were poorly infectious. These virions could enter the target cells, but failed to initiate reverse transcription. Notably, this restriction phenotype was also present in CEM.NKR and 293T heterokaryons.These results clearly indicate that CEM.NKR cells express a HIV inhibitory gene(s. Further characterization of this novel gene product(s will reveal a new antiretroviral mechanism that directly inactivates wild type HIV-1.

  7. Genotoxicity of 11 heavy metals detected as food contaminants in two human cell lines.

    Science.gov (United States)

    Kopp, B; Zalko, D; Audebert, M

    2018-04-01

    Heavy metals, such as arsenic (As), antimony (Sb), barium (Ba), cadmium (Cd), cobalt (Co), germanium (Ge), lead (Pb), nickel (Ni), tellurium (Te), and vanadium (V) are widely distributed in the environment and in the food chain. Human exposure to heavy metals through water and food has been reported by different international agencies. Although some of these heavy metals are essential elements for human growth and development, they may also be toxic at low concentrations due to indirect mechanisms. In this study, the genotoxic and cytotoxic properties of 15 different oxidation statuses of 11 different heavy metals were investigated using high-throughput screening (γH2AX assay) in two human cell lines (HepG2 and LS-174T) representative of target organs (liver and colon) for food contaminants. Base on their lowest observed adverse effect concentration, the genotoxic potency of each heavy metal in each cell line was ranked in decreasing order, NaAsO 2  > CdCl 2  > PbCl 2 (only in LS-174T cells) > As 2 O 5  > SbCl 3  > K 2 TeO 3  > As 2 O 3 . No significant genotoxicity was observed with the other heavy metals tested. Cell viability data indicate that several heavy metals (As, Cd, Co, Ni, Sb, and Te) induce cytotoxicity at high concentrations, whereas an increase in the number of cells was observed for lead concentrations >100 µM in both cell lines tested, suggesting that lead stimulates cell growth. All these results highlight the possible human health hazards associated with the presence of heavy metals present in food. Environ. Mol. Mutagen. 59:202-210, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. γ-radiation induces cellular sensitivity and aberrant methylation in human tumor cell lines.

    Science.gov (United States)

    Kumar, Ashok; Rai, Padmalatha S; Upadhya, Raghavendra; Vishwanatha; Prasada, K Shama; Rao, B S Satish; Satyamoorthy, Kapettu

    2011-11-01

    Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression. Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2'-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16(INK4a)) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay. γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16(INK4a) and ATM gene promoters. These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and

  9. Biologic characteristics of the side population of human small cell lung cancer cell line H446.

    Science.gov (United States)

    Wang, Bo; Yang, Huan; Huang, Yu-Zheng; Yan, Ru-Hong; Liu, Fen-Ju; Zhang, Jun-Ning

    2010-03-01

    Recently, the theory of cancer stem cells (CSCs) has presented new targets and orientations for tumor therapy. The major difficulties in researching CSCs include their isolation and purification. The aim of this study is to identify and characterize the side population (SP) cells in small cell lung cancer (SCLC) cell line H446, which lays the foundation for the isolation and purification of CSCs. Fluorescence-activated cell sorting (FACS) was used to sort SP and non-SP (NSP) cells from H446. Both subgroups were cultivated to survey the capacity to form into suspended tumor cell spheres. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR were used to evaluate the expression levels of the mRNA of CD133, ABCG2, and nucleostemin in both subgroups. The capacity of proliferation and the differences in drug resistance of both subgroups and unsorted cells were tested by the MTT method. The differentiation ability of both subgroups was determined by FACS. Proliferation was determined by subcutaneous tumor formation in nude mice. The percent of Hoechst 33342 negative cells was about (5.1 +/- 0.2)% in H446 by fluorescence microscopy. The percent of SP cells was (6.3 +/- 0.1)% by flow cytometry. SP cells had a stronger capability of forming into tumor spheres than NSP cells. The mRNA expression levels of ABCG2, CD133, and nucleostemin in SP cells were 21.60 +/- 0.26, 7.10 +/- 0.14, and 1.02 +/- 0.08 folds higher than that in NSP cells (P 0.05, respectively). In vivo, SP cells showed better proliferative ability and tougher viability when treated with drugs. SP cells can differentiate into NSP cells, but NSP cells cannot differentiate into SP cells. SP cells had a greater ability to form tumors. The H446 cell line contained some SP cells with stem cell properties. CD133 and ABCG2 may be cancer stem cell markers of SCLC.

  10. Altered expression of asparagine synthetase mRNA in human leukemic and carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goodwin, L.O.; Guzowski, D.E.; Millan, C.A. [North Shore Univ. Hospital/Cornell Univ. Medical College, Manhasset, NY (United States)] [and others

    1994-09-01

    Asparagine synthetase (AS) is the enzyme responsible for the ATP-dependant conversion of aspartic acid to asparagine. The AS gene is expressed constitutively in most mammalian cells, including cells of the lymphoid lineage, as a 2 kb mRNA. In some leukemic phenotypes, AS expression is abrogated, resulting in no detectable enzyme activity. These cells are rendered sensitive to killing by L-asparaginase, which destroys extracellular asparagine. Prolonged treatment of leukemic cells with this agent can lead to resistance and the reappearance of AS activity, suggesting derepression of the AS gene, which has been shown to be regulated by intracellular levels of asparagine. Modulation of AS expression by asparagine employs cis and trans-acting elements involved in transcriptional and translational regulation. We have cloned and sequenced the human AS gene and surrounding sequence elements as well as the full-length cDNA. Using probes specific to the third and fourth exons of AS, we have identified an additional higher molecular weight mRNA (2.7 kb) in Northern blots derived from a chronic myelogenous leukemia and a colon carcinoma but not in normal lymphocytic or other human cell lines. We speculate that elements present in the cancer-derived mRNAs may be involved in the derepression of AS activity. This hypothesis is being evaluated by RNase protection assays using RNA isolated from a variety of human cell lines to characterize and elucidate the nature of this additional AS encoded message.

  11. Exogenous wild type p53 gene affects radiosensitivity of human lung adenocarcinoma cell line under hypoxia

    International Nuclear Information System (INIS)

    Wang Jianhua; Wang Feng; Liu Yongping; Zhang Yaping; Ni Yan; Li Shirong

    2008-01-01

    Objective: To evaluate the effect of exogenous wild type p53 (wtp53) gene on radiosensitivity of human lung adenocarcinoma cell line under hypoxia. Methods: Human lung adenocarcinoma cell line A549 was transfected with adenovirus carrying recombinant exogenous wtp53. Four irradiation groups were studied: normal cell (Group A), wtp53 transfected cell (Group B), normal cell under hypoxia (Group C) and wtp53 transfected cell under hypoxia(Group D). Cells were irradiated with 9 MeV electron beams. Cellular survival fraction was analyzed. Multi-target single-hit model was used to plot the survival curve. D 0 , D q , oxygen enhancement ratio (OER), sensitizing enhancement ratio (SER) and other parameters were used to evaluate the effects of wtp53 gene on radiosensitivity of A549. The cell apoptotic rate of each group was examined by flow cytometry. Results: OER was 1.75 and 0.81 before and after wtp53 transfection. SER was 1.77 in oxic circumstance and 3.84 under hypoxia. The cell apoptotic rate of Group A and B was lower than Group C and D (F=7.92, P=0.048), with Group A lower than B and Group C lower than D (F=82.50, P=0.001). But Group B and D were similar(t=2.04, P=0.111). Conclusions: Hypoxia can increase the radiation resistance of lung adenocarcinoma cell line A549. The wtp53 can promote apoptosis and improve tumor radiosensitivity, especially under hypoxia. (authors)

  12. Lipofection of plasmid DNA into human mast cell lines using lipid nanoparticles generated by microfluidic mixing.

    Science.gov (United States)

    Duguay, Brett A; Huang, Kate Wei-Chen; Kulka, Marianna

    2018-04-18

    Mast cells are important immune cells that have significant roles in mediating allergy and asthma. Therefore, studying the molecular mechanisms regulating these and other processes in mast cells is important to elucidate. Methods such as lipofection, transduction, and electroporation are often employed to dissect these mechanisms by disrupting gene expression in mast cell lines. However, as with other leukocytes, human mast cells (HMCs) are often refractory to the delivery of plasmids by lipofection. In this study, we investigated the utility of lipid nanoparticles (LNPs) containing the ionizable cationic lipids 1,2-dioleoyloxy-3-dimethylaminopropane, 1,2-dioleyloxy-3-dimethylaminopropane, or 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane for the delivery of plasmid DNA into HMC lines. Herein, we demonstrate for the first time the use of LNPs to achieve significant and reproducible levels of plasmid DNA transfection in HMC-1.2 and laboratory of allergic diseases 2 (LAD2) cells. These levels reached 53.2% and 16.0% in HMC-1.2 and LAD2 cells, respectively; and outperformed Lipofectamine 3000 in both cases. Moreover, cell viability in the transfected cells remained above 65% for all LNP conditions tested. Together, these observations illustrate the efficacy of this technique for mast cell researchers and further support the use of LNPs for nucleic acid delivery into leukocytes. ©2018 Society for Leukocyte Biology.

  13. [Establishment of human multidrug-resistant lung carcinoma cell line (D6/MVP)].

    Science.gov (United States)

    Ma, Sheng-lin; Feng, Jian-guo; Gu, Lin-hui; Ling, Yu-tian

    2003-03-01

    To establish human multidrug-resistant lung carcinoma cell line (D6/MVP) with its characteristics studied. Intermittent administration of high-dose MMC, VDS and DDP (MVP) was used to induce human lung carcinoma cell line (D6) to a multidrug-resistant variety (D6/MVP). MTT assay was used to study the multidrug resistance of D6/MVP to multianticarcinogen. Flow cytometry was used to study the cell cycle distribution and the expression of P-gp, multidrug resistance-associated protein (MRP) and GSH/GST. 1. D6/MVP was resistant to many anti-tumor agents, with the IC(50) 13.3 times higher and the drug resistance 2 - 6 times higher than D6, 2. The multiplication time of D6/MVP was prolonged and the cell number of S-phase decreased while that of G1- and G(2)-phase increased and 3. The expression of P-gp and MRP was enhanced significantly (96.2% vs 51.7%), but the expression of GSH/GST kept stable. D6/MVP is a multidrug-resistant cell line possessing the basic characteristics of drug-resistance.

  14. Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines.

    Science.gov (United States)

    Offerdahl, Danielle K; Dorward, David W; Hansen, Bryan T; Bloom, Marshall E

    2017-01-15

    The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60-100nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20-30nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. Published by Elsevier Inc.

  15. Imiquimod activates p53-dependent apoptosis in a human basal cell carcinoma cell line.

    Science.gov (United States)

    Huang, Shi-Wei; Chang, Shu-Hao; Mu, Szu-Wei; Jiang, Hsin-Yi; Wang, Sin-Ting; Kao, Jun-Kai; Huang, Jau-Ling; Wu, Chun-Ying; Chen, Yi-Ju; Shieh, Jeng-Jer

    2016-03-01

    The tumor suppressor p53 controls DNA repair, cell cycle, apoptosis, autophagy and numerous other cellular processes. Imiquimod (IMQ), a synthetic toll-like receptor (TLR) 7 ligand for the treatment of superficial basal cell carcinoma (BCC), eliminates cancer cells by activating cell-mediated immunity and directly inducing apoptosis and autophagy in cancer cells. To evaluate the role of p53 in IMQ-induced cell death in skin cancer cells. The expression, phosphorylation and subcellular localization of p53 were detected by real-time PCR, luciferase reporter assay, cycloheximide chase analysis, immunoblotting and immunocytochemistry. Using BCC/KMC1 cell line as a model, the upstream signaling of p53 activation was dissected by over-expression of TLR7/8, the addition of ROS scavenger, ATM/ATR inhibitors and pan-caspase inhibitor. The role of p53 in IMQ-induced apoptosis and autophagy was assessed by genetically silencing p53 and evaluated by a DNA content assay, immunoblotting, LC3 puncta detection and acridine orange staining. IMQ induced p53 mRNA expression and protein accumulation, increased Ser15 phosphorylation, promoted nuclear translocation and up-regulated its target genes in skin cancer cells in a TLR7/8-independent manner. In BCC/KMC1 cells, the induction of p53 by IMQ was achieved through increased ROS production to stimulate the ATM/ATR-Chk1/Chk2 axis but was not mediated by inducing DNA damage. The pharmacological inhibition of ATM/ATR significantly suppressed IMQ-induced p53 activation and apoptosis. Silencing of p53 significantly decreased the IMQ-induced caspase cascade activation and apoptosis but enhanced autophagy. Mutant p53 skin cancer cell lines were more resistant to IMQ-induced apoptosis than wildtype p53 skin cancer cell lines. IMQ induced ROS production to stimulate ATM/ATR pathways and contributed to p53-dependent apoptosis in a skin basal cell carcinoma cell line BCC/KMC1. Copyright © 2015 Japanese Society for Investigative Dermatology

  16. Publishing SNP genotypes of human embryonic stem cell lines: policy statement of the International Stem Cell Forum Ethics Working Party.

    Science.gov (United States)

    Knoppers, Bartha M; Isasi, Rosario; Benvenisty, Nissim; Kim, Ock-Joo; Lomax, Geoffrey; Morris, Clive; Murray, Thomas H; Lee, Eng Hin; Perry, Margery; Richardson, Genevra; Sipp, Douglas; Tanner, Klaus; Wahlström, Jan; de Wert, Guido; Zeng, Fanyi

    2011-09-01

    Novel methods and associated tools permitting individual identification in publicly accessible SNP databases have become a debatable issue. There is growing concern that current technical and ethical safeguards to protect the identities of donors could be insufficient. In the context of human embryonic stem cell research, there are no studies focusing on the probability that an hESC line donor could be identified by analyzing published SNP profiles and associated genotypic and phenotypic information. We present the International Stem Cell Forum (ISCF) Ethics Working Party's Policy Statement on "Publishing SNP Genotypes of Human Embryonic Stem Cell Lines (hESC)". The Statement prospectively addresses issues surrounding the publication of genotypic data and associated annotations of hESC lines in open access databases. It proposes a balanced approach between the goals of open science and data sharing with the respect for fundamental bioethical principles (autonomy, privacy, beneficence, justice and research merit and integrity).

  17. A UV-sensitive human clonal cell line, RSa, which has low repair activity

    International Nuclear Information System (INIS)

    Suzuki, N.; Fuse, A.

    1981-01-01

    The repair activity of a human transformed cell line, RSa, which was found to be highly sensitive to the lethal effects of 254 mm far-ultraviolet radiation, was compared with that of HeLa cells by evaluating the range of UV-induced incorporation of [methyl- 3 H]thymidine ([ 3 H]dThd) or 5-[6- 3 H]bromodeoxyuridine ([ 3 H]BrdUrd) into deoxyribonucleic acid. Direct scintillation counting was used for measuring the extent of unscheduled DNA synthesis (UDS) in UV-irradiated cells, which were treated with hydroxyurea or with arginine deprivation. More quantitative measurements were made by using the density labeling and equilibrium centrifugation method for assaying repair replication. All the amounts of UDS and repair replication in RSa cells were markedly below those in HeLa cells. The possible relationships of the low repair activity to abnormally high UV sensitivity in RSa cells are discussed. (orig.)

  18. Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

    OpenAIRE

    Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Na?ra; Rau, Fr?d?rique; Jollet, Arnaud; Edom-Vovard, Fr?d?rique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois

    2017-01-01

    International audience; Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded rep...

  19. Cytotoxic effect of Argentine medicinal plant extracts on human hepatocellular carcinoma cell line.

    Science.gov (United States)

    Ruffa, M J; Ferraro, G; Wagner, M L; Calcagno, M L; Campos, R H; Cavallaro, L

    2002-03-01

    Methanolic extracts from Achyrocline satureioides (Dc.) Lam, Aristolochia macroura Gomez, Lithraea molleoides (Vell.) Engl., Schinus molle L., unlike those from Celtis spinosa Spreng, Chenopodium ambrosioides L., Petiveria alliacea L., and Plantago major L. showed cytotoxic activity against a human hepatocellular carcinoma cell line, Hep G2. Schinus molle L. was the most active (IC50=50+/-7 microg/ml). These results call for further studies of these extracts.

  20. Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

    Science.gov (United States)

    Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F.

    2017-01-01

    ABSTRACT Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. PMID:28188264

  1. Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds

    Directory of Open Access Journals (Sweden)

    Ludovic Arandel

    2017-04-01

    Full Text Available Myotonic dystrophy type 1 (DM1 and type 2 (DM2 are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations.

  2. Immortalized human myotonic dystrophy muscle cell lines to assess therapeutic compounds.

    Science.gov (United States)

    Arandel, Ludovic; Polay Espinoza, Micaela; Matloka, Magdalena; Bazinet, Audrey; De Dea Diniz, Damily; Naouar, Naïra; Rau, Frédérique; Jollet, Arnaud; Edom-Vovard, Frédérique; Mamchaoui, Kamel; Tarnopolsky, Mark; Puymirat, Jack; Battail, Christophe; Boland, Anne; Deleuze, Jean-Francois; Mouly, Vincent; Klein, Arnaud F; Furling, Denis

    2017-04-01

    Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations. © 2017. Published by The Company of Biologists Ltd.

  3. Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S. [Institute of Genetics, Taipei (Taiwan, Province of China)

    1994-09-01

    Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.

  4. Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

    Science.gov (United States)

    Quang, Tara; Marquez, Maribel; Blanco, Giselle; Zhao, Yuanxiang

    2014-01-01

    Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

  5. A Rapid Culture Technique Produces Functional Dendritic-Like Cells from Human Acute Myeloid Leukemia Cell Lines

    Directory of Open Access Journals (Sweden)

    Jian Ning

    2011-01-01

    Full Text Available Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML cells as progenitors from which functional dendritic-like antigen presenting cells (DLC were generated, that constitutively express tumour antigens for recognition by CD8+ T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+ T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

  6. Inhibitory Effects of Probiotic Lactobacillus on the Growth of Human Colonic Carcinoma Cell Line HT-29.

    Science.gov (United States)

    Chen, Zhung-Yuan; Hsieh, You-Miin; Huang, Chun-Chih; Tsai, Cheng-Chih

    2017-01-10

    This study was conducted to investigate the inhibitory effect of Lactobacillus cells and supernatants on the growth of the human colon cancer cell line HT-29. Our study results indicated that the PM153 strain exhibits the best adhesion ability and the highest survival in the gastrointestinal tract simulation experiment. Furthermore, after an 8-h co-culture of PM153 and HT-29 cells, the PM153 strain can induce the secretion of nitric oxide from the HT-29 cells. In addition, after the co-culture of the BCRC17010 strain (10⁸ cfu/mL) and HT-29 cells, the Bax/Bcl-2 ratio in the HT-29 cells was 1.19, which showed a significant difference from the other control and LAB groups ( p strain exerts a pro-apoptotic effect on the HT-29 cells. Upon co-culture with HT-29 cells for 4, 8 and 12 h, the BCRC14625 strain (10⁸ cfu/mL) demonstrated a significant increase in lactate dehydrogenase (LDH) activity ( p strains have ability to inhibit the growth of the colorectal cancer cell line HT-29 Bax/Bcl-2 pathway or NO production. In summary, we demonstrated that the BCRC17010 strain, good abilities of adhesion and increased LDH release, was the best probiotic potential for inhibition of HT-29 growth amongst the seven LAB strains tested in vitro.

  7. Androgen responsiveness of the new human endometrial cancer cell line MFE-296.

    Science.gov (United States)

    Hackenberg, R; Beck, S; Filmer, A; Hushmand Nia, A; Kunzmann, R; Koch, M; Slater, E P; Schulz, K D

    1994-04-01

    MFE-296 endometrial cancer cells express androgen receptors in vitro. These cells, which are tumorigenic in nude mice, are derived from a moderately differentiated human endometrial adenocarcinoma. They express vimentin and the cytokeratins 7, 8, 18, and 19. Karyotyping revealed near-tetraploidy for most of the cells. No marker chromosomes were observed. DNA analyses confirmed the genetic identity of the cell line and the patient from whom the cell line was derived. Proliferation of MFE-296 cells was inhibited by the progestin R5020 and the androgen dihydrotestosterone (DHT). The inhibition of proliferation by DHT was antagonized by the antiandrogen Casodex, demonstrating the involvement of the androgen receptor. Androgen binding was determined at 22,000 binding sites per cell using a whole-cell assay (KD = 0.05 nM) and 30 fmol/mg protein with the dextran charcoal method; 7 fmol/mg protein of progesterone receptors were found, whereas estrogen receptors were below 5 fmol/mg protein. The androgen receptor was functionally intact, as demonstrated by transfection experiments with a reporter-gene construct, containing an androgen-responsive element. In MFE-296 cells the content of the androgen receptor was up-regulated by its own ligand.

  8. Galectin-9 enhances cytokine secretion, but suppresses survival and degranulation, in human mast cell line.

    Directory of Open Access Journals (Sweden)

    Reiji Kojima

    Full Text Available Galectin-9 (Gal-9, a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.

  9. Biflorin induces cytotoxicity by DNA interaction in genetically different human melanoma cell lines.

    Science.gov (United States)

    Ralph, Ana Carolina Lima; Calcagno, Danielle Queiroz; da Silva Souza, Luciana Gregório; de Lemos, Telma Leda Gomes; Montenegro, Raquel Carvalho; de Arruda Cardoso Smith, Marília; de Vasconcellos, Marne Carvalho

    2016-08-01

    Cancer is a public health problem and the second leading cause of death worldwide. The incidence of cutaneous melanoma has been notably increasing, resulting in high aggressiveness and poor survival rates. Taking into account the antitumor activity of biflorin, a substance isolated from Capraria biflora L. roots that is cytotoxic in vitro and in vivo, this study aimed to demonstrate the action of biflorin against three established human melanoma cell lines that recapitulate the molecular landscape of the disease in terms of genetic alterations and mutations, such as the TP53, NRAS and BRAF genes. The results presented here indicate that biflorin reduces the viability of melanoma cell lines by DNA interactions. Biflorin causes single and double DNA strand breaks, consequently inhibiting cell cycle progression, replication and DNA repair and promoting apoptosis. Our data suggest that biflorin could be considered as a future therapeutic option for managing melanoma. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Two distinct affinity binding sites for IL-1 on human cell lines

    International Nuclear Information System (INIS)

    Bensimon, C.; Wakasugi, N.; Tagaya, Y.; Takakura, K.; Yodoi, J.; Tursz, T.; Wakasugi, H.

    1989-01-01

    We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125 I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated

  11. Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2.

    Science.gov (United States)

    Priddle, Helen; Allegrucci, Cinzia; Burridge, Paul; Munoz, Maria; Smith, Nigel M; Devlin, Lyndsey; Sjoblom, Cecilia; Chamberlain, Sarah; Watson, Sue; Young, Lorraine E; Denning, Chris

    2010-04-01

    The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

  12. Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

    Institute of Scientific and Technical Information of China (English)

    Yi Feng; Zhong-Min Tian; Ming-Xi Wan; Zhao-Bin Zheng

    2007-01-01

    AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.METHODS: Total proteins from human hepatocarcinomacell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite.Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)and database searching.RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin,endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein Ⅰ,peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

  13. Establishment and cell cycle distribution pattern of a radioresistant subline from human lung cancer D6 cell line

    International Nuclear Information System (INIS)

    Wei Qichun; Zheng Shu

    2003-01-01

    Objective: To establish a radioresistant cell subline from a human D6 lung cancer cell line and investigate the mechanism of radioresistance. Methods: D6 human NSCLC cells were exposed to X-rays generated by a linear accelerator(650 cGy per fraction). After a total exposure dose of 5200 cGy, a monoclone was obtained. The radiosensitivity and cell cycle distribution of this clone, together with its parent D6 cells, were measured by clonogenic assay and flow cytometry. Results: The new clone, namely D 6 -R subline, had a higher D 0 (D 0 =2.08 Gy) and a broader initial shoulder(Dq=1.64 Gy, N=2.20) than those of the parent D6 cell line (D 0 =1.84 Gy, Dq=0.34 Gy, N=1.20), being 1.65-fold increase in radioresistance as regards to the SF 2 . The D6-R subline also showed higher percentage of cells in S phase(53.4% vs 37.8%), but lower percentages in G 1 (44.1% vs 57.2%) and G 2 /M(2.5% vs 5%) phases. Conclusion: The new subline D6-R is more radioresistant as compare to its parent D6 cell line, and has a different cell cycle distribution

  14. Dose rate effect models for biological reaction to ionizing radiation in human cell lines

    International Nuclear Information System (INIS)

    Magae, Junji; Ogata, Hiromitsu

    2008-01-01

    Full text: Because of biological responses to ionizing radiation are dependent on irradiation time or dose rate as well as dose, simultaneous inclusion of dose and dose rate is required to evaluate the risk of long term irradiation at low dose rates. We previously published a novel statistical model for dose rate effect, modified exponential (MOE) model, which predicts irradiation time-dependent biological response to low dose rate ionizing radiation, by analyzing micronucleus formation and growth inhibition in a human osteosarcoma cell line, exposed to wide range of doses and dose rates of gamma-rays. MOE model demonstrates that logarithm of median effective dose exponentially increases in low dose rates, and thus suggests that the risk approaches to zero at infinitely low dose rate. In this paper, we extend the analysis in various kinds of human cell lines exposed to ionizing radiation for more than a year. We measured micronucleus formation and [ 3 H]thymidine uptake in human cell lines including an osteosarcoma, a DNA-dependent protein kinase-deficient glioma, a SV40-transformed fibroblast derived from an ataxia telangiectasia patient, a normal fibroblast, and leukemia cell lines. Cells were exposed to gamma-rays in irradiation room bearing 50,000 Ci of cobalt-60. After the irradiation, they were cultured for 24 h in the presence of cytochalasin B to block cytokinesis, and cytoplasm and nucleus were stained with DAPI and prospidium iodide. The number of binuclear cells bearing a micronucleus was counted under a fluorescence microscope. For proliferation inhibition, cells were cultured for 48 h after the irradiation and [ 3 H] thymidine was pulsed for 4 h before harvesting. We statistically analyzed the data for quantitative evaluation of radiation risk. While dose and dose rate relationship cultured within one month followed MOE model in cell lines holding wild-type DNA repair system, dose rate effect was greatly impaired in DNA repair-deficient cell lines

  15. SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic neurons in Parkinson's disease.

    Science.gov (United States)

    Xie, Hong-rong; Hu, Lin-sen; Li, Guo-yi

    2010-04-20

    To evaluate the human neuroblastoma SH-SY5Y cell line as an in vitro model of dopaminergic (DAergic) neurons for Parkinson's disease (PD) research and to determine the effect of differentiation on this cell model. The data of this review were selected from the original reports and reviews related to SH-SY5Y cells published in Chinese and foreign journals (Pubmed 1973 to 2009). After searching the literature, 60 articles were selected to address this review. The SH-SY5Y cell line has become a popular cell model for PD research because this cell line posses many characteristics of DAergic neurons. For example, these cells express tyrosine hydroxylase and dopamine-beta-hydroxylase, as well as the dopamine transporter. Moreover, this cell line can be differentiated into a functionally mature neuronal phenotype in the presence of various agents. Upon differentiation, SH-SY5Y cells stop proliferating and a constant cell number is subsequently maintained. However, different differentiating agents induce different neuronal phenotypes and biochemical changes. For example, retinoic acid induces differentiation toward a cholinergic neuronal phenotype and increases the susceptibility of SH-SY5Y cells to neurotoxins and neuroprotective agents, whereas treatment with retinoic acid followed by phorbol ester 12-O-tetradecanoylphorbol-13-acetate results in a DAergic neuronal phenotype and decreases the susceptibility of cells to neurotoxins and neuroprotective agents. Some differentiating agents also alter kinetics of 1-methyl-4-phenyl-pyridinium (MPP(+)) uptake, making SH-SY5Y cells more similar to primary mesencephalic neurons. Differentiated and undifferentiated SH-SY5Y cells have been widely used as a cell model of DAergic neurons for PD research. Some differentiating agents afford SH-SY5Y cells with more potential for studying neurotoxicity and neuroprotection and are thus more relevant to experimental PD research.

  16. Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.

    Science.gov (United States)

    Bonin, L R; Madden, K; Shera, K; Ihle, J; Matthews, C; Aziz, S; Perez-Reyes, N; McDougall, J K; Conroy, S C

    1999-03-01

    The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2

  17. [Streptococcus group B--association with Aerobic vaginitis and ability to human cell lines activation].

    Science.gov (United States)

    Romanik, Małgorzata; Kafel, Joanna; Lagergård, Teresa; Martirosian, Gayane

    2007-01-01

    The aim of this study was to estimate: the frequency of aerobic vaginitis, susceptibility of the GBS isolated from vagina of non-pregnant women with and without cervicitis to selected antibiotics and chemotherapeutics and the proinflammatory cytokines production by HeLa, THP-I, U - 937 cells after stimulation by vaginal GBS. Our results indicated low frequency of the aerobic vaginitis -4.5% among non-pregnant young women and ability of the vaginal GBS to release proinflammatory cytokines by human cell lines in vitro.

  18. Microculture-based chemosensitivity testing: a feasibility study comparing freshly explanted human melanoma cells with human melanoma cell lines.

    Science.gov (United States)

    Marshall, E S; Finlay, G J; Matthews, J H; Shaw, J H; Nixon, J; Baguley, B C

    1992-03-04

    The culture of cancer cells has many applications in chemosensitivity testing and new drug development. Our goal was to adapt simple semiautomated microculture methods for testing the chemosensitivity of melanoma cells freshly recovered from patients' tumors. Cells were cultured on a substrate of agarose and exposed continuously to cytotoxic drugs, the effects of which were measured by determining the uptake of [3H]thymidine 4-7 days later. Immunocytochemical staining of cells cultured with 5-bromo-2'-deoxyuridine demonstrated that tumor cells were responsible for the measured thymidine incorporation. The effects of cytotoxic drugs were calculated as logarithmic 50% inhibitory concentrations and expressed as divergences from the mean in a log-mean graph. The inhibitory effects of amsacrine, etoposide, doxorubicin, cisplatin, mitomycin C, and fluorouracil were tested. Tumors differed widely in their sensitivity to these drugs, although sensitivity to the three topoisomerase-II-directed agents was highly correlated. Cells from two non-neoplastic hematopoietic progenitor cell lines (FT and 32D) showed chemosensitivity patterns distinct from those in the melanoma cells, indicating tissue selectivity. Two established melanoma cell lines, MM-96 and FME, were tested under the same conditions and showed sensitivity typical of at least some fresh specimens. These results support the validity of melanoma cell lines as models of freshly resected melanoma cells. If successfully applied to other tumor types, such semiautomated approaches could find wide application in routine hospital laboratories for the chemosensitivity testing of patients' tumor cells.

  19. The molecule HLA-G: radiosensitivity indicator of a human melanoma cell line

    International Nuclear Information System (INIS)

    Michelin, S.C.; Gallegos, C.E.; Dubner, D.L.; Baffa Trasci, S.; Favier, B.; Carosella, E.D.

    2010-01-01

    The physiological and pathological relevance of the HLA-G molecule (non-classical Human Leukocyte Antigen) has been motif of important research studies. Its distribution is restricted to only few tissues. HLA-G takes part in the implantation after in vitro fecundation, in graft tolerance, in auto-immune diseases, and in tumoral immune escape. Its expression has been demonstrated in more than 30% of tumors of 15 different histological types. Gamma radiation modulates HLA-G expression at the cell surface. However, its involvement in tumoral radiosensitivity has not been demonstrated yet. The objective of this work was to demonstrate if the HLA-G molecule intervenes in the radiosensibility of human melanoma cells cultured in vitro. For this purpose we used the human melanoma cell line M8, which was transfected with the plasmid containing the HLA-G gene (M8 HLA-G+) or with the plasmid alone, without the HLA-G gene (M8 pc DNA). Both cell lines were irradiated with 0, 2, 5 y 10 Gy and in all cases survival frequency was determined with the clonogenic assay. We observed a significant reduction in M8 HLA-G+ survival with respect to M8 pc DNA for all irradiation doses and was independent of doses. These results, if confirmed in other histological types, could postulate the HLA-G molecule as a tumoral radiosensitivity marker. The specific mechanism involved in the radiosensibility modification exerted by HLA-G has not been elucidated yet. (authors) [es

  20. Identification of proteins sensitive to thermal stress in human neuroblastoma and glioma cell lines.

    Directory of Open Access Journals (Sweden)

    Guilian Xu

    Full Text Available Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y and glial (CCF-STTG1 lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48 residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.

  1. Cytokinetic effects of razoxane and relevance to radiopotentiation in a human adenocarcinoma cell line

    International Nuclear Information System (INIS)

    Norimah Yusof.

    1982-01-01

    Razoxane (+-1,2-bis (3,5-dioxopiperazin-1-yl)propane) or ICRF 159 has been shown to enhance the X-radiation sensitivity of a human colorectal adenocarcinoma cell line (HT29R) in culture if the cells were incubated with the drug for more than ten hours prior irradiation. Razoxane was shown not to interfere with repair of sublethal of potentially lethal damage of radiation. The radiosensitizing effect is therefore attributed to a reduced ability to accumulate such damage. As the effect was shown greater in exponential phase, which consists of greater proportion of dividing cells than plateau phase cells, it is believed that the drug (0.1 - 100 μg/ml) potentiates the effect of X-radiation in HT29R cells by pertubation in the cell cycle kinetics. Razoxane at low and high concentrations (5 and 100 μg/ml) was shown to delay the cell progression on cells entering mitosis. Chromosomes of drug-treated cells seemed to be unable to condense at the premitosis stage and cells with chromosomes resembling those in prophase were allowed to proceed through mitosis but progressed very slowly so that accumulation of mitotic cells were seen. Cells were than allowed to complete mitosis but frequently failed to undergo cytokinesis and subsequently produced many giant multinucleate cells. It is likely that the cells in mitosis and multinucleate cells are more radiosensitive. However, the mitotic delay was found to be reversible following progressive drug inactivation in the cells. Cells appeared to return to normal cell progression rate and division while the radiopotentiating effect was disappearing. The mechanism of drug action in inhibiting chromosome condensation was shown to involve the drug interference with histone H 1 phosphorylation. The inhibition of H 1 phosphorylation might reduce the cross-linking of the histone on DNA strands and thus preventing them from participating in supercoiling of the chromosome. (author)

  2. Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

    Science.gov (United States)

    Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

    2017-02-01

    More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    International Nuclear Information System (INIS)

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-01-01

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  4. Behavior of a Spontaneously Arising Human Retinal Pigment Epithelial Cell Line Cultivated on Thin Alginate Film.

    Science.gov (United States)

    Najafabadi, Hoda Shams; Soheili, Zahra-Soheila; Ganji, Shahla Mohammad

    2015-01-01

    A cell line spontaneously derived from human retinal pigment epithelium (hRPE) was cultured on alginate film gelatinized with different concentrations of neurobasal cell culture medium (NCCM) to assess its growth and morphological behavior on this naturally occurring polysaccharide. Neonatal human globes were used to isolate hRPE cells. They were cultured in Dulbecco's modified Eagle's-medium-and-Ham's-F12-medium-(DMEM/F12) supplemented with 10% fetal bovine serum (FBS). Cultures were continuously studied using phase contrast microscopy. After the nineth passage, cells were characterized through immunocytochemical analysis for Oct4, Chx10, and Pax6 and Ki67 markers. In each well of a 6-well microplate, 1 and 2% weight/volume (w/v) alginate in deionized water was added and gelatinized using 1× and 10× NCCM. hRPE cells were cultured at a density of 2 × 105 cells/well in alginate-coated microplates. After 5 days, hRPE colonies were harvested and re-plated on polystyrene substrates. Morphology and growth of hRPE cultures were determined during the next 2 weeks. The first few passages of the cultures were purely hRPE cells that revealed typical morphological features of the pigmented epithelium. They made spaces, devoid of cells, between hRPE cell monolayer and fill in the unoccupied spaces. They grew faster than native RPE cells and rapidly overgrew. Immunocytochemical test revealed that the founded cells expressed Chx10, Pax6, Ki67 and Oct4. The hRPE cells survived unlimitedly on alginate film and formed giant adjoining colonies. After re-plating, hRPE colonies adhered quickly on polystyrene and displayed native hRPE morphological features. Alginate film can support the survival and growth of hRPE cells and induce the cells to re-organize in tissue-like structures.

  5. Presence of estrogen receptors in human myeloid monocytic cells (THP-1 cell line).

    Science.gov (United States)

    Cutolo, M; Villaggio, B; Bisso, A; Sulli, A; Coviello, D; Dayer, J M

    2001-01-01

    To test THP-1 cells for the presence of estrogen receptors (ER) since studies have demonstrated in vivo and in vitro, the influence of estrogens on cells involved in immune response (i.e. macrophages), and since it has been demonstrated that human myeloid monocytic THP-1 cells acquire phenotypic and functional macrophage-like features after incubation with several cytokines or pharmacological agents. Stimulation of THP-1 cells with phorbol myristate acetate (PMA) to prompt their differentiation into macrophage-like cells and evaluation of the possible induction of ER. The expression of ER was analyzed by immunocytochemical assay, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. After stimulation by PMA, the human myeloid monocytic THP-1 cells showed the presence of ER, together with markers of monocytic cell differentiation such as CD68, CD54 and HLA-DR. Estrogen effects may be exerted directly through ER on monocytes/macrophages. PMA-treated THP-1 cells may constitute a useful in vitro model to determine the effects of estrogens on macrophage-like cells and their implications in the inflammatory and immune processes.

  6. Anticancer potential of Hericium erinaceus extracts against particular human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Younis AM

    2017-06-01

    Full Text Available Cancer is a leading cause of death worldwide. Cancer resulted in 8.2 million human deaths in 2012. It is expected that annual cancer cases will rise from 14 million in 2013 to 22 million within the next two decades. Mushrooms are extensively used as nutritional supplements in many countries. Moreover, mushrooms have many medicinal properties, including anticancer activity. In this study, the anticancer activity of different polar and non-polar extracts of Hericium erinaceus were evaluated against different human cancer cell lines including human liver carcinoma (Hep G2, the human colonic epithelial carcinoma (HCT 116, the human cervical cancer cells (HeLa and the human breast adenocarcinoma (MCF-7 using 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay. Furthermore, as a control, the cytotoxicity effect of the different extracts were tested against isolated mouse hepatocytes. It was observed that the extracts by water and methanol from fresh and lyophilized fruiting bodies of H. erinaceus had the strongest anticancer effect. In contrast, the extracts by ether and ethyl acetate from mycelia and broth of H. erinaceus showed lower anticancer activity against the tested carcinoma cell lines. The highest anticancer activity was recorded for aqueous extract of lyophilized fruiting bodies with half maximal inhibitory concentration (IC50 values of 6.1±0.2, 5.1±0.1, 5.7±0.2 and 5.8±0.3 µg/ml against Hep G2, HCT 116, HeLa and MCF-7 cells, respectively with non-significant effect on the normal mouse hepatocytes. To summarise, polar extracts of H. erinaceus can be good sources for isolating natural anticancer compounds. I recommend further chemical studies to isolate the active principles of the extract of H. erinaceus evaluated in the present.

  7. Anti-proliferative effects of Bifidobacterium adolescentis SPM0212 extract on human colon cancer cell lines

    International Nuclear Information System (INIS)

    Lee, Do Kyung; Jang, Seok; Kim, Mi Jin; Kim, Jung Hyun; Chung, Myung Jun; Kim, Kyung Jae; Ha, Nam Joo

    2008-01-01

    Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as anti-tumor activity. The aim of the present work was to study the growth inhibition of tumor cells by butanol extract of Bifidobacterium adolescentis isolated from healthy young Koreans. The anti-proliferative activity of B. adolescentis isolates was assessed by XTT assays on three human colon cancer cell lines (Caco-2, HT-29, and SW480). The effects of B. adolescentis SPM0212 butanol extract on tumor necrosis factor-α (TNF-α) and nitric oxide (NO) production were tested using the murine macrophage RAW 264.7 cell line. The butanol extract of B. adolescentis SPM0212 dose-dependently inhibited the growth of Caco-2, HT-29, and SW480 cells by 70%, 30%, and 40%, respectively, at 200 μg/mL. Additionally, the butanol extract of B. adolescentis SPM0212 induced macrophage activation and significantly increased the production of TNF-α and NO, which regulate immune modulation and are cytotoxic to tumor cells. The butanol extract of B. adolescentis SPM0212 increased activity of the host immune system and may improve human health by helping to prevent colon cancer as a biological response modifier

  8. Analysis of human protein replacement stable cell lines established using snoMEN-PR vector.

    Directory of Open Access Journals (Sweden)

    Motoharu Ono

    Full Text Available The study of the function of many human proteins is often hampered by technical limitations, such as cytotoxicity and phenotypes that result from overexpression of the protein of interest together with the endogenous version. Here we present the snoMEN (snoRNA Modulator of gene ExpressioN vector technology for generating stable cell lines where expression of the endogenous protein can be reduced and replaced by an exogenous protein, such as a fluorescent protein (FP-tagged version. SnoMEN are snoRNAs engineered to contain complementary sequences that can promote knock-down of targeted RNAs. We have established and characterised two such partial protein replacement human cell lines (snoMEN-PR. Quantitative mass spectrometry was used to analyse the specificity of knock-down and replacement at the protein level and also showed an increased pull-down efficiency of protein complexes containing exogenous, tagged proteins in the protein replacement cell lines, as compared with conventional co-expression strategies. The snoMEN approach facilitates the study of mammalian proteins, particularly those that have so far been difficult to investigate by exogenous expression and has wide applications in basic and applied gene-expression research.

  9. Effects of recombinant human epidermal growth factor on the proliferation and radiation survival of human fibroblast cell lines in vitro

    International Nuclear Information System (INIS)

    Kim, Hyun Sook; Kang, Ki Mun; Na, Jae Boem; Chai, Gyu Young; Lee, Sang Wook

    2006-01-01

    To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. Number of fibroblast was significant more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing

  10. Expression and migratory analysis of 5 human uveal melanoma cell lines for CXCL12, CXCL8, CXCL1, and HGF

    Directory of Open Access Journals (Sweden)

    Di Cesare Sebastian

    2007-01-01

    Full Text Available Abstract Background The aim of this study was to characterize the presence and roles of CXCL12, CXCL8, CXCL1, and HGF in five human uveal melanoma cell lines, using different methods, in order to ascertain their significance in this disease. Methods Five human uveal melanoma cell lines (92.1, SP6.5, MKT-BR, OCM-1, and UW-1 of known proliferative, invasive, and metastatic potential were used in this experiment. A migration assay was used in order to assess the responsiveness of each cell line towards the four chosen chemotactic factors. Immunohistochemistry was then performed for all five cell lines (cytospins using antibodies directed toward CXCL1, CXCL8 and their receptors CXCR2 and CXCR1 respectively. Quantitative real-time PCR was then performed on all five cell lines in order to establish the presence of these four chemotactic factors. Results All five human uveal melanoma cell lines migrated towards the four chosen chemotactic factors at a level greater than that of the negative control. Chemokines CXCL1 and CXCL8 resulted in the greatest number of migrating cells in all five of our cell lines. Immunohistochemistry confirmed the expression of CXCL1, CXCL8, and their receptors CXCR2 and CXCR1 in all five of the cell lines. Quantitative real-time PCR results established expression of CXCL8, CXCL1, and HGF in all 5 cell lines tested. CXCL1 and CXCL8 are highly expressed in SP6.5 and UW-1. None of the five cell lines expressed any detectable levels of CXCL12. Conclusion The migratory ability of the 5 human uveal melanoma cell lines was positively influenced by the four chemotactic factors tested, namely CXCL12, CXCL8, CXCL1, and HGF. Self-expression of chemotactic factors CXCL8, CXCL1, and HGF may indicate an autocrine system, which perhaps contributes to the cells' metastatic ability in vivo.

  11. [Apoptosis of human lung carcinoma cell line GLC-82 induced by high power electromagnetic pulse].

    Science.gov (United States)

    Cao, Xiao-zhe; Zhao, Mei-lan; Wang, De-wen; Dong, Bo

    2002-09-01

    Electromagnetic pulse (EMP) could be used for sterilization of food and the efficiency is higher than 2450 MHz continuous microwave done. This study was designed to evaluate the effect of electromagnetic pulse (EMP) on apoptosis of human lung carcinoma cell line GLC-82, so that to explore and develop therapeutic means for cancer. The injury changes in GLC-82 cells after irradiated with EMP (electric field intensity was 60 kV/m, 5 pulses/2 min) were analyzed by cytometry, MTT chronometry, and flow cytometry. The immunohistochemical SP staining was used to determine the expressions of bcl-2 protein and p53 protein. The stained positive cells were analyzed by CMIAS-II image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. EMP could obviously inhibited proliferation and activity of lung carcinoma cell line GLC-82. The absorbance value (A570) of MTT decreased immediately, at 0 h, 1 h, and 6 h after the GLC-82 cells irradiated by EMP as compared with control group. The highest apoptosis rate was found to reach 13.38% by flow cytometry at 6 h after EMP irradiation. Down-regulation of bcl-2 expression and up-regulation of p53 expression were induced by EMP. EMP promotes apoptosis of GLC-82 cells. At same time, EMP can down-regulate bcl-2 expression and up-regulate p53 expression in GLC-82 cells. The bcl-2 and the p53 protein may involve the apoptotic process.

  12. Lethality of radiation-induced chromosome aberrations in human tumour cell lines with different radiosensitivities.

    Science.gov (United States)

    Coco-Martin, J M; Ottenheim, C P; Bartelink, H; Begg, A C

    1996-03-01

    In order to find an explanation for the eventual disappearance of all chromosome aberrations in two radiosensitive human tumour cell lines, the type and stability of different aberration types was investigated in more detail. To classify the aberrations into unstable and stable types, three-colour fluorescence in situ hybridization was performed, including a whole-chromosome probe, a pancentromere probe, and a stain for total DNA. This technique enables the appropriate classification of the aberrations principally by the presence (stable) or not (unstable) of a single centromere per chromosome. Unstable-type aberrations were found to disappear within 7 days (several divisions) in the two radiosensitive and the two radioresistant tumour lines investigated. Stable-type aberrations were found to remain at an approximately constant level over the duration of the experiment (14 days; 8-10 divisions) in the two radioresistant lines. In contrast, the majority of these stable-type aberrations had disappeared by 14 days in the two radiosensitive lines. The previous findings of disappearance of total aberrations in radiosensitive cells was therefore not due to a reduced induction of stable-type aberrations, but the complete disappearance of cells with this aberration type. These results could not be explained by differences in apoptosis or G1 blocks. Two possible explanations for these unexpected findings involve non-random induction of unstable-type aberrations, or lethality of stable-type aberrations. The results suggest caution in the use of stable-type aberration numbers as a predictor for radiosensitivity.

  13. Inhibitory Effects of Probiotic Lactobacillus on the Growth of Human Colonic Carcinoma Cell Line HT-29

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    Zhung-Yuan Chen

    2017-01-01

    Full Text Available This study was conducted to investigate the inhibitory effect of Lactobacillus cells and supernatants on the growth of the human colon cancer cell line HT-29. Our study results indicated that the PM153 strain exhibits the best adhesion ability and the highest survival in the gastrointestinal tract simulation experiment. Furthermore, after an 8-h co-culture of PM153 and HT-29 cells, the PM153 strain can induce the secretion of nitric oxide from the HT-29 cells. In addition, after the co-culture of the BCRC17010 strain (109 cfu/mL and HT-29 cells, the Bax/Bcl-2 ratio in the HT-29 cells was 1.19, which showed a significant difference from the other control and LAB groups (p < 0.05, which therefore led to the inference that the BCRC17010 strain exerts a pro-apoptotic effect on the HT-29 cells. Upon co-culture with HT-29 cells for 4, 8 and 12 h, the BCRC14625 strain (109 cfu/mL demonstrated a significant increase in lactate dehydrogenase (LDH activity (p < 0.05, causing harm to the HT-29 cell membrane; further, after an 8-h co-culture with the HT-29 cells, it induced the secretion of nitric oxide (NO from the HT-29 cells. Some lactic acid bacteria (LAB strains have ability to inhibit the growth of the colorectal cancer cell line HT-29 Bax/Bcl-2 pathway or NO production. In summary, we demonstrated that the BCRC17010 strain, good abilities of adhesion and increased LDH release, was the best probiotic potential for inhibition of HT-29 growth amongst the seven LAB strains tested in vitro.

  14. Effect of Evodiamine on Inducing Apoptosis of Human Gastric Cancer Cell Line SGC-7901 in Vitro

    Directory of Open Access Journals (Sweden)

    LIU Shao-ping

    2014-09-01

    Full Text Available Objective: To explore the proliferation inhibition and apoptosis-inducing effect of evodiamine in human gastric cancer SGC-7901 cells. Methods: After 48 or 24 h exposure to different concentrations of evodiamine, cell proliferation was analyzed using tetrazolium blue (MTT assay while apoptosis and cell-cycle phase distribution using flow cytometry. Results: In 0.01~30.00 μg/mL range of concentrations, evodiamine inhibited the proliferation of SGC-7901 cells in dose-dependent manner, and the overall mean IC50 was (3.79±0.16 μg/mL; the apoptosis rate was increased from 3.4% to 7.0%, 13.8% and 36.3% at concentrations of 0, 0.5, 1.5 and 30 μg/mL of evodiamine, respectively; the percentage of cells accumulated in G2/M phase was increased from 17.26% to 98.92% in the cells treated with evodiamine for 24 h in 0.01~30.00 μg/mL range of concentrations. Conclusion: Evodiamine can inhibit the proliferation, induce apoptosis in human gastric cancer cell line SGC-7901 in vitro and arrest the cell cycle at the G2/M phase.

  15. Radiation of different human melanoma cell lines increased expression of RHOB. Level of this tumor suppressor gene in different cell lines

    International Nuclear Information System (INIS)

    Notcovich, C.; Molinari, B.; Duran, H.; Delgado González, D.; Sánchez Crespo, R.

    2013-01-01

    Previous results of our group show that a correlation exists between intrinsic radiosensitivity of human melanoma cells and cell death by apoptosis. RhoB is a small GTPase that regulates cytoskeletal organization. Besides, is related to the process of apoptosis in cells exposed to DNA damage as radiation. Also, RhoB levels decrease in a wide variety of tumors with the tumor stage, being considered a tumor suppressor gene due to its antiproliferative and proapoptotic effect. The aim of this study was to analyze the expression of RhoB in different human melanoma cell lines in relation to melanocytes, and evaluate the effect of gamma radiation on the expression of RhoB. We used the A375, SB2 and Meljcell lines, and the derived from melanocytes Pig1. It was found for all three tumor lines RhoB expression levels significantly lower than those of Pig1 (p <0.05), as assessed by semiquantitative RT-PCR . When tumor cells were irradiated to a dose of 2Gyinduction was observed at 3 hours RhoB irradiation. RhoB expression increased in all lines relative to non-irradiated control, showing a greater induction ( p< 0.05) for the more radiosensitive line SB2, consistent with apoptosis in response to radiation. The results allow for the first time in melanoma demonstrate that RhoB, as well as in other tumor types, has a lower expression in tumor cells than their normal counterparts. Moreover, induction in the expression of RhoB in irradiated cells may be associated with the process of radiation-induced apoptosis. The modulation of RhoB could be a new tool to sensitize radioresistant melanoma. (author)

  16. Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Hirako, Yoshiaki, E-mail: s47526a@cc.nagoya-u.ac.jp [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan); Yonemoto, Yuki; Yamauchi, Tomoe [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan); Nishizawa, Yuji; Kawamoto, Yoshiyuki [Department of Biomedical Sciences, Chubu University, 1200 Matsumoto-cho, Kasugai 487-8501 (Japan); Owaribe, Katsushi [Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan)

    2014-06-10

    Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases. - Highlights: • A defined condition promoted accumulation of hemidesmosomes in human cultured cells. • A fraction isolated from the cells contained eight major polypeptides. • The polypeptides were the five major hemidesmosome proteins and laminin-332. • The cultured cells deposited laminin-332 in its unprocessed form under the condition. • We report a method to prepare a fraction highly enriched in hemidesmosome proteins.

  17. Solutions for the Cell Cycle in Cell Lines Derived from Human Tumors

    Directory of Open Access Journals (Sweden)

    B. Zubik-Kowal

    2006-01-01

    Full Text Available The goal of the paper is to compute efficiently solutions for model equations that have the potential to describe the growth of human tumor cells and their responses to radiotherapy or chemotherapy. The mathematical model involves four unknown functions of two independent variables: the time variable t and dimensionless relative DNA content x. The unknown functions can be thought of as the number density of cells and are solutions of a system of four partial differential equations. We construct solutions of the system, which allow us to observe the number density of cells for different t and x values. We present results of our experiments which simulate population kinetics of human cancer cells in vitro. Our results show a correspondence between predicted and experimental data.

  18. A zinc-resistant human epithelial cell line is impaired in cadmium and manganese import

    International Nuclear Information System (INIS)

    Rousselet, Estelle; Richaud, Pierre; Douki, Thierry; Chantegrel, Jocelyne Garcia; Favier, Alain; Bouron, Alexandre; Moulis, Jean-Marc

    2008-01-01

    A human epithelial cell line (HZR) growing with high zinc concentrations has been analyzed for its ability to sustain high cadmium concentrations. Exposure to up to 200 μM of cadmium acetate for 24 h hardly impacted viability, whereas most of parental HeLa cells were killed by less than 10 μM of cadmium. Upon challenge by 35 fold higher cadmium concentrations than HeLa cells, HZR cells did not display increased DNA damage, increased protein oxidation, or changed intracellular cadmium localization. Rather, the main cause of resistance against cadmium was by avoiding cadmium entry into cells, which differs from that against zinc as the latter accumulates inside cells. The zinc-resistant phenotype of these cells was shown to also impair extracellular manganese uptake. Manganese and cadmium competed for entry into HeLa cells. Probing formerly identified cadmium or manganese transport systems in different animal cells did not evidence any significant change between HeLa and HZR cells. These results reveal zinc adaptation influences manganese and cadmium cellular traffic and they highlight previously unknown connections among homeostasis of divalent metals

  19. Characterization of the replication timing program of 6 human model cell lines

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    Djihad Hadjadj

    2016-09-01

    Full Text Available During the S-phase, the DNA replication process is finely orchestrated and regulated by two programs: the spatial program that determines where replication will start in the genome (Cadoret et al. (2008 Oct 14, Cayrou et al. (2011 Sep, Picard et al. (2014 May 1 [1–3], and the temporal program that determines when during the S phase different parts of the genome are replicated and when origins are activated. The temporal program is so well conserved for each cell type from independent individuals [4] that it is possible to identify a cell type from an unknown sample just by determining its replication timing program. Moreover, replicative domains are strongly correlated with the partition of the genome into topological domains (determined by the Hi-C method, Lieberman-Aiden et al. (2009 Oct 9, Pope et al. (2014 Nov 20 [5,6]. On the one hand, replicative areas are well defined and participate in shaping the spatial organization of the genome for a given cell type. On the other hand, studies on the timing program during cell differentiation showed a certain plasticity of this program according to the stage of cell differentiation Hiratani et al. (2008 Oct 7, 2010 Feb [7,8]. Domains where a replication timing change was observed went through a nuclear re-localization. Thus the temporal program of replication can be considered as an epigenetic mark Hiratani and Gilbert (2009 Feb 16 [9]. We present the genomic data of replication timing in 6 human model cell lines: U2OS (GSM2111308, RKO (GSM2111309, HEK 293T (GSM2111310, HeLa (GSM2111311, MRC5-SV (GSM2111312 and K562 (GSM2111313. A short comparative analysis was performed that allowed us to define regions common to the 6 cell lines. These replication timing data can be taken into account when performing studies that use these model cell lines.

  20. Mechanisms of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplicon

    NARCIS (Netherlands)

    Eijdems, E. W.; de Haas, M.; Coco-Martin, J. M.; Ottenheim, C. P.; Zaman, G. J.; Dauwerse, H. G.; Breuning, M. H.; Twentyman, P. R.; Borst, P.; Baas, F.

    1995-01-01

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

  1. HL60 human premyelocitic cell line as a model system for bystander response

    International Nuclear Information System (INIS)

    Dini, V.; Tabocchini, M.A.; Belli, M.; Simone, G.; Di Carlo, B.; Sapora, O.; Superiore di Sanita, Rome

    2007-01-01

    Complete text of publication follows. Objective: to evaluate HL60 human premyelocitic cell line as a model system to study bystander response. Methods: HL60 cell line, isolated from the blood of a patient affected by premyelocitic leukemia, has 45-46 chromosomes with abnormalities mainly on chromosomes 5, 8 and X and can undergo chemical-induced in vitro differentiation. Differentiation gives rise to granulocytes, monocytes or macrophages depending on the drug used. We define as proliferative (AP) cells those in log phase of growth with less than 10 passages from thawing and as differentiated (D) cells those treated with 10 nM TPA (phorbol ester) for 72 hours. Phorbol ester treatment induces differentiation to monocytes and macrophages. Differentiation has been evaluated through the expression of differentiation cluster membrane antigens (CD95, CD9 and CD14). Results: AP cells resulted positive for CD95 and negative for CD9 and CD14, while D cells resulted positive for CD9 and negative for CD95 and CD14. Our data on AP and D cells showed that: (i) the level of intracellular reactive oxygen and nitrogen species (ROS and RNS) is lower in D cells compared to AP cells; (ii) radiation induced DNA damage (single and double strand breaks, SSB and DSB, as measured with the comet assay technique) is lower in D cells than in AP cells. This different radiosensitivity can be related to the higher degree of compactness of nuclear structure in D cells. Radiation induced bystander effect (BE) was analyzed with the medium transfer technique. The medium from irradiated, with 0.5 Gy of γ-rays, AP cells was collected after 0, 2, 4 and 24 hours from irradiation and added to non irradiated log phase cells. The frequency of micronuclei formation in bystander cells was measured by using the cytokinesis block technique by adding cytochalasin B to the non irradiated culture together with the irradiated medium. Preliminary data indicate about 1.4-fold increase in micronuclei formation in

  2. Gallic Acid Protects 6-OHDA Induced Neurotoxicity by Attenuating Oxidative Stress in Human Dopaminergic Cell Line.

    Science.gov (United States)

    Chandrasekhar, Y; Phani Kumar, G; Ramya, E M; Anilakumar, K R

    2018-04-18

    Gallic acid is one of the most important polyphenolic compounds, which is considered an excellent free radical scavenger. 6-Hydroxydopamine (6-OHDA) is a neurotoxin, which has been implicated in mainly Parkinson's disease (PD). In this study, we investigated the molecular mechanism of the neuroprotective effects of gallic acid on 6-OHDA induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that 6-OHDA induced cytotoxicity in SH-SY5Y cells was suppressed by pre-treatment with gallic acid. The percentage of live cells (90%) was high in the pre-treatment of gallic acid when compared with 6-OHDA alone treated cell line. Moreover, gallic acid was very effective in attenuating the disruption of mitochondrial membrane potential, elevated levels of intracellular ROS and apoptotic cell death induced by 6-OHDA. Gallic acid also lowered the ratio of the pro-apoptotic Bax protein and the anti-apoptotic Bcl-2 protein in SH-SY5Y cells. 6-OHDA exposure was up-regulated caspase-3 and Keap-1 and, down-regulated Nrf2, BDNF and p-CREB, which were sufficiently reverted by gallic acid pre-treatment. These findings indicate that gallic acid is able to protect the neuronal cells against 6-OHDA induced injury and proved that gallic acid might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.

  3. Expression of the transcription factor Evi-1 in human erythroleukemia cell lines and in leukemias.

    Science.gov (United States)

    Fontenay-Roupie, M; Bouscary, D; Melle, J; Viguié, F; Picard, F; Guesnu, M; Dreyfus, F

    1997-02-01

    The Evi-1 proto-oncogene is a zinc finger DNA binding protein. Although activation of the Evi-1 gene has been associated with chromosomal rearrangements of the 3q25-q28 region, ectopic expression of Evi-1 could also be observed in acute myelogenous leukemias and myelodysplastic syndromes without cytogenetic abnormalities of the 3q26 locus. In this study, human erythroleukemic cell lines were screened for the expression of Evi-1 mRNA by northern blotting. Evi-1 was expressed in all the erythroid cell lines, whether undifferentiated (K 562, HEL, LAMA 84) or exhibiting spontaneous terminal erythroid differentiation (KU 812, JK-1). Evi-1 mRNA levels were constant or elevated in hemoglobin-synthesizing KU 812 or K 562 cells in response to erythropoietin or hemin treatment, respectively. In human acute myeloblastic leukemias (AML), 11/30 expressed Evi-1 by RT-PCR. Among these cases, 4/6 erythroleukemias without abnormalities of the 3q25-q28 region were found positive. The presence of acidophilic erythroblasts (15-47% of bone marrow cells) accounted for the existence of a terminal erythroid differentiation in all Evi-1-positive AML M6, whereas one negative case was poorly differentiated and referred to as AML M6 variant. These results suggest that Evi-1 mRNA expression can coexist with erythroid differentiation.

  4. Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line.

    Science.gov (United States)

    Freiburghaus, C; Janicke, B; Lindmark-Månsson, H; Oredsson, S M; Paulsson, M A

    2009-06-01

    Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 microM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G(1)/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time.

  5. NANOG reporter cell lines generated by gene targeting in human embryonic stem cells

    DEFF Research Database (Denmark)

    Fischer, Yvonne; Ganic, Elvira; Ameri, Jacqueline

    2010-01-01

    Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role...

  6. Nanomelatonin triggers superior anticancer functionality in a human malignant glioblastoma cell line

    Science.gov (United States)

    Yadav, Sanjeev Kumar; Srivastava, Anup Kumar; Dev, Atul; Kaundal, Babita; Choudhury, Subhasree Roy; Karmakar, Surajit

    2017-09-01

    Melatonin (MEL) has promising medicinal value as an anticancer agent in a variety of malignancies, but there are difficulties in achieving a therapeutic dose due to its short half-life, low bioavailability, poor solubility and extensive first-pass metabolism. In this study chitosan/tripolyphosphate (TPP) nanoparticles were prepared by an ionic gelation method to overcome the therapeutic challenges of melatonin and to improve its anticancer efficacy. Characterization of the melatonin-loaded chitosan (MEL-CS) nanoformulation was performed using transmission and scanning electron microscopies, dynamic light scattering, Fourier transform infrared spectroscopy, Raman spectroscopy and x-ray diffraction. In vitro release, cellular uptake and efficacy studies were tested for their enhanced anticancer potential in human U87MG glioblastoma cells. Confocal studies revealed higher cellular uptake of MEL-CS nanoparticles and enhanced anticancer efficacy in human malignant glioblastoma cancer cells than in healthy non-malignant human HEK293T cells in mono- and co-culture models. Our study has shown for the first time that MEL-CS nanocomposites are therapeutically more effective as compared to free MEL at inducing functional anticancer efficacy in the human brain tumour U87MG cell line.

  7. ERC/mesothelin is expressed in human gastric cancer tissues and cell lines.

    Science.gov (United States)

    Ito, Tomoaki; Kajino, Kazunori; Abe, Masaaki; Sato, Koichi; Maekawa, Hiroshi; Sakurada, Mutsumi; Orita, Hajime; Wada, Ryo; Kajiyama, Yoshiaki; Hino, Okio

    2014-01-01

    ERC/mesothelin is expressed in mesothelioma and other malignancies. The ERC/mesothelin gene (MSLN) encodes a 71-kDa precursor protein, which is cleaved to yield 31-kDa N-terminal (N-ERC/mesothelin) and 40-kDa C-terminal (C-ERC/mesothelin) proteins. N-ERC/mesothelin is a soluble protein and has been reported to be a diagnostic serum marker of mesothelioma and ovarian cancer. Gastric cancer tissue also expresses C-ERC/mesothelin, but the significance of serum N-ERC levels for diagnosing gastric cancer has not yet been studied. We examined the latter issue in the present study as well as C-ERC/mesothelin expression in human gastric cancer tissues and cell lines. We immunohistochemically examined C-ERC/mesothelin expression in tissue samples from 50 cases of gastric cancer, and we also assessed the C-ERC/mesothelin expression in 6 gastric cancer cell lines (MKN-1, MKN-7, MKN-74, NUGC-3, NUGC-4 and TMK-1) using reverse transcription-polymerase chain reaction, flow cytometry, immunohistochemistry and immunoblotting. We also examined the N-ERC/mesothelin concentrations in the supernatants of cultured cells and in the sera of gastric cancer patients using an enzyme-linked immunosorbent assay (ELISA). N-ERC/mesothelin was detected in the supernatants of 3 gastric cancer cell lines (MKN-1, NUGC-4 and TMK-1) by ELISA, but its concentration in the sera of gastric cancer patients was almost same as that observed in the sera of the normal controls. In the gastric cancer tissues, C-ERC/mesothelin expression was associated with lymphatic invasion. N-ERC/mesothelin was secreted into the supernatants of gastric cancer cell lines, but does not appear to be a useful serum marker of gastric cancer.

  8. Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines. Mitochondrial implication in cell death.

    Science.gov (United States)

    Galeano, Eva; Nieto, Elena; García-Pérez, Ana Isabel; Delgado, M Dolores; Pinilla, Montserrat; Sancho, Pilar

    2005-10-01

    Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2microM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20microM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O2*- accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.

  9. Generation of a human induced pluripotent stem cell line from urinary cells of a patient with primary congenital glaucoma using integration free Sendai technology.

    Science.gov (United States)

    Zhang, Jingxue; Wu, Shen; Hu, Man; Liu, Qian

    2018-04-09

    We have generated a human induced pluripotent stem cell (iPSC) line derived from urinary cells of a 10years old patient with primary congenital glaucoma (PCG). The cells were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system and shown to have full differentiation potential. The line is available and registered in the human pluripotent stem cell registry as BIOi001-A. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  10. BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7

    International Nuclear Information System (INIS)

    Goupille, Olivier; Penglong, Tipparat; Lefèvre, Carine; Granger, Marine; Kadri, Zahra; Fucharoen, Suthat; Maouche-Chrétien, Leila; Leboulch, Philippe; Chrétien, Stany

    2012-01-01

    Highlights: ► UT7 erythroleukemia cells are known to be refractory to differentiate. ► Brief JQ1 treatment initiates the first steps of erythroid differentiation program. ► Engaged UT7 cells then maturate in the presence of erythropoietin. ► Sustained JQ1 treatment inhibits both proliferation and erythroid differentiation. -- Abstract: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

  11. BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7

    Energy Technology Data Exchange (ETDEWEB)

    Goupille, Olivier [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Penglong, Tipparat [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Thalassemia Research Center and Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Thailand); Lefevre, Carine; Granger, Marine; Kadri, Zahra [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Fucharoen, Suthat [Thalassemia Research Center and Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Thailand); Maouche-Chretien, Leila [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Leboulch, Philippe [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Genetics Division, Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chretien, Stany, E-mail: stany.chretien@cea.fr [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer UT7 erythroleukemia cells are known to be refractory to differentiate. Black-Right-Pointing-Pointer Brief JQ1 treatment initiates the first steps of erythroid differentiation program. Black-Right-Pointing-Pointer Engaged UT7 cells then maturate in the presence of erythropoietin. Black-Right-Pointing-Pointer Sustained JQ1 treatment inhibits both proliferation and erythroid differentiation. -- Abstract: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

  12. Regulation of low density lipoprotein receptor function in a human hepatoma cell line

    International Nuclear Information System (INIS)

    Leichtner, A.M.; Krieger, M.; Schwartz, A.L.

    1984-01-01

    Low density lipoprotein (LDL) processing was investigated in a human hepatoma-derived cell line, Hep G2. Hep G2 cells bound, internalized and degraded LDL via a saturable, high affinity pathway similar to that present in other mammalian cells. Although 80% of the uptake and degradation of 125 I-LDL was inhibited by 40-fold excess native LDL, the same concentration of methylated LDL, which cannot bind to LDL receptors, had virtually no effect on processing. When added at low concentrations, the lysosomotropic agent, chloroquine, inhibited degradation without affecting the rate of lipoprotein internalization. Receptor activity was decreased 60% by preincubation of the cells in medium containing a source of cholesterol (LDL or unesterified cholesterol) and increased 1.7-fold by preincubation with compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. The Hep G2 cell line may prove a useful system both for the further study of hepatic lipoprotein metabolism and for the evaluation of new antihypercholesterolemic agents

  13. Variation in sensitizing effect of caffeine in human tumour cell lines after γ-irradiation

    International Nuclear Information System (INIS)

    Valenzuela, M.T.; Almodovar, M.R. de; Mateos, S.; McMillan, T.J.

    2000-01-01

    We have investigated whether the protective role of the G2 checkpoint has increasing importance when the p53-dependent G1 checkpoint is inactivated. We have studied the differential effect of caffeine by clonogenic assays and flow cytometry in three human tumour cell lines with different functionality of p53 protein. The radiosensitizing effect of caffeine (2 mM) expressed itself as a significant decrease in surviving fraction at 2 Gy and a significant increase in α-values in RT112 and TE671, both with non-functional p53. However, no radiosensitizing effect was seen in cells with a normal p53 function (MCF-7 BUS). Two millimoles of caffeine also caused important changes in the cell cycle progression after irradiation. MCF-7 BUS showed a G1 arrest after irradiation and an early G2 arrest but those cells that reached the second G2 did not arrest significantly. In contrast, TE671 exhibited radiosensitization by caffeine, no G1 arrest, a G2 arrest in those cells irradiated in G2, no significant accumulation in the second G2 but an overall delay in release from the first cell cycle, which could be abrogated by caffeine. RT112 was similar to TE671 except that the emphasis in a G2 arrest was shifted from the block in cells irradiated in G2 to those irradiated at other cell cycle phases. The data presented confirm that p53 status can be a significant determinant of the efficacy of caffeine as radiosensitizer in these tumour cell lines, and document the importance of the G2 checkpoint in this effect. (author)

  14. Radiation response of drug-resistant variants of a human breast cancer cell line

    International Nuclear Information System (INIS)

    Lehnert, S.; Greene, D.; Batist, G.

    1989-01-01

    The radiation response of drug-resistant variants of the human tumor breast cancer cell line MCF-7 has been investigated. Two sublines, one resistant to adriamycin (ADRR) and the other to melphalan (MLNR), have been selected by exposure to stepwise increasing concentrations of the respective drugs. ADRR cells are 200-fold resistant to adriamycin and cross-resistant to a number of other drugs and are characterized by the presence of elevated levels of selenium-dependent glutathione peroxidase and glutathione-S-transferase. MLNR cells are fourfold resistant to melphalan and cross-resistant to some other drugs. The only mechanism of drug resistance established for MLNR cells to date is an enhancement of DNA excision repair processes. While the spectrum of drug resistance and the underlying mechanisms differ for the two sublines, their response to radiation is qualitatively similar. Radiation survival curves for ADRR and MLNR cells differ from that for wild-type cells in a complex manner with, for the linear-quadratic model, a decrease in the size of alpha and an increase in the size of beta. There is a concomitant decrease in the size of the alpha/beta ratio which is greater for ADRR cells than for MLNR cells. Analysis of results using the multitarget model gave values of D0 of 1.48, 1.43, and 1.67 Gy for MCF-7 cells are not a consequence of cell kinetic differences between these sublines. Results of split-dose experiments indicated that for both drug-resistant sublines the extent of sublethal damage repair reflected the width of the shoulder on the single-dose survival curve. For MCF-7 cells in the stationary phase of growth, the drug-resistant sublines did not show cross-resistance to radiation; however, delayed subculture following irradiation of stationary-phase cultures increased survival to a greater extent for ADRR and MLNR cells than for wild-type cells

  15. Potentiation of luteolin cytotoxicity by flavonols fisetin and quercetin in human chronic lymphocytic leukemia cell lines.

    Science.gov (United States)

    Sak, Katrin; Kasemaa, Kristi; Everaus, Hele

    2016-09-14

    Despite numerous studies chronic lymphocytic leukemia (CLL) still remains an incurable disease. Therefore, all new compounds and novel strategies which are able to eradicate CLL cells should be considered as valuable clues for a potential future remedy against this malignancy. In the present study, the cytotoxic profiles of natural flavonoids were described in two human CLL cell lines, HG-3 and EHEB, indicating the flavone luteolin as the most potent flavonoid with half-maximal inhibitory constants (IC50) of 37 μM and 26 μM, respectively. Luteolin significantly increased the apoptotic cell population in both cell lines by increasing the activities of caspases-3 and -9 and triggering the intrinsic apoptotic pathway. Two flavonols, fisetin and quercetin, were somewhat less efficient in suppressing cellular viability, whereas baicalein, chrysin, (+)-catechin and hesperetin exerted only a small or no response at doses as high as 100 μM. Both fisetin and quercetin were able to augment the cytotoxic activity of luteolin in both cell lines by reducing the IC50 values up to four fold. As a result of this, luteolin displayed cytotoxicity activity already at low micromolar concentrations that could potentially be physiologically achievable through oral ingestion. No other tested flavonoids were capable of sensitizing CLL cells to luteolin pointing to a specific binding of fisetin and quercetin to the cellular targets which interfere with the signaling pathways induced by luteolin. Although further molecular studies to unravel this potentiating mechanism are certainly needed, this phenomenon could contribute to future remedies for prevention and treatment of chronic lymphocytic leukemia.

  16. Translation of LINE-1 DNA elements in vitro and in human cells

    International Nuclear Information System (INIS)

    Leibold, D.M.; Swergold, G.D.; Thayer, R.E.; Singer, M.F.; Fanning, T.G.; Dombroski, B.A.

    1990-01-01

    The LINE-1(L1) family of interspread DNA sequences found throughout the human genome (L1 Homo sapiens, L1Hs) includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. The authors report that an antiserum against the polypeptide encoded by the L1Hs 5' open reading frame (ORF1) detects, in human cells, an endogenous ORF1 protein as well as the ORG1 product of an appropriate transfecting recombinant vector. The endogenous polypeptide is most abundant in teratocarcinoma and choriocarcinoma cells, among those cell lines tested; it appears to be a single species of ∼38 kDa. In contrast, RNAs synthesized in vitro from cDNAs representing full-length, polyadenylylated cytoplasmic L1Hs RNA yield, upon in vitro translation, ORF1 products of slightly different sizes. This is consistent with the fact that the various cDNAs are different and represent transcription of different genomic L1Hs elements. In vitro studies additionally suggest that translation of ORF1 is initiated at the first AUG codon. Finally, in no case was an ORF1-ORF2 fusion protein detected

  17. Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications

    Directory of Open Access Journals (Sweden)

    Dana M. Cairns

    2016-09-01

    Full Text Available Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain.

  18. Achillea millefolium L. hydroethanolic extract inhibits growth of human tumor cell lines by interfering with cell cycle and inducing apoptosis.

    Science.gov (United States)

    Pereira, Joana M; Peixoto, Vanessa; Teixeira, Alexandra; Sousa, Diana; Barros, Lillian; Ferreira, Isabel C F R; Vasconcelos, M Helena

    2018-06-05

    The cell growth inhibitory activity of the hydroethanolic extract of Achillea millefolium was studied in human tumor cell lines (NCI-H460 and HCT-15) and its mechanism of action was investigated. The GI 50 concentration was determined with the sulforhodamine B assay and cell cycle and apoptosis were analyzed by flow cytometry following incubation with PI or Annexin V FITC/PI, respectively. The expression levels of proteins involved in cell cycle and apoptosis were analyzed by Western blot. The extracts were characterized regarding their phenolic composition by LC-DAD-ESI/MS. 3,5-O-Dicaffeoylquinic acid, followed by 5-O-caffeoylquinic acid, were the main phenolic acids, while, luteolin-O-acetylhexoside and apigenin-O-acetylhexoside were the main flavonoids. This extract decreased the growth of the tested cell lines, being more potent in HCT-15 and then in NCI-H460 cells. Two different concentrations of the extract (75 and 100 μg/mL) caused alterations in cell cycle profile and increased apoptosis levels in HCT-15 and NCI-H460 cells. Moreover, the extract caused an increase in p53 and p21 expression in NCI-H460 cells (which have wt p53), and reduced XIAP levels in HCT-15 cells (with mutant p53). This work enhances the importance of A. millefolium as source of bioactive phenolic compounds, particularly of XIAP inhibitors. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Establishment of functioning human corneal endothelial cell line with high growth potential.

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    Tadashi Yokoi

    Full Text Available Hexagonal-shaped human corneal endothelial cells (HCEC form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na(+- and K(+-dependent ATPase (Na(+/K(+-ATPase. Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs in the Rb pathway (p16-CDK4/CyclinD1-pRb. In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7 and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin. Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7, THCEH (Cyclin and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7 and THCEH (Cyclin. THCEH (Cyclin expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na(+/K(+-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7. This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.

  20. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

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    Heema Hewitson

    2016-03-01

    Full Text Available The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. 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. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  1. Structurally modified curcumin analogs inhibit STAT3 phosphorylation and promote apoptosis of human renal cell carcinoma and melanoma cell lines.

    Directory of Open Access Journals (Sweden)

    Matthew A Bill

    Full Text Available The Janus kinase-2 (Jak2-signal transducer and activator of transcription-3 (STAT3 pathway is critical for promoting an oncogenic and metastatic phenotype in several types of cancer including renal cell carcinoma (RCC and melanoma. This study describes two small molecule inhibitors of the Jak2-STAT3 pathway, FLLL32 and its more soluble analog, FLLL62. These compounds are structurally distinct curcumin analogs that bind selectively to the SH2 domain of STAT3 to inhibit its phosphorylation and dimerization. We hypothesized that FLLL32 and FLLL62 would induce apoptosis in RCC and melanoma cells and display specificity for the Jak2-STAT3 pathway. FLLL32 and FLLL62 could inhibit STAT3 dimerization in vitro. These compounds reduced basal STAT3 phosphorylation (pSTAT3, and induced apoptosis in four separate human RCC cell lines and in human melanoma cell lines as determined by Annexin V/PI staining. Apoptosis was also confirmed by immunoblot analysis of caspase-3 processing and PARP cleavage. Pre-treatment of RCC and melanoma cell lines with FLLL32/62 did not inhibit IFN-γ-induced pSTAT1. In contrast to FLLL32, curcumin and FLLL62 reduced downstream STAT1-mediated gene expression of IRF1 as determined by Real Time PCR. FLLL32 and FLLL62 significantly reduced secretion of VEGF from RCC cell lines in a dose-dependent manner as determined by ELISA. Finally, each of these compounds inhibited in vitro generation of myeloid-derived suppressor cells. These data support further investigation of FLLL32 and FLLL62 as lead compounds for STAT3 inhibition in RCC and melanoma.

  2. Analysis of transcript and protein overlap in a human osteosarcoma cell line

    Directory of Open Access Journals (Sweden)

    Emanuelsson Olof

    2010-12-01

    Full Text Available Abstract Background An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC and immunofluorescence microscopy (IF. Results A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2% were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies. Conclusion This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.

  3. A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

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    Ute Hofmann

    2014-06-01

    Full Text Available In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells expressing green fluorescent protein (GFP under the control of the stress-inducible HSP70B’ promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B’. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B’ gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B’ promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

  4. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

    Science.gov (United States)

    Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

    2011-01-01

    Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

  5. Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

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    Lotta E Andersson

    Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

  6. Decreased immunoglobulin production by a human lymphoid cell line following melphalan treatment

    Energy Technology Data Exchange (ETDEWEB)

    Griffin, G.D. (Oak Ridge National Lab., TN); Owen, B.A.; Atchley, C.E.; Novelli, G.D.; Solomon, A.

    1982-11-01

    The effect of melphalan on immunoglobulin G (IgG) production by a human lymphoblastoid cell line (BF) was studied. The amount of secreted IgG and the percentage of cells containing cytoplasmic IgG were measured by immunoassay and cytofluorometry, respectively. Dose-response studies indicated that melphalan concentrations of 2 x 10/sup -8/ M had no effect, while concentrations of 8 x 10/sup -7/ M were totally toxic, after 72-h exposures to the drug. Statistically significant, persistent, alterations in both synthesis and secretion of IgG by BF cells were observed following treatment for 72 h with 4 x 10/sup -7/ M melphalan, and there was an increase in population-doubling time from 24 to 72 h in these drug-treated cells. The percentage of IgG-containing cells in melphalan-treated cultures was significantly decreased as compared to control cultures. IgG secretion was also decreased in these cultures, and the variation in IgG secretion as a function of cellular growth was significantly altered following melphalan treatment. Decreased IgG production following melphalan treatment may be related to altered cell cycle kinetics. Based on immunological analysis, there was no evident alteration in the IgG secreted by melphalan-treated cells, nor did melphalan treatment produce a cellular population lacking IgG entirely.

  7. Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line

    International Nuclear Information System (INIS)

    Martins-Neves, Sara R; Lopes, Áurio O; Carmo, Anália do; Paiva, Artur A; Simões, Paulo C; Abrunhosa, Antero J; Gomes, Célia MF

    2012-01-01

    Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs) have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma

  8. Therapeutic implications of an enriched cancer stem-like cell population in a human osteosarcoma cell line

    Directory of Open Access Journals (Sweden)

    Martins-Neves Sara R

    2012-04-01

    Full Text Available Abstract Background Osteosarcoma is a bone-forming tumor of mesenchymal origin that presents a clinical pattern that is consistent with the cancer stem cell model. Cells with stem-like properties (CSCs have been identified in several tumors and hypothesized as the responsible for the relative resistance to therapy and tumor relapses. In this study, we aimed to identify and characterize CSCs populations in a human osteosarcoma cell line and to explore their role in the responsiveness to conventional therapies. Methods CSCs were isolated from the human MNNG/HOS cell line using the sphere formation assay and characterized in terms of self-renewal, mesenchymal stem cell properties, expression of pluripotency markers and ABC transporters, metabolic activity and tumorigenicity. Cell's sensitivity to conventional chemotherapeutic agents and to irradiation was analyzed and related with cell cycle-induced alterations and apoptosis. Results The isolated CSCs were found to possess self-renewal and multipotential differentiation capabilities, express markers of pluripotent embryonic stem cells Oct4 and Nanog and the ABC transporters P-glycoprotein and BCRP, exhibit low metabolic activity and induce tumors in athymic mice. Compared with parental MNNG/HOS cells, CSCs were relatively more resistant to both chemotherapy and irradiation. None of the treatments have induced significant cell-cycle alterations and apoptosis in CSCs. Conclusions MNNG/HOS osteosarcoma cells contain a stem-like cell population relatively resistant to conventional chemotherapeutic agents and irradiation. This resistant phenotype appears to be related with some stem features, namely the high expression of the drug efflux transporters P-glycoprotein and BCRP and their quiescent nature, which may provide a biological basis for resistance to therapy and recurrence commonly observed in osteosarcoma.

  9. Correlation between the parameters of radiosensitivity in human cancer cell lines

    International Nuclear Information System (INIS)

    Park, Woo Yoon; Kim, Won Dong; Min, Kyung Soo

    1998-01-01

    We conducted clonogenic assay using human cancer cell lines (MKN-45, PC-14, Y-79, HeLa) to investigate a correlation between the parameters of radiosensitivity. Human cancer cell lines were irradiated with single doses of 1, 2, 3, 5, 7 and 10Gy for the study of radiosensitivity and sublethal damage repair capacity was assessed with two fractions of 5Gy separated with a time interval of 0, 1, 2, 3, 4, 6 and 24 hours. Surviving fraction was assessed with clonogenic assay using Sperman-Karber method and mathematical analysis of survival curves was done with linear-quadratic (LQ), multitarget-single hit(MS) model and mean inactivation dose(D). Surviving fractions at 2Gy(SF2) were variable among the cell lines, ranged from 0.174 to 0.85. The SF2 of Y-79 was lowest and that of PC-14 was highest(p<0.05, t-test). LQ model analysis showed that the values of α for Y-79, MKN-45, HeLa and PC-14 were 0.603, 0.356, 0.275 and 0.102 respectively, and those of β were 0.005, 0.016, 0.025 and 0.027 respectively. Fitting to MS model showed that the values of Do for Y-79, MKN-45, HeLa and PC-14 were 1.59, 1.84, 1.88 and 2.52 respectively, and those of n were 0.97, 1.46, 1.52 and 1.69 respectively. The Ds calculated by Gauss-Laguerre method were 1.62, 2.37, 2.61 and 3.95 respectively. So the SF2 was significantly correlated with α, Do and D. Their Pearson correlation coefficiencics were -0.953 and 0.993, 0.999 respectively(p<0.05). Sublethal damage repair was saturated around 4 hours and recovery ratios (RR) at plateau phase ranged from 2 to 3.79. But RR was not correlated with SF2, α, β, Do, D. The intrinsic radiosensitivity was very different among the tested human cell lines. Y-79 was the most sensitive and PC-14 was the least sensitive. SF2 was well correlated with α, Do, and D. RR was high for MKN-45 and HeLa but had nothing to do with radiosensitivity parameters. These basic parameters can be used as baseline data for various in vitro radiobiological experiments

  10. Radiosensitivity of mesothelioma cell lines

    International Nuclear Information System (INIS)

    Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

    1996-01-01

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

  11. In vitro radiosensitization by oxaliplatin and 5-fluorouracil in a human colon cancer cell line

    International Nuclear Information System (INIS)

    Kjellstroem, Johan; Kjellen, Elisabeth; Johnsson, Anders

    2005-01-01

    The current study was designed to compare the radiosensitizing effects of oxaliplatin and 5-fluorouracil (5FU) in a human colon cancer cell line. A human colon cancer cell line (S1) was treated with various doses of oxaliplatin, 5FU, radiation, and combinations thereof. Various clinically used schedules were mimicked. 5FU was either incubated during 1 h ('bolus') or 24 h ('continuous infusion'). When combining oxaliplatin and 5FU, an isobologram analysis revealed synergistic effects, regardless of 5FU schedule. The IC 10 and IC 50 -doses for the drugs where then combined with radiotherapy. With equitoxic drug doses (IC 50 ), radiosensitization was observed in the following order: oxaliplatin>5FU 24 h>5FU 1 h exposure. The degree of potentiation corresponded to approximately 0.8 Gy, 0.7 Gy, and 0.2 Gy, respectively. In this experimental setting, oxaliplatin seemed to be a better radiosensitizer than 5FU, and longer incubation time with 5FU was better than short exposure

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

    DEFF Research Database (Denmark)

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

    2002-01-01

    Mutations in presenilin 1 (PS1) are the major cause of autosomal dominant Alzheimer's disease. We have measured the voltage-gated K+ current in the human neuroblastoma cell line SH-SY5Y using whole-cell patch-clamp. When cells were stably transfected to over-express PS1, no change in K+ current...

  13. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    Science.gov (United States)

    Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

    2012-01-01

    Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

  14. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

    Directory of Open Access Journals (Sweden)

    Juliana Branco Novo

    2012-01-01

    Full Text Available Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher’s patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr− cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa and secreted (63–69 kDa form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

  15. Generation of a constitutively expressing Tetracycline repressor (TetR human embryonic stem cell line BJNhem20-TetR

    Directory of Open Access Journals (Sweden)

    Ronak Shetty

    2016-03-01

    Full Text Available Human embryonic stem cell line BJNhem20-TetR was generated using non-viral method. The construct pCAG-TetRnls was transfected using microporation procedure. BJNhem20-TetR can subsequently be transfected with any vector harbouring a TetO (Tet operator sequence to generate doxycycline based inducible line. For example, in human embryonic stem cells, the pSuperior based TetO system has been transfected into a TetR containing line to generate OCT4 knockdown cell line (Zafarana et al., 2009. Thus BJNhem20-TetR can be used as a tool to perturb gene expression in human embryonic stem cells.

  16. Effects of TGF-β signaling blockade on human A549 lung adenocarcinoma cell lines.

    Science.gov (United States)

    Xu, Cheng-Cheng; Wu, Lei-Ming; Sun, Wei; Zhang, Ni; Chen, Wen-Shu; Fu, Xiang-Ning

    2011-01-01

    Transforming growth factor β (TGF-β) is overexpressed in a wide variety of cancer types including lung adenocarcinoma (LAC), and the TGF-β signaling pathway plays an important role in tumor development. To determine whether blockade of the TGF-β signaling pathway can inhibit the malignant biological behavior of LAC, RNA interference (RNAi) technology was used to silence the expression of TGF-β receptor, type II (TGFβRII) in the LAC cell line, A549, and its effects on cell proliferation, invasion and metastasis were examined. Three specific small interfering RNAs (siRNAs) designed for targeting human TGFβRII were transfected into A549 cells. The expression of TGFβRII was detected by Western blot analysis. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The invasion and metastasis of A549 cells were investigated using the wound healing and Matrigel invasion assays. The expression of PI3K, phosphorylated Smad2, Smad4, Akt, Erk1/2, P38 and MMPs was detected by Western blot analysis. The TGFβRII siRNA significantly reduced the expression of TGFβRII in A549 cells. The knockdown of TGFβRII in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis. In addition to the Smad-dependent pathway, independent pathways including the Erk MAPK, PI3K/Akt and p38 MAPK pathways, as well as the expression of MMPs and VEGF, were inhibited. In conclusion, TGF-β signaling is required for LAC progression. Therefore, the blockade of this signaling pathway by the down-regulation of TGFβRII using SiRNA may provide a potential gene therapy for LAC.

  17. The discussion of method for survey the radiosensitivity of human glioma cell line SHG-44

    International Nuclear Information System (INIS)

    Li Li; Xu Changshao; Zhou Juying; Xu Xiaoting; Luo Jialin

    2005-01-01

    Objective: To investigate if thiazolyl blue colorimetric assay (MTT) and cell counting kit-8 (CCK-8) can replace clone forming assay for survey the radiosensitivity of SHG-44. Methods; Three assays was applied to examine the growth inhibition of human glioma cell line SHG-44 in eight dose groups of 0, 1, 2, 3, 4, 6, 8 and 10 Gy, and statistical research was applied to analyze the correlation between survival fraction and various doses. Results: Dose was associated with survival fraction in these three assays at some range of irradiation doseage (dose≤3 Gy). If out of the range, the relation is poor. CCK-8 has no rather superiority than MTT. Conclusion: By now clone forming assay is still the 'gold standard'. In some cases, MTT and other assays can give us some reference, but these assays still can not replace clone forming assay. (authors)

  18. Radiobiological studies with a series of human cell lines of varying glutathione content

    International Nuclear Information System (INIS)

    Astor, M.B.

    1984-01-01

    The effect of decreased levels of intracellular glutathione (GSH) on the radiosensitivity of aerated and hypoxic cells was studied using human skin fibroblasts obtained from patients affected with the inborn error of metabolism, 5-oxoprolinuria. The oxygen enhancement ratios (OER) were determined for four cell lines obtained from a single family, SR and SUR (heterozygous parents) and GM3877 and GM3878 (affected homozygous siblings). Glutathione values ranged from 7.4 to 130% of control values. Only GM3877 with a GSH value 7.4% of control exhibited a reduced OER of 1.9 compared with a control value of 3. These results suggest that a reduction in OER is observed only when GSH levels reach extremely low values. (author)

  19. Radiobiological studies with a series of human cell lines of varying glutathione content

    Energy Technology Data Exchange (ETDEWEB)

    Astor, M.B. (Columbia Univ., New York (USA). Radiological Research Lab.)

    1984-08-01

    The effect of decreased levels of intracellular glutathione (GSH) on the radiosensitivity of aerated and hypoxic cells was studied using human skin fibroblasts obtained from patients affected with the inborn error of metabolism, 5-oxoprolinuria. The oxygen enhancement ratios (OER) were determined for four cell lines obtained from a single family, SR and SUR (heterozygous parents) and GM3877 and GM3878 (affected homozygous siblings). Glutathione values ranged from 7.4 to 130% of control values. Only GM3877 with a GSH value 7.4% of control exhibited a reduced OER of 1.9 compared with a control value of 3. These results suggest that a reduction in OER is observed only when GSH levels reach extremely low values.

  20. Iron phosphate nanoparticles for food fortification: Biological effects in rats and human cell lines.

    Science.gov (United States)

    von Moos, Lea M; Schneider, Mirjam; Hilty, Florentine M; Hilbe, Monika; Arnold, Myrtha; Ziegler, Nathalie; Mato, Diogo Sales; Winkler, Hans; Tarik, Mohamed; Ludwig, Christian; Naegeli, Hanspeter; Langhans, Wolfgang; Zimmermann, Michael B; Sturla, Shana J; Trantakis, Ioannis A

    2017-05-01

    Nanotechnology offers new opportunities for providing health benefits in foods. Food fortification with iron phosphate nanoparticles (FePO 4 NPs) is a promising new approach to reducing iron deficiency because FePO 4 NPs combine high bioavailability with superior sensory performance in difficult to fortify foods. However, their safety remains largely untested. We fed rats for 90 days diets containing FePO 4 NPs at doses at which iron sulfate (FeSO 4 ), a commonly used food fortificant, has been shown to induce adverse effects. Feeding did not result in signs of toxicity, including oxidative stress, organ damage, excess iron accumulation in organs or histological changes. These safety data were corroborated by evidence that NPs were taken up by human gastrointestinal cell lines without reducing cell viability or inducing oxidative stress. Our findings suggest FePO 4 NPs appear to be as safe for ingestion as FeSO 4 .

  1. Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

    Science.gov (United States)

    Nakahata, Adriana Miti; Mayer, Barbara; Neth, Peter; Hansen, Daiane; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

    2013-03-01

    In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies

  2. Cadmium telluride quantum dots induce apoptosis in human breast cancer cell lines.

    Science.gov (United States)

    Naderi, Saeed; Zare, Hakimeh; Taghavinia, Nima; Irajizad, Azam; Aghaei, Mahmoud; Panjehpour, Mojtaba

    2018-05-01

    Semiconductor quantum dots (QDs), especially those containing cadmium, have undergone marked improvements and are now widely used nanomaterials in applicable biological fields. However, great concerns exist regarding their toxicity in biomedical applications. Because of the lack of sufficient data regarding the toxicity mechanism of QDs, this study aimed to evaluate the cytotoxicity of three types of QDs: CdTe QDs, high yield CdTe QDs, and CdTe/CdS core/shell QDs on two human breast cancer cell lines MDA-MB468 and MCF-7. The breast cancer cells were treated with different concentrations of QDs, and cell viability was evaluated via MTT assay. Hoechst staining was applied for observation of morphological changes due to apoptosis. Apoptotic DNA fragmentation was visualized by the agarose gel electrophoresis assay. Flow cytometric annexin V/propidium iodide (PI) measurement was used for apoptosis detection. A significant decrease in cell viability was observed after QDs treatment ( p < 0.05). Apoptotic bodies and chromatin condensation was observed by Hoechst staining. DNA fragmentation assay demonstrated a DNA ladder profile in the exposed cells and also annexin V/PI flow cytometry confirmed apoptosis in a dose-dependent manner. Our results revealed that CdTe, high yield CdTe, and CdTe/CdS core/shell QDs induce apoptosis in breast cancer cell lines in a dose-dependent manner. This study would help realizing the underlying cytotoxicity mechanism, at least partly, of CdTe QDs and may provide information for the development of nanotoxicology and safe use of biological applications of QDs.

  3. Chylomicron remnant-vitamin A metabolism by the human hepatoma cell line HepG2

    International Nuclear Information System (INIS)

    Lenich, C.M.

    1985-01-01

    The binding and metabolism of [ 3 H] vitamin A-containing chylomicron remnants (CMR) by the human hepatoma cell line Hep G2 was studied. Mesenteric lymph chylomicrons (CM) were collected from [ 3 H] retinol-fed rats and incubated with lipoprotein-lipase to obtain CMR. At 4 0 C, specific CMR binding was inhibited by excess unlabeled CMR. Specific binding predominated at low concentrations and approached saturation while total binding continued to increase over an extensive concentration range (0.45-32 μg triglyceride/ml). CMR uptake at 37 0 C was greater than that of CM and at least 100 times more efficient than the fluid-phase pinocytosis of sucrose. CMR binding increased as the extent of lipolysis obtained by incubation with lipoprotein-lipase increased. Addition of human apolipoprotein E enhanced both CMR and CM binding. After internalization, Hep G2 cells hydrolyzed CMR-[ 3 H]retinyl esters and radiolabeled metabolites accumulated as a function of time and temperature. As a function of the concentration of [ 3 H] VA initially cell-bound, retinol and retinyl esters accumulated as the major cell-associated metabolites. By contrast, retinol was the major metabolite in the medium only at low VA concentrations as other more polar metabolites accumulated at higher concentrations (> 110 pmol VA/mg cell protein). The accumulation of CMR-VA metabolites in the medium was reduced when cells were preincubated in retinol-supplemented media. Also, the specific activity of retinol in the medium closely resembled that in the cell indicating that CMR-VA mixed with the cellular store prior to its secretion

  4. Effects of disulfiram on apoptosis in PANC-1 human pancreatic cancer cell line.

    Science.gov (United States)

    Dastjerdi, M Nikbakht; Babazadeh, Z; Rabbani, M; Gharagozloo, M; Esmaeili, A; Narimani, M

    2014-01-01

    Pancreatic carcinoma is currently considered as a rapidly progressive and fatal disease, and is typically diagnosed late in its natural course. It is characterized by a poor diagnosis and lack of response to conventional therapy. Recent studies have suggested that disulfiram (DSF), a member of the dithiocarbamate family, may have antitumor activity. This study aimed to evaluate the in vitro effect of DSF on apoptosis in human pancreatic cancerous cell line (PANC-1). PANC-1 cells were cultured and treated with DSF at doses of 5, 10, 13 μM for 24 h and apoptosis was measured. Methylation specific PCR (MS-PCR) and real-time quantitative PCR were carried out to detect the methylation pattern and to estimate the mRNA expression levels of RASSF1A, p21 and Bax. MS-PCR analysis demonstrated that no unmethylated band was apeared in PANC-1 cell line after DSF treatments. The real-time quantitative PCR results showed no significant mRNA expression for RASSF1A (p>0.05); whereas p21 and Bax expression were significantly (pPANC-1 through p21 and Bax pathway but not through RASSF1A.

  5. Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines.

    Directory of Open Access Journals (Sweden)

    Nicoletta Potenza

    Full Text Available Hepatocellular carcinoma (HCC is a multi-factorial cancer with a very poor prognosis; therefore, there are several investigations aimed at the comprehension of the molecular mechanisms leading to development and progression of HCC and at the definition of new therapeutic strategies. We have recently evaluated the expression of selenoproteins in HCC cell lines in comparison with normal hepatocytes. Recent results have shown that some of them are down- and others up-regulated, including the selenoprotein K (SELK, whose expression was also induced by sodium selenite treatment on cells. However, so far very few studies have been dedicated to a possible effect of microRNAs on the expression of selenoproteins and their implication in HCC. In this study, the analysis of SELK 3'UTR by bioinformatics tools led to the identification of eight sites potentially targeted by human microRNAs. They were then subjected to a validation test based on luciferase reporter constructs transfected in HCC cell lines. In this functional screening, miR-544a was able to interact with SELK 3'UTR suppressing the reporter activity. Transfection of a miR-544a mimic or inhibitor was then shown to decrease or increase, respectively, the translation of the endogenous SELK mRNA. Intriguingly, miR-544a expression was found to be modulated by selenium treatment, suggesting a possible role in SELK induction by selenium.

  6. Trichostatin A, a histone deacetylase inhibitor, potentiated cytotoxic effect of lionizing radiation in human head and neck cancer cell lines

    International Nuclear Information System (INIS)

    Kim, Jin Ho; Shin, Jin Hee; Chie, Eui Kyu; Wu, Hong Gyun; Kim, Jae Sung; Kim, Il Han; Ha, Sung Whan; Park, Charn Il; Kang, Wee Saing

    2004-01-01

    We have previously reported that human glioblastoma cells are sensitized to radiation-induced death after their exposure to trichostatin A (TSA), a histone deacetylase inhibitor (HDAC-I), prior to the irradiation. We aimed to measure the magnitude of the radiosensitizing effect of TSA in human head and neck cancer cell lines. human head and neck cancer cell lines, HN-3 and HN-9, were exposed to 0, 50, 100, and 200 nM TSA for 18 hr prior to irradiation. Then, the TSA-treated cells were irradiated with 0, 2, 4, 6, and 8 Gy, and cell survival was measured by clonogenic assay. Pre-irradiation exposure to TSA was found to radiosensitize HN-3 and HN-9 cell lines. In HN-9 cells, the fraction surviving after 2 Gy (SF2) was significantly reduced by treatment of TSA at concentration as low as 50 nM. However, a treatment with 200 nM TSA was required to significantly decrease SF2 in the HN-3 cell line. SER of pre-irradiation treatment with 200 nM TSA was 1.84 in HN-3 and 7.24 in HN-9, respectively. Our results clearly showed that human head and neck cancer cell lines can be sensitized to ionizing radiation by pre-irradiation inhibition of histone deacetylase (HDAC) using TSA, and that this potentiation might well be a general phenomenon

  7. Cdx2 Polymorphism Affects the Activities of Vitamin D Receptor in Human Breast Cancer Cell Lines and Human Breast Carcinomas

    Science.gov (United States)

    Di Benedetto, Anna; Korita, Etleva; Goeman, Frauke; Sacconi, Andrea; Biagioni, Francesca; Blandino, Giovanni; Strano, Sabrina; Muti, Paola; Mottolese, Marcella; Falvo, Elisabetta

    2015-01-01

    Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D) and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954) human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative). These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression. PMID:25849303

  8. Cdx2 polymorphism affects the activities of vitamin D receptor in human breast cancer cell lines and human breast carcinomas.

    Directory of Open Access Journals (Sweden)

    Claudio Pulito

    Full Text Available Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR. It regulates the action of hormone responsive genes and is involved in cell cycle regulation, differentiation and apoptosis. VDR is a critical component of the vitamin D pathway and different common single nucleotide polymorphisms have been identified. Cdx2 VDR polymorphism can play an important role in breast cancer, modulating the activity of VDR. The objective of this study is to assess the relationship between the Cdx2 VDR polymorphism and the activities of VDR in human breast cancer cell lines and carcinomas breast patients. Cdx2 VDR polymorphism and antiproliferative effects of vitamin D treatment were investigated in a panel of estrogen receptor-positive (MCF7 and T-47D and estrogen receptor-negative (MDA-MB-231, SUM 159PT, SK-BR-3, BT549, MDA-MB-468, HCC1143, BT20 and HCC1954 human breast cancer cell lines. Furthermore, the potential relationship among Cdx2 VDR polymorphism and a number of biomarkers used in clinical management of breast cancer was assessed in an ad hoc set of breast cancer cases. Vitamin D treatment efficacy was found to be strongly dependent on the Cdx2 VDR status in ER-negative breast cancer cell lines tested. In our series of breast cancer cases, the results indicated that patients with variant homozygote AA were associated with bio-pathological characteristics typical of more aggressive tumours, such as ER negative, HER2 positive and G3. Our results may suggest a potential effect of Cdx2 VDR polymorphism on the efficacy of vitamin D treatment in aggressive breast cancer cells (estrogen receptor negative. These results suggest that Cdx2 polymorphism may be a potential biomarker for vitamin D treatment in breast cancer, independently of the VDR receptor expression.

  9. Effect of capping agents on the cytotoxicity of silver nanoparticles in human normal and cancer skin cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Netchareonsirisuk, Ponsawan [Chulalongkorn University, Program in Biotechnology, Faculty of Science (Thailand); Puthong, Songchan [Chulalongkorn University, Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering (Thailand); Dubas, Stephan [Chulalongkorn University, Petroleum and Petrochemical College (Thailand); Palaga, Tanapat [Chulalongkorn University, Department of Microbiology, Faculty of Science (Thailand); Komolpis, Kittinan, E-mail: kittinan.k@chula.ac.th [Chulalongkorn University, Antibody Production Research Unit, Institute of Biotechnology and Genetic Engineering (Thailand)

    2016-11-15

    Silver nanoparticles (AgNPs) are among the most widely used nanomaterials in medical and consumer products. However, safety in the uses of AgNPs is still controversial. The toxicity of AgNPs toward various cell types has been reported to depend on the surface properties of the nanoparticles. In this study, the effect of AgNPs with the average size of 5–15 nm on the viability of the CCD-986SK human normal skin fibroblast cell line and A375 human malignant melanoma cell line was evaluated. Comparative toxicity studies, based on MTT assay, were performed by using either sodium alginate or poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA) as capping agent in the nanoparticle preparation. The cytotoxicity tests revealed that AgNO{sub 3} alone was highly toxic to both cell types while both alginate and PSSMA alone were not toxic. AgNPs capped with alginate were selectively toxic to the cancer cell line but not to the normal cell line while AgNPs capped with PSSMA were toxic to both cancer and normal cell lines. Judging from the 50 % inhibition concentration (IC{sub 50}), it was found that the cancer cell line was more sensitive to AgNPs than the normal cell line. Study on the mode of cell death by annexin V and propidium iodide staining revealed that AgNPs induced more apoptotic cell death (84–90 %) than necrosis (8–12 %) in the skin cancer cell line. These results suggest that the toxicity of AgNPs depended on the type of capping agent and the type of cell line.

  10. Generation of OCIAD1 inducible overexpression human embryonic stem cell line: BJNhem20-OCIAD1-Tet-On

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    Deeti K. Shetty

    2016-03-01

    Full Text Available Human embryonic stem cell line BJNhem20-OCIAD1-Tet-On was generated using non-viral method. The constructs pCAG-Tet-On and pTRE-Tight vector driving OCIAD1 expression were transfected using microporation procedure. pCAG-Tet-On cells can be used for inducible expression of any coding sequence cloned into pTRE-Tight vector. For example, in human embryonic stem cells, Tet-On system has been used to generate SOX2 overexpression cell line (Adachi et al., 2010.

  11. Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

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    M.A. Sukoyan

    2002-05-01

    Full Text Available Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

  12. Effects of Pulsed Electromagnetic Field on Differentiation of HUES-17 Human Embryonic Stem Cell Line

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    Yi-Lin Wu

    2014-08-01

    Full Text Available Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP, stage-specific embryonic antigen-3 (SSEA-3, SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells.

  13. Apoptosis and radiosensitivity induced by N-acety1 phytosphingosine, in human cancer cell line

    International Nuclear Information System (INIS)

    Kim, Y. H.; Kim, K. S.; Han, Y. S.; Jeon, S. J.; Song, J. Y.; Jung, I. S.; Hong, S. H.; Yun, Y. S.; Park, J. S.

    2004-01-01

    Ceramide is a key lipid molecule in signal transduction with a role in various regulatory pathways including differentiation, proliferation and especially apoptosis. Ionizing radiation-induced apoptosis is associated with accumulation of ceramide, and the sphingomyelinase deficiency results in radioresistance. We investigated the exogenous treatment of N-acetyl-phytosphingosine (NAPS), an analogue of N-acetyl-sphingosine (C 2 -Ceramide), and C 2 -ceramide exert apoptotic effect on human T cell lymphoma Jurkat cells and breast cancer cell line MDA-MB-231. NAPS and C 2 -Ceramide has cytotoxic effect in time- and dose-dependent manner, and increased caspase-3, 8 activity. However, NAPS induced apoptosis more effectively, and increased caspase activity induced by NAPS is more higher than C 2 -ceramide. Moreover, NAPS decreased clonogenicity of irradiated cells and increased radiation-induced apoptosis significantly. Increased cell death by irradiation in the presence of NAPS is owing to the increase of caspase activity. These data suggest that NAPS might be used for lead as a new type of radiosensitizing agent increasing radiation-induced apoptosis

  14. Efficient Generation of NKX6-1+ Pancreatic Progenitors from Multiple Human Pluripotent Stem Cell Lines

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    M. Cristina Nostro

    2015-04-01

    Full Text Available Human pluripotent stem cells (hPSCs represent a renewable source of pancreatic beta cells for both basic research and therapeutic applications. Given this outstanding potential, significant efforts have been made to identify the signaling pathways that regulate pancreatic development in hPSC differentiation cultures. In this study, we demonstrate that the combination of epidermal growth factor (EGF and nicotinamide signaling induces the generation of NKX6-1+ progenitors from all hPSC lines tested. Furthermore, we show that the size of the NKX6-1+ population is regulated by the duration of treatment with retinoic acid, fibroblast growth factor 10 (FGF10, and inhibitors of bone morphogenetic protein (BMP and hedgehog signaling pathways. When transplanted into NOD scid gamma (NSG recipients, these progenitors differentiate to give rise to exocrine and endocrine cells, including monohormonal insulin+ cells. Together, these findings provide an efficient and reproducible strategy for generating highly enriched populations of hPSC-derived beta cell progenitors for studies aimed at further characterizing their developmental potential in vivo and deciphering the pathways that regulate their maturation in vitro.

  15. Construction, characterization, and complementation of a conditional-lethal DNA topoisomerase IIalpha mutant human cell line.

    Science.gov (United States)

    Carpenter, Adam J; Porter, Andrew C G

    2004-12-01

    DNA Topoisomerase IIalpha (topoIIalpha) is a DNA decatenating enzyme, abundant constituent of mammalian mitotic chromosomes, and target of numerous antitumor drugs, but its exact role in chromosome structure and dynamics is unclear. In a powerful new approach to this important problem, with significant advantages over the use of topoII inhibitors or RNA interference, we have generated and characterized a human cell line (HTETOP) in which >99.5% topoIIalpha expression can be silenced in all cells by the addition of tetracycline. TopoIIalpha-depleted HTETOP cells enter mitosis and undergo chromosome condensation, albeit with delayed kinetics, but normal anaphases and cytokineses are completely prevented, and all cells die, some becoming polyploid in the process. Cells can be rescued by expression of topoIIalpha fused to green fluorescent protein (GFP), even when certain phosphorylation sites have been mutated, but not when the catalytic residue Y805 is mutated. Thus, in addition to validating GFP-tagged topoIIalpha as an indicator for endogenous topoIIalpha dynamics, our analyses provide new evidence that topoIIalpha plays a largely redundant role in chromosome condensation, but an essential catalytic role in chromosome segregation that cannot be complemented by topoIIbeta and does not require phosphorylation at serine residues 1106, 1247, 1354, or 1393.

  16. Abnormal A-type lamin organization in a human lung carcinoma cell line

    NARCIS (Netherlands)

    Machiels, BM; Broers, JL; Raymond, Y; de Leij, Louis; Kuijpers, HJH; Caberg, NEH; Ramaekers, Frans C. S.

    We have studied the expression of lamins A and C (A-type lamins) in a lung carcinoma cell line using type-specific monoclonal antibodies, Using immunofluorescence and immunoblotting studies it was noted that several irregularities in lamin expression exist in the cell line GLC-A1, derived from an

  17. 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

  18. The effect of taurine chloramine on human osteosarcoma cell-lines

    International Nuclear Information System (INIS)

    Pilz, M.

    2010-01-01

    Osteosarcoma is the most frequent nonhaematogenic primary malignant tumor of the bone. The rather rare tumour occurs mainly in children and adolescents. This osteogenic tumour is a high-aggressive mesenchymal neoplasm typically producing osteoid. The surgical resection of the complete tumour must be carried out with wide or radical margins to prevent recurrence. Even though the combination of surgery with chemotherapy has noticeably improved the survival rate of osteosarcoma patients, the application of anticancer drugs is still associated with significant adverse reactions, for example the common acquisition of drug-resistant phenotypes, indicating the need of new chemotherapeutical substances. Taurine is a sulfur-containing β-amino acid, which is present in high concentrations in mammalian cells and plasma, in granulocytes and lymphocytes. Large quantities of taurine are released by stimulated neutrophiles and rapidly chlorinated by the reaction with hypochlorous acid (HOCl) generating taurine monochloramine (NCT). NCT is noted to play quite a few roles in the modulation of the immune response and sizeably decreases the production of plenty proinflammatory mediators from adherent as well as non-adherent leucocytes. NCT inhibits the intracellular transcription of nitric-oxide synthase and the production of nitric oxide and switches cell death from the less advantageous necrosis to the more physiologically beneficial apoptosis. Aim of this study was to research, if different concentrations of NCT are capable to induce apoptosis in the human osteosarcoma cell lines HOS, MG-63 and SAOS-2. Therefore the cell proliferation assay EZ4U, a fluorescence staining with acridine-orange, an ELISA for the detection of DNA-fragments and a JC-1 mitochondrial FACS-analysis were performed. The results of these four independent experiments show, that NCT has a pro-apoptotic effect on these osteosarcoma cell lines. (author) [de

  19. Phytol isolated from watermelon (Citrullus lanatus) sprouts induces cell death in human T-lymphoid cell line Jurkat cells via S-phase cell cycle arrest.

    Science.gov (United States)

    Itoh, Tomohiro; Ono, Akito; Kawaguchi, Kaori; Teraoka, Sayaka; Harada, Mayo; Sumi, Keitaro; Ando, Masashi; Tsukamasa, Yasuyuki; Ninomiya, Masayuki; Koketsu, Mamoru; Hashizume, Toshiharu

    2018-05-01

    The phytol isolated from watermelon (Citrullus lanatus) sprouts inhibited the growth of a human T-cell leukemia line Jurkat cell and suppressed tumor progression in a xenograft model of human lung adenocarcinoma epithelial cell line A549 in nude mice. To elucidate the mechanisms underlying the phytol-induced cell death in the present study, we examined the changes in cell morphology, DNA fragmentation, and intracellular reactive oxygen species (ROS) levels and performed flow cytometric analysis to evaluate cell cycle stage. There were no significant changes in apoptosis, autophagy, and necrosis marker in cells treated with the phytol. But, we found, for the first time, that phytol remarkably induced S-phase cell cycle arrest accompanied with intracellular ROS production. Western blot analyses showed that phytolinduced S-phase cell cycle arrest was mediated through the decreased expression of cyclins A and D and the downregulations of MAPK and PI3K/Akt. The tumor volume levels in mice treated with phytol were lower than those of non-treatment groups, and it showed very similar suppression compared with those of mice treated with cyclophosphamide. Based on the data of in vitro and in vivo studies and previous studies, we suggest phytol as a potential therapeutic compound for cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

  20. Synthesis and secretion of platelet-derived growth factor by human breast cancer cell lines

    International Nuclear Information System (INIS)

    Bronzert, D.A.; Pantazis, P.; Antoniades, H.N.; Kasid, A.; Davidson, N.; Dickson, R.B.; Lippman, M.E.

    1987-01-01

    The authors report that human breast cancer cells secrete a growth factor that is biologically and immunologically similar to platelet-derived growth factor (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or competence activity that is capable of inducing incorporation of [ 3 H] thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 0 C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of ≅30 kDa on NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO 4 /polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and 16-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like growth factor by breast cancer cell lines may be important in mediating paracrine stimulation of tumor growth

  1. Endogenous and exogenous porphyrins as photosensitizers in the Hep-2 human carcinoma cell line.

    Science.gov (United States)

    Alvarez, M G; Milanesio, M; Rivarola, V; Durantini, E; Batlle, A; Fukuda, H

    2009-07-01

    The photodynamic activity of three photosensitizers (PS): AL-induced PPIX, the porphyrin derivative 5-(4-trimethylammoniumphenyl)-10, 5, 20-tris (2,4,6- trimethoxyphenyl) porphyrin (CP) and the molecular dyad porphyrin-C(60) (P-C(60)), the last two incorporated into liposomal vesicles, was evaluated on Hep-2 human larynx carcinoma cell line. ALA-induced accumulation of the endogenous PS PPIX, reached saturation values between 5 and 24 h incubation time; the maximal PPIX content was 5.7 nmol/106 cells. The same intracellular level was accumulated when the cationic porphyrin CP was used, while the amount of P-C(60) attained was 1.5 nmol/106 cells. Under violet-blue exciting light, the fluorescence of PPIX and P-C(60) was found in the cytoplasm showing a granular appearance indicating lysosomal localization. CP was mainly detected as a filamentous pattern characteristic of mitochondrial localization. No dark cytotoxicity was observed using 1mM ALA, 5 microM CP and 1 microM P-C(60) after 24 h incubation. Cell morphology was analyzed using Hoechst-33258, toluidine blue staining, TUNEL assay and DNA fragmentation, 24 h after irradiation with 54 J/cm2. When photosensitized with ALA and P-C(60), chromatine condensation characteristic of apoptotic cell death was found; instead, 58 % of necrotic cells were observed with CP. The results show that in the Hep-2 cells, of the three PS analyzed, the molecular dyad P-C(60) was more efficient than CP and PPIX, and confirm that PDT can induce different mechanisms of cell death depending on the PS and the irradiation dose.

  2. Radiosensitivity evaluation of Human tumor cell lines by detecting 4977bp deletion in mitochondrial DNA

    International Nuclear Information System (INIS)

    Zhang Yipei

    2009-01-01

    Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using the assay of mtDNA4977bp deletion. Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction(SF), the ratio of mtDNA4977bp deletion and DNA damage were detected by MTT assay and nested PCR technique respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4and 8Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. PCR method:The differences on mtDNA 4977bp deletion in mitochondrial DNA among HepG 2 , EC-9706 and MCF-7 were not significant after 1Gy and 4Gy γ-ray irradiation. The ratio of 4977bp deletion in mitochondrial DNA of HepG 2 and EC-9706 increased while that of MCF-7 decreased after 8Gy irradiation. The ratio of mtDNA 4977bp deletion of HepG 2 and EC-9706 was higher significantly than that of MCF-7, which implies that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF -7. Conclusion: As a new biological marker, mtDNA4977bp deletion may be hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

  3. Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment

    International Nuclear Information System (INIS)

    Gery, Bernard; Little, John B.; Coppey, Jacques

    1996-01-01

    Purpose: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. Methods and Materials: The cell system consists of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. Results: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10 5 fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. Conclusions: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumors fragments

  4. Development of an in vitro skin sensitization test using human cell lines; human Cell Line Activation Test (h-CLAT). II. An inter-laboratory study of the h-CLAT.

    Science.gov (United States)

    Sakaguchi, H; Ashikaga, T; Miyazawa, M; Yoshida, Y; Ito, Y; Yoneyama, K; Hirota, M; Itagaki, H; Toyoda, H; Suzuki, H

    2006-08-01

    Recent regulatory changes have placed a major emphasis on in vitro safety testing and alternative models. In regard to skin sensitization tests, dendritic cells (DCs) derived from human peripheral blood have been considered in the development of new in vitro alternatives. Human cell lines have been also reported recently. In our previous study, we suggested that measuring CD86 and/or CD54 expression on THP-1 cells (human monocytic leukemia cell line) could be used as an in vitro skin sensitization method. An inter-laboratory study among two laboratories was undertaken in Japan in order to further develop an in vitro skin sensitization model. In the present study, we used two human cell lines: THP-1 and U-937 (human histiocytic lymphoma cell line). First we optimized our test protocol (refer to the related paper entitled "optimization of the h-CLAT protocol" within this journal) and then we did an inter-laboratory validation with nine chemicals using the optimized protocol. We measured the expression of CD86 and CD54 on the above cells using flow cytometry after a 24h and 48h exposure to six known allergens (e.g., DNCB, pPD, NiSO(4)) and three non-allergens (e.g., SLS, tween 80). For the sample test concentration, four doses (0.1x, 0.5x, 1x, and 2x of the 50% inhibitory concentration (IC(50))) were evaluated. IC(50) was calculated using MTT assay. We found that allergens/non-allergens were better predicted using THP-1 cells compared to U-937 cells following a 24 h and a 48 h exposure. We also found that the 24h treatment time tended to have a better accuracy than the 48 h treatment time for THP-1 cells. Expression of CD86 and CD54 were good predictive markers for THP-1 cells, but for U-937 cells, expression of CD86 was a better predictor than CD54, at the 24h and the 48 h treatment time. The accuracy also improved when both markers (CD86 and CD54) were used as compared with a single marker for THP-1 cells. Both laboratories gave a good prediction of allergen

  5. Design, Synthesis and Evaluation of N13-Substituted Evodiamine Derivatives against Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Senchuan Song

    2013-12-01

    Full Text Available Attempting to improve the anticancer activity and solubility of evodiamine in simulated gastric fluid (SGF and simulated intestinal fluid (SIF solutions, thirty-eight N13-substituted evodiamine derivatives were designed, synthesized and tested for antitumor activities against six kinds of human cancer cell lines, namely prostate cancer (DU-145 and PC-3, lung cancer (H460, breast cancer (MCF-7, colon cancer (HCT-5 and glioblastoma (SF-268. The solubility of these compounds in SGF and SIF solutions was evaluated, and apoptosis induced by 2-2, 2-3, 2-16 and 3-2 was determined. The results showed: (1 among all compounds examined, 2-16 showed the highest antitumor activity and a broader spectrum of activity, with IC50 values ranging from 1–2 µM; (2 their solubility was obviously improved; (3 2-3, 2-16 and 3-2 had a significant impact inducing apoptosis in some cancer cell lines. The preliminary structure-activity relationships of these derivatives were discussed.

  6. Interaction of PLP with GFP-MAL2 in the human oligodendroglial cell line HOG.

    Directory of Open Access Journals (Sweden)

    Raquel Bello-Morales

    Full Text Available The velocity of the nerve impulse conduction of vertebrates relies on the myelin sheath, an electrically insulating layer that surrounds axons in both the central and peripheral nervous systems, enabling saltatory conduction of the action potential. Oligodendrocytes are the myelin-producing glial cells in the central nervous system. A deeper understanding of the molecular basis of myelination and, specifically, of the transport of myelin proteins, will contribute to the search of the aetiology of many dysmyelinating and demyelinating diseases, including multiple sclerosis. Recent investigations suggest that proteolipid protein (PLP, the major myelin protein, could reach myelin sheath by an indirect transport pathway, that is, a transcytotic route via the plasma membrane of the cell body. If PLP transport relies on a transcytotic process, it is reasonable to consider that this myelin protein could be associated with MAL2, a raft protein essential for transcytosis. In this study, carried out with the human oligodendrocytic cell line HOG, we show that PLP colocalized with green fluorescent protein (GFP-MAL2 after internalization from the plasma membrane. In addition, both immunoprecipitation and immunofluorescence assays, indicated the existence of an interaction between GFP-MAL2 and PLP. Finally, ultrastructural studies demonstrated colocalization of GFP-MAL2 and PLP in vesicles and tubulovesicular structures. Taken together, these results prove for the first time the interaction of PLP and MAL2 in oligodendrocytic cells, supporting the transcytotic model of PLP transport previously suggested.

  7. Combined Effects of Nonylphenol and Bisphenol A on the Human Prostate Epithelial Cell Line RWPE-1

    Directory of Open Access Journals (Sweden)

    Weidong Gan

    2015-04-01

    Full Text Available The xenoestrogens nonylphenol (NP and bisphenol A (BPA are regarded as endocrine disrupting chemicals (EDCs which have widespread occurrence in our daily life. In the present study, the purpose was to analyze the combined effects of NP and BPA on the human prostate epithelial cell line RWPE-1 using two mathematical models based on the Loewe additivity (LA theory and the Bliss independence (BI theory. RWPE-1 cells were treated with NP (0.01–100 µM and BPA (1–5000 µM in either a single or a combined format. A cell viability assay and lactate dehydrogenase (LDH leakage rate assay were employed as endpoints. As predicted by the two models and based on the cell viability assay, significant synergism between NP and BPA were observed. However, based on the LDH assay, the trends were reversed. Given that environmental contaminants are frequently encountered simultaneously, these data indicated that there were potential interactions between NP and BPA, and the combined effects of the chemical mixture might be stronger than the additive values of individual chemicals combined, which should be taken into consideration for the risk assessment of EDCs.

  8. KCC2a expression in a human fetal lens epithelial cell line.

    Science.gov (United States)

    Lauf, Peter K; Di Fulvio, Mauricio; Srivastava, Vinita; Sharma, Neelima; Adragna, Norma C

    2012-01-01

    The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin. Copyright © 2012 S. Karger AG, Basel.

  9. Modulation of gene expression in a human cell line caused by poliovirus, vaccinia virus and interferon

    Directory of Open Access Journals (Sweden)

    Hoddevik Gunnar

    2007-03-01

    Full Text Available Abstract Background The project was initiated to describe the response of a human embryonic fibroblast cell line to the replication of two different viruses, and, more specifically, to look for candidate genes involved in viral defense. For this purpose, the cells were synchronously infected with poliovirus in the absence or presence of interferon-alpha, or with vaccinia virus, a virus that is not inhibited by interferon. By comparing the changes in transcriptosome due to these different challenges, it should be possible to suggest genes that might be involved in defense. Results The viral titers were sufficient to yield productive infection in a majority of the cells. The cells were harvested in triplicate at various time-points, and the transcriptosome compared with mock infected cells using oligo-based, global 35 k microarrays. While there was very limited similarities in the response to the different viruses, a large proportion of the genes up-regulated by interferon-alpha were also up-regulated by poliovirus. Interferon-alpha inhibited poliovirus replication, but there were no signs of any interferons being induced by poliovirus. The observations suggest that the cells do launch an antiviral response to poliovirus in the absence of interferon. Analyses of the data led to a list of candidate antiviral genes. Functional information was limited, or absent, for most of the candidate genes. Conclusion The data are relevant for our understanding of how the cells respond to poliovirus and vaccinia virus infection. More annotations, and more microarray studies with related viruses, are required in order to narrow the list of putative defence-related genes.

  10. Establishment, characterization, chemosensitivity, and radiosensitivity of two different cell lines derived from a human breast cancer biopsy

    International Nuclear Information System (INIS)

    Gioanni, J.; Courdi, A.; Lalanne, C.M.; Fischel, J.L.; Zanghellini, E.; Lambert, J.C.; Ettore, F.; Namer, M.

    1985-01-01

    In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro

  11. Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells.

    Science.gov (United States)

    Chang, Yuewen; Zhao, Yongfang; Zhan, Hongsheng; Wei, Xiaoen; Liu, Tianjin; Zheng, Bo

    2014-02-01

    Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

  12. Effects of radiation and porphyrin on mitosis and chromosomes in human hematopoietic cell lines

    International Nuclear Information System (INIS)

    Tan, J.C.; Huang, C.C.; Fiel, R.J.

    1976-01-01

    The effect on mitosis of a human hematopoietic cell line RPMI-1788 treated with a metal chelate (Zn ++ ) of meso-tetra (p-carboxyphenyl) porphine (Zn-TCPP) alone at various concentrations or in combination with gamma-irradiation at various doses were studied. The results showed that both Zn-TCPP and radiation were effective in interfering with normal mitosis and that the effect of radiation was relatively more effective. Data also suggest interacting effects between Zn-TCPP and gamma-irradiation. At low doses of radiation, Zn-TCPP potentiated the effect of radiation. The reverse seemed to be true at a high dose of radiation. The effects of two porphyrins (Zn-TCPP and hematoporphyrin) and radiation on chromosomes were also studied. Chromosomal aberrations characteristic of radiation were observed. The porphyrins were found not to be effective chromosome-breaking agents under the experimental conditions tested

  13. Nyctanthes arbortristis mediated synthesis of silver nanoparticles: Cytotoxicity assay against THP-1 human leukemia cell lines

    Science.gov (United States)

    Kumari, Priti; Kumari, Niraj; Jha, Anal K.; Singh, K. P.; Prasad, K.

    2018-05-01

    Green synthesis, characterizations and applications of nanoparticles have become an important branch of nanotechnology now a day. In this paper, green synthesis of silver nanoparticles (AgNPs) using the aqueous extract of Nyctanthes arbortristis as a reducing and stabilizing agent, has been discussed. Present synthetic method is very handy, cost-effective and reproducible. Formation of AgNPs was characterized by X-ray diffraction, dynamic light scattering, scanning electron microscopy and UV-visible spectroscopy techniques. The phytochemicals responsible for nano-transformation were principally flavonoids, phenols and glycosides present in the leaves. Further, the dose dependent cytotoxicity assay of biosynthesized AgNPs against THP-1 human leukemia cell lines showed the encouraging results.

  14. The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line

    DEFF Research Database (Denmark)

    Suzuki, Harukazu; Forrest, Alistair R R; van Nimwegen, Erik

    2009-01-01

    , we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks......Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites...... involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process....

  15. Radiosensitivity of different human tumor cells lines grown as multicellular spheroids determined from growth curves and survival data

    International Nuclear Information System (INIS)

    Schwachoefer, J.H.C.; Crooijmans, R.P.; van Gasteren, J.J.; Hoogenhout, J.; Jerusalem, C.R.; Kal, H.B.; Theeuwes, A.G.

    1989-01-01

    Five human tumor cell lines were grown as multicellular tumor spheroids (MTS) to determine whether multicellular tumor spheroids derived from different types of tumors would show tumor-type dependent differences in response to single-dose irradiation, and whether these differences paralleled clinical behavior. Multicellular tumor spheroids of two neuroblastoma, one lung adenocarcinoma, one melanoma, and a squamous cell carcinoma of the oral tongue, were studied in terms of growth delay, calculated cell survival, and spheroid control dose50 (SCD50). Growth delay and cell survival analysis for the tumor cell lines showed sensitivities that correlated well with clinical behavior of the tumor types of origin. Similar to other studies on melanoma multicellular tumor spheroids our spheroid control dose50 results for the melanoma cell line deviated from the general pattern of sensitivity. This might be due to the location of surviving cells, which prohibits proliferation of surviving cells and hence growth of melanoma multicellular tumor spheroids. This study demonstrates that radiosensitivity of human tumor cell lines can be evaluated in terms of growth delay, calculated cell survival, and spheroid control dose50 when grown as multicellular tumor spheroids. The sensitivity established from these evaluations parallels clinical behavior, thus offering a unique tool for the in vitro analysis of human tumor radiosensitivity

  16. Adaptive regulation of taurine and beta-alanine uptake in a human kidney cell line from the proximal tubule

    DEFF Research Database (Denmark)

    Jessen, H; Jacobsen, Christian

    1997-01-01

    1. The underlying mechanisms involved in the adaptive regulation of beta-amino acid uptake in the human proximal tubule were examined by use of an immortalized human embryonic kidney epithelial cell line (IHKE). 2. The results indicated that the adaptive response to maintain whole-body taurine...

  17. Dig1 protects against cell death provoked by glyphosate-based herbicides in human liver cell lines

    Directory of Open Access Journals (Sweden)

    Travert Carine

    2010-10-01

    Full Text Available Abstract Background Worldwide used pesticides containing different adjuvants like Roundup formulations, which are glyphosate-based herbicides, can provoke some in vivo toxicity and in human cells. These pesticides are commonly found in the environment, surface waters and as food residues of Roundup tolerant genetically modified plants. In order to know their effects on cells from liver, a major detoxification organ, we have studied their mechanism of action and possible protection by precise medicinal plant extracts called Dig1. Methods The cytotoxicity pathways of four formulations of glyphosate-based herbicides were studied using human hepatic cell lines HepG2 and Hep3B, known models to study xenobiotic effects. We monitored mitochondrial succinate dehydrogenase activity and caspases 3/7 for cell mortality and protection by Dig1, as well as cytochromes P450 1A1, 1A2, 3A4 and 2C9 and glutathione-S-transferase to approach the mechanism of actions. Results All the four Roundup formulations provoke liver cell death, with adjuvants having stronger effects than glyphosate alone. Hep3B are 3-5 times more sensitive over 48 h. Caspases 3/7 are greatly activated in HepG2 by Roundup at non-cytotoxic levels, and some apoptosis induction by Roundup is possible together with necrosis. CYP3A4 is specifically enhanced by Roundup at doses 400 times less than used in agriculture (2%. CYP1A2 is increased to a lesser extent together with glutathione-S-transferase (GST down-regulation. Dig 1, non cytotoxic and not inducing caspases by itself, is able to prevent Roundup-induced cell death in a time-dependant manner with an important efficiency of up to 89%, within 48 h. In addition, we evidenced that it prevents Caspases 3/7 activation and CYP3A4 enhancement, and not GST reduction, but in turn it slightly inhibited CYP2C9 when added before Roundup. Conclusion Roundup is able to provoke intracellular disruption in hepatic cell lines at different levels, but a

  18. Comparison of herpes simplex (HSV) proteins synthesized in Vero, HEP-2 and human megakaryocyte-like cell lines

    International Nuclear Information System (INIS)

    Soslau, G.; Pastorino, M.B.; Morgan, D.A.; Brodsky, I.; Howett, M.K.

    1986-01-01

    The natural human host blood cell capable of supporting herpes virus replication has yet to be defined. They found that a recently cultured human megakaryocyte-like (Meg) cell line can support HSV 1 and 2 replication as demonstrated by growth inhibition, CPE, virus production and HSV DNA synthesis. The HSV proteins synthesized and post-translationally modified in Vero and HEp-2 infected cells were compared to the protein species produced in the infected Meg cell since differences may influence antigenic properties and host range. Host cell protein synthesis was greatly reduced in all three cell lines within hours post infection (pi). However, maximum viral protein synthesis occurs between 4 and 24 hrs pi with the Meg cells as compared to 24-48 hrs pi with the other cell lines. The immunoprecipitated 35 S-methionine labeled HSV protein gel patterns for each infected cell line are qualitatively and quantitatively very different from each other. Dramatic differences were also observed when infected cells were labeled with 32 P-ATP (in vitro method) or 32 Pi (in vivo method). Finally, analysis of 3 H-mannose labeled HSV glycoproteins demonstrates that the post-translational modifications of these proteins are significantly altered in the Meg cell as compared to the Vero and HEp-2 cells

  19. In Vitro Cytotoxic Effects of Cuscuta chinensis Whole Extract on Human Acute Lymphoblastic Leukemia Cell Line

    Directory of Open Access Journals (Sweden)

    Fatemeh Zeraati

    2010-12-01

    Full Text Available Background: One of the major paths for drug development isthe study of bioactivities of natural products. Therefore, theaim of this study was to compare the cytotoxic effects ofaqueous extract of whole Cuscuta chinensis Lam., which is atraditional medicinal herb commonly used in Iran and otheroriental countries, on the human caucasian acute lymphoblasticleukemia (CCRF-CEM and another human lymphocyte,Jurkat (JM cell lines.Methods: In vitro cytotoxic screening with various concentrations(0, 0.1, 1, 10, 25 and 50 μg/ml of the extract wasperformed using microscope and methyl tetrazolium bromidetest (MTT.Results: The minimum effective concentration of the plantextract was 1 μg/ml, and increasing the dose to 10 μg/mlinduced increasingly stronger effects. The inhibitory concentration50% (IC50 of the extract against CCRF wasabout 3 μg/ml in 24 hours and 2.5 μg/ml in 48 hrs. In contrast,the extract did not have cytotoxic effect for the JMcells at these doses.Conclusion: The findings of the present study suggest that C.chinensis is toxic against CCRF-CEM and JM tumor cells.Whether or not such effects can be employed for the treatmentof such tumors must await future studies.Iran J Med Sci 2010; 35(4: 310-314.

  20. N-Linked Glycosylation is an Important Parameter for Optimal Selection of Cell Lines Producing Biopharmaceutical Human IgG

    NARCIS (Netherlands)

    van Berkel, Patrick H. C.; Gerritsen, Jolanda; Perdok, Gerrard; Valbjorn, Jesper; Vink, Tom; van de Winkel, Jan G. J.; Parren, Paul W. H. I.

    2009-01-01

    We studied the variations in N-linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO-KISV cells. The glycans

  1. Carboplatin- and cisplatin-induced potentiation of moderate-dose radiation cytotoxicity in human lung cancer cell lines

    NARCIS (Netherlands)

    Groen, H. J.; Sleijfer, S.; Meijer, C.; Kampinga, H. H.; Konings, A. W. T.; de Vries, E. G. E.; Mulder, N. H.

    1995-01-01

    The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC(4)) and -resistant (GLC(4)-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the

  2. Comprehensive prediction in 78 human cell lines reveals rigidity and compactness of transcription factor dimers

    Science.gov (United States)

    Jankowski, Aleksander; Szczurek, Ewa; Jauch, Ralf; Tiuryn, Jerzy; Prabhakar, Shyam

    2013-01-01

    The binding of transcription factors (TFs) to their specific motifs in genomic regulatory regions is commonly studied in isolation. However, in order to elucidate the mechanisms of transcriptional regulation, it is essential to determine which TFs bind DNA cooperatively as dimers and to infer the precise nature of these interactions. So far, only a small number of such dimeric complexes are known. Here, we present an algorithm for predicting cell-type–specific TF–TF dimerization on DNA on a large scale, using DNase I hypersensitivity data from 78 human cell lines. We represented the universe of possible TF complexes by their corresponding motif complexes, and analyzed their occurrence at cell-type–specific DNase I hypersensitive sites. Based on ∼1.4 billion tests for motif complex enrichment, we predicted 603 highly significant cell-type–specific TF dimers, the vast majority of which are novel. Our predictions included 76% (19/25) of the known dimeric complexes and showed significant overlap with an experimental database of protein–protein interactions. They were also independently supported by evolutionary conservation, as well as quantitative variation in DNase I digestion patterns. Notably, the known and predicted TF dimers were almost always highly compact and rigidly spaced, suggesting that TFs dimerize in close proximity to their partners, which results in strict constraints on the structure of the DNA-bound complex. Overall, our results indicate that chromatin openness profiles are highly predictive of cell-type–specific TF–TF interactions. Moreover, cooperative TF dimerization seems to be a widespread phenomenon, with multiple TF complexes predicted in most cell types. PMID:23554463

  3. Transient HEXA expression in a transformed human fetal Tay-Sachs disease neuroglial cell line

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, M.J.; Hechtman, P.; Kaplan, F. [McGill Univ., Quebec (Canada)] [and others

    1994-09-01

    Tay-Sachs disease (TSD) is a severe neurodegenerative disorder characterized by the accumulation of GM{sub 2} ganglioside in the neurons of the central cortex. The recessively inherited disorder results from deficiency of hexosaminidase A (Hex A), a heterodimer of an {alpha} and {beta} subunit encoded by the HEXA and HEXB genes. Expression of HEXA mutations in COS cells has several disadvantages including high endogenous hexosaminidase activity. We report a new transient expression system with very low endogenous Hex A activity. An SV40-transformed fetal TSD neuroglial cell line was assessed for transient expression of the HEXA gene. pCMV{alpha}, a vector incorporating the cytomegalovirus promoter with the human {alpha}-subunit cDNA insert, proved to be the most efficient expression vector. Transfection of 4x10{sup 6} cells with 5-20 {mu}g of plasmid resulted in 100 to 500-fold Hex A activity (4MUGS hydrolysis) relative to mock-transfected cells. Use of pCMV{beta}-Gal as a control for transfection efficiency indicated that 10-20% of cells were transfected. Hex A specific activity increased for at least 72 h post-transfection. This new transient expression system should greatly improve the characterization of mutations in which low levels of HEXA expression result in milder clinical phenotypes and permit studies on enzymatic properties of mutant forms of Hex A. Since the cells used are of CNS origin and synthesize gangliosides, it should also be possible to study, in culture, the metabolic phenotype associated with TSD.

  4. Enantioselective cytotoxicity of the insecticide bifenthrin on a human amnion epithelial (FL) cell line

    International Nuclear Information System (INIS)

    Liu Huigang; Zhao Meirong; Zhang Cong; Ma Yun; Liu Weiping

    2008-01-01

    Synthetic pyrethroids (SPs) are used in preference to organochlorines and organophosphates due to their high efficiency, low toxicity to mammals, and ready biodegradability. Previous studies reported that enantioselective toxicity of SPs occurs in aquatic toxicity. Several studies have indicated that SPs could lead to oxidative damage in humans or animals which was associated with their toxic effects. Little is known about the differences in the effects of chronic toxicity induced by individual stereoisomers of chiral SPs. The present study was therefore undertaken to evaluate the enantioselectivity in cytotoxicity, genotoxicity caused by bifenthrin (BF) on human amnion epithelial (FL) cell lines and pesticidal activity on target organism. The cell proliferation and cytoflow analysis indicated that 1S-cis-BF presented more toxic effects than 1R-cis-BF above the concentration of 7.5 mg L -1 (p > 0.05). FL cells incubated with 1S-cis-BF exhibited a dose-dependent accumulation of intracellular reactive oxygen species (ROS). In the comet assay, the number of cells with damaged DNA incubated with 1S-cis-BF was more than that with 1R-cis-BF (p 50 values of enantiomer to the target pest on Pieris rapae L. show that 1R-cis-BF was 300 times more active than 1S-cis-BF. These results indicate that the enantioselective toxicity and activity of BF between non-target organism and target organism was reversal. These implications together suggest that assessment of the environmental safety and new pesticides development with chiral centers should consider enantioselectivity

  5. Generation of functional neuromuscular junctions from human pluripotent stem cell lines

    Directory of Open Access Journals (Sweden)

    Katja ePuttonen

    2015-12-01

    Full Text Available Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs, yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC or embryonic stem cell (ESC lines as monolayers without basic fibroblast factor (bFGF and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs. The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca2+ concentration in sarcoplasm, while electrical stimulation evoked Ca2+ transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical MEPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.

  6. Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Michelin, S.; Gallegos, C.E.; Dubner, D. [Radiopathology Laboratory, Nuclear Regulatory Authority, Buenos Aires (Argentina); Favier, B.; Carosella, E.D. [CEA, I2BM, Hopital Saint-Louis, IUH, Service de Recherches en Hemato-Immunologie, Paris (France)

    2009-07-01

    Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of {gamma}-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that {gamma}-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

  7. Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line

    International Nuclear Information System (INIS)

    Michelin, S.; Gallegos, C.E.; Dubner, D.; Favier, B.; Carosella, E.D.

    2009-01-01

    Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of γ-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of down regulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that γ-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule. (authors)

  8. Generation of human induced pluripotent stem cell line from a patient with a long QT syndrome type 2

    Directory of Open Access Journals (Sweden)

    Azra Fatima

    2016-03-01

    Full Text Available We report here the generation of human iPS cell line UKKi009-A from dermal fibroblasts of a patient carrying heterozygous mutation c.3035-3045delTCCCTCGATGC, p.Leu1012Pro (fs*55 in KCNH2 gene leading to long QT syndrome type 2 (LQT2. We used the Sleeping Beauty transposon-based plasmids expressing OSKM along with microRNAs 307/367 to reprogram the fibroblasts. The iPS cells possess pluripotent stem cell characteristics and differentiate to cell lineages of all three germ layers. This cell line can serve as a source for in vitro modeling of LQT2. This cell line is distributed by the European Collection of Authenticated Cell Cultures (ECACC.

  9. In vitro antioxidant and anticancer activity of young Zingiber officinale against human breast carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Iqbal Asif

    2011-09-01

    Full Text Available Abstract Background Ginger is one of the most important spice crops and traditionally has been used as medicinal plant in Bangladesh. The present work is aimed to find out antioxidant and anticancer activities of two Bangladeshi ginger varieties (Fulbaria and Syedpuri at young age grown under ambient (400 μmol/mol and elevated (800 μmol/mol CO2 concentrations against two human breast cancer cell lines (MCF-7 and MDA-MB-231. Methods The effects of ginger on MCF-7 and MDA-MB-231 cell lines were determined using TBA (thiobarbituric acid and MTT [3-(4,5-dimethylthiazolyl-2,5-diphenyl-tetrazolium bromide] assays. Reversed-phase HPLC was used to assay flavonoids composition among Fulbaria and Syedpuri ginger varieties grown under increasing CO2 concentration from 400 to 800 μmol/mol. Results Antioxidant activities in both varieties found increased significantly (P ≤ 0.05 with increasing CO2 concentration from 400 to 800 μmol/mol. High antioxidant activities were observed in the rhizomes of Syedpuri grown under elevated CO2 concentration. The results showed that enriched ginger extract (rhizomes exhibited the highest anticancer activity on MCF-7 cancer cells with IC50 values of 34.8 and 25.7 μg/ml for Fulbaria and Syedpuri respectively. IC50 values for MDA-MB-231 exhibition were 32.53 and 30.20 μg/ml for rhizomes extract of Fulbaria and Syedpuri accordingly. Conclusions Fulbaria and Syedpuri possess antioxidant and anticancer properties especially when grown under elevated CO2 concentration. The use of ginger grown under elevated CO2 concentration may have potential in the treatment and prevention of cancer.

  10. Origin of Microcells in the Human Sarcoma Cell Line HT-1080

    Directory of Open Access Journals (Sweden)

    Indulis Buiķis

    1999-01-01

    Full Text Available The aim of this study was to investigate the development of microcells in the human sarcoma cell line HT‐1080 after interference with thiophosphamidum. We found that damaged interphase macrocells located at the projection of the nucleolus may form one or several microcells. The micronuclei of the microcells intensively incorporate the thymidine analogue 5‐bromo‐2'‐deoxyuridine and strongly express argyrophilic nucleolar organiser region proteins. At an early phase of the development, the micronuclei contain fragmented DNA, but in subsequent phases, the micronuclei accumulate polymeric DNA, simultaneously with an increase in their size. After desintegration of the damaged macrocell, the microcells appear in the intercellular space. The microcells can enter mitosis and they strongly express the lung resistance protein. Electron microscopic observations suggest that coiled bodies are involved in the development of the microcells. Since the observed path of microcell formation differs from apoptotic cell fragmentation into apoptotic bodies, we propose a new term for this microcell development: sporosis. We suggest that self‐renewal of the tumour stem cells is likely based on sporosis.

  11. Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

    Science.gov (United States)

    Waddell, D; Ullman, B

    1983-04-10

    From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

  12. Characterization of endothelin receptors on a human neuroblastoma cell line: evidence for the ETA subtype.

    Science.gov (United States)

    Wilkes, L C; Boarder, M R

    1991-11-01

    1. Specific binding sites for synthetic endothelin (ET) isoforms were studied on intact cells of the SK-N-MC cell line, derived from a human neuroblastoma. 2. [125I]-ET-1 (2.5 x 10(-11) M) specifically bound to a single class of binding sites on these cells (Hill coefficient of 1.06 +/- 0.04, n = 3) with an apparent Kd of 1.4 +/- 0.3 x 10(-9) M and a Bmax of 3.1 +/- 1.0 pmol mg-1 protein. [125I]-ET-3 (2.5 x 10(-11) M), did not specifically bind to SK-N-MC cells. 3. The binding of [125I]-ET-1 was competitively inhibited by other ET isoforms, the order of potency being ET-1 greater than sarafotoxin S6b greater than ET-3. 4. Association of 1 nM [125I]-ET-1 at 37 degrees C reached apparent equilibrium at 60-80 min, with half-maximal binding being achieved at 12 min. 5. Dissociation was measured after both 10 min and 60 min of association with 64% and 30% respectively of specifically bound [125I]-ET-1 dissociating. The actual amounts of [125I]-ET-1 dissociated were similar in both cases. 6. Incubation of [125I]-ET-3 with SK-N-MC cells at 37 degrees C for 60 min did not result in significant degradation of this peptide. However, [125I]-ET-1 was broken down by incubation with SK-N-MC cells, the pattern of degradation of dissociable [125I]-ET-1 (and that found in the supernatant) being different from that of non-dissociable [125I]-ET-1. 7. ET-1 concentration-dependently induced an increase in total inositol phosphate accumulation in subconfluent (but not in confluent) cultures of SK-N-MC cells (EC50 = 6.43 +/- 1.9 x 1010M). ET-3 was without effect. 8. These results show that ET-1 specifically binds to SK-N-MC cells with the characteristics of an ETA receptor. Our earlier finding that adrenal chromaffin cells express an ETB receptor indicates the existence of multiple ET receptor types on neuronal cells.

  13. Purinergic receptors and calcium signalling in human pancreatic duct cell lines

    DEFF Research Database (Denmark)

    Hansen, Mette R; Krabbe, Simon; Novak, Ivana

    2008-01-01

    pancreatic duct cell lines PANC-1 and CFPAC-1. Expression of P2 receptors was examined using RT-PCR and immunocytochemistry. Both cell lines, and also Capan-1 cells, express RNA transcripts for the following receptors: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11-14 and P2X1, P2X2, P2X4, P2X5, P2X6 and P2X7. Using Fura-2...... and single-cell imaging we tested effects of various nucleotide analogues on intracellular Ca(2+) signals in PANC-1 and CFPAC-1 cells. The cell lines responded to all nucleotides with the following efficiency: UTP >or= ATP = ATPgammaS > BzATP. ATP, UTP and ATPgammaS elicited oscillatory responses. Bz...

  14. Iso-suillin from Suillus flavus Induces Apoptosis in Human Small Cell Lung Cancer H446 Cell Line.

    Science.gov (United States)

    Zhao, Jun-Xia; Zhang, Qing-Shuang; Chen, Ying; Yao, Sheng-Jie; Yan, Yong-Xin; Wang, Ying; Zhang, Jin-Xiu; Wang, Li-An

    2016-05-20

    The suillin isoform iso-suillin is a natural substance isolated from a petroleum ether extract of the fruiting bodies of the mushroom Suillus flavus. Previous studies have found its inhibition effect on some cancer cells, and we aimed to study its effects on human small cell lung cancer H446 cell line. Cell viability was measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cellular morphological changes (apoptosis and necrosis) were evaluated using an electron microscope and Hoechst 33258 staining detected by the inverted microscope. Flow cytometry was used to detect cell apoptosis, cell cycle distribution, and mitochondrial membrane potential. Protein expression was determined by Western blotting analysis. Here, we describe the ability of iso-suillin to inhibit the growth of H446 cells in time- and dose-dependent way. Iso-suillin had no obvious impact on normal human lymphocyte proliferation at low concentrations (9.09, 18.17, or 36.35 μmol/L) but promoted lymphocyte proliferation at a high concentration (72.70 μmol/L). After treatment of different concentrations of iso-suillin (6.82, 13.63, or 20.45 μmol/L), the apoptosis rate of H446 cells increased with increasing concentrations of iso-suillin (16.70%, 35.54%, and 49.20%, respectively, all P iso-suillin could induce H446 cell apoptosis through the mitochondrial pathway and the death-receptor pathway. Therefore, iso-suillin might have a potential application as a novel drug for lung cancer treatment.

  15. Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2012-01-01

    Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

  16. Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines.

    Science.gov (United States)

    Massi, Paola; Vaccani, Angelo; Ceruti, Stefania; Colombo, Arianna; Abbracchio, Maria P; Parolaro, Daniela

    2004-03-01

    Recently, cannabinoids (CBs) have been shown to possess antitumor properties. Because the psychoactivity of cannabinoid compounds limits their medicinal usage, we undertook the present study to evaluate the in vitro antiproliferative ability of cannabidiol (CBD), a nonpsychoactive cannabinoid compound, on U87 and U373 human glioma cell lines. The addition of CBD to the culture medium led to a dramatic drop of mitochondrial oxidative metabolism [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide test] and viability in glioma cells, in a concentration-dependent manner that was already evident 24 h after CBD exposure, with an apparent IC(50) of 25 microM. The antiproliferative effect of CBD was partially prevented by the CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; SR2) and alpha-tocopherol. By contrast, the CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716; SR1), capsazepine (vanilloid receptor antagonist), the inhibitors of ceramide generation, or pertussis toxin did not counteract CBD effects. We also show, for the first time, that the antiproliferative effect of CBD was correlated to induction of apoptosis, as determined by cytofluorimetric analysis and single-strand DNA staining, which was not reverted by cannabinoid antagonists. Finally, CBD, administered s.c. to nude mice at the dose of 0.5 mg/mouse, significantly inhibited the growth of subcutaneously implanted U87 human glioma cells. In conclusion, the nonpsychoactive CBD was able to produce a significant antitumor activity both in vitro and in vivo, thus suggesting a possible application of CBD as an antineoplastic agent.

  17. Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

    International Nuclear Information System (INIS)

    Jinno-Oue, Atsushi; Shimizu, Nobuaki; Hamada, Nobuyuki; Wada, Seiichi; Tanaka, Atsushi; Shinagawa, Masahiko; Ohtsuki, Takahiro; Mori, Takahisa; Saha, Manujendra N.; Hoque, Ariful S.; Islam, Salequl; Kogure, Kimitaka; Funayama, Tomoo; Kobayashi, Yasuhiko

    2010-01-01

    Purpose: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). Methods and Materials: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated β-galactosidase (SA-β-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21 WAF1/CIP1 in the irradiated cells was analyzed by Western blotting. Results: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells ( WAF1/CIP1 was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Conclusions: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.

  18. Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

    International Nuclear Information System (INIS)

    Gu Yongpeng; Li Hongzhen; Miki, Jun; Kim, Kee-Hong; Furusato, Bungo; Sesterhenn, Isabell A.; Chu, Wei-Sing; McLeod, David G.; Srivastava, Shiv; Ewing, Charles M.; Isaacs, William B.; Rhim, Johng S.

    2006-01-01

    In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents

  19. Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

    Science.gov (United States)

    Armour, Kathryn L.; Smith, Cheryl S.; Ip, Natasha C. Y.; Ellison, Cara J.; Kirton, Christopher M.; Wilkes, Anthony M.; Williamson, Lorna M.; Clark, Michael R.

    2014-01-01

    We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance. PMID:25302805

  20. Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

    Directory of Open Access Journals (Sweden)

    Kathryn L Armour

    Full Text Available We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

  1. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    Energy Technology Data Exchange (ETDEWEB)

    Sustarsic, Elahu G. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Junnila, Riia K. [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Kopchick, John J., E-mail: kopchick@ohio.edu [Edison Biotechnology Institute, 1 Watertower Drive, Athens, OH (United States); Department of Biological Sciences, Ohio University, Athens, OH (United States); Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH (United States)

    2013-11-08

    Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on

  2. Establishment and characterization of a new cell line derived from human colorectal laterally spreading tumor

    Science.gov (United States)

    Wang, Xin-Ying; Lai, Zhou-Sheng; Yeung, Chung-Man; Wang, Ji-De; Deng, Wen; Li, Hoi Yee; Han, Yu-Jing; Kung, Hsiang-Fu; Jiang, Bo; Lin, Marie Chia-mi

    2008-01-01

    AIM: To study the molecular mechanism of laterally spreading tumor (LST), a cell line [Laterally Spreading Tumor-Rectum 1 (LST-R1)] was derived and the characteristics of this cell line were investigated. METHODS: A new cell line (LST-R1) originated from laterally spreading tumor was established. Properties of the cell line were characterized using scanning and transmission electron microscopy, immunohistochemistry method, cytogenetic analysis and nude mice xenograft experiments. In vitro invasion assay, cDNA microarray and Western blotting were used to compare the difference between the LST-R1 and other colorectal cancer cell lines derived from prudent colon cancer. RESULTS: Our study demonstrated that both epithelial special antigen (ESA) and cytokeratin-20 (CK20) were expressed in LST-R1. The cells presented microvilli and tight junction with large nuclei. The karyotypic analysis showed hyperdiploid features with structural chromosome aberrations. The in vivo tumorigenicity was also demonstrated in nude mice xenograft experiments. The invasion assay suggested this cell line has a higher invasive ability. cDNA microarray and Western blotting show the loss of the expression of E-cadherin in LST-R1 cells. CONCLUSION: We established and characterized a colorectal cancer cell line, LST-R1 and LST-R1 has an obvious malignant tendency, which maybe partially attributed to the changes of the expression of some adhesion molecules, such as E-cadherin. It is also a versatile tool for exploring the original and progressive mechanisms of laterally spreading tumor and the early colon cancer genesis. PMID:18300345

  3. Huh-7 cell line as an alternative cultural model for the production of human like erythropoietin (EPO

    Directory of Open Access Journals (Sweden)

    Kausar Humera

    2011-11-01

    Full Text Available Abstract Background and Aims Erythropoietin (EPO is a glycoprotein hormone which is required to regulate the production of red blood cells. Deficiency of EPO is known to cause anemia in chronically infected renal patients and they require regular blood transfusion. Availability of recombinant EPO has eliminated the need for blood transfusion and now it is extensively used for the treatment of anemia. Glycosylation of erythropoietin is essential for its secretion, stability, protein conformation and biological activity. However, maintenance of human like glycosylation pattern during manufacturing of EPO is a major challenge in biotechnology. Currently, Chinese hamster ovary (CHO cell line is used for the commercial production of erythropoietin but this cell line does not maintain glycosylation resembling human system. With the trend to eliminate non-human constituent from biopharmaceutical products, as a preliminary approach, we have investigated the potential of human hepatoma cell line (Huh-7 to produce recombinant EPO. Materials and methods Initially, the secretory signal and Kozak sequences was added before the EPO mature protein sequence using overlap extension PCR technique. PCR-amplified cDNA fragments of EPO was inserted into mammalian expression vector under the control of the cytomegalovirus (CMV promoter and transiently expressed in CHO and Huh-7 cell lines. After RT-PCR analysis, ELISA and Western blotting was performed to verify the immunochemical properties of secreted EPO. Results Addition of secretory signal and Kozak sequence facilitated the extra-cellular secretion and enhanced the expression of EPO protein. Significant expression (P Conclusion Huh-7 cell line has a great potential to produce glycosylated EPO, suggesting the use of this cell line to produce glycoproteins of the therapeutic importance resembling to the natural human system.

  4. Tributyltin or triphenyltin inhibits aromatase activity in the human granulosa-like tumor cell line KGN.

    Science.gov (United States)

    Saitoh, M; Yanase, T; Morinaga, H; Tanabe, M; Mu, Y M; Nishi, Y; Nomura, M; Okabe, T; Goto, K; Takayanagi, R; Nawata, H

    2001-11-23

    The superimposition of male sex organs (penis and vas deferens) in a female gastropod, called imposex, is widely attributed to the exposure to tributyltin (TBT) compounds, used world-wide in antifouling paints for ships. It has been hypothesized that the TBT-induced imposex is mediated by an increasing androgen level relative to the estrogen level, namely a decreased conversion of androgens to estrogens (i.e., aromatization). In the present study, we tested this hypothesis by examining the effects of TBT or triphenyltin (TPT) on the aromatase activity in a cultured human granulosa-like tumor cell line, KGN, which was recently established by our group. Treatment with more than 1000 ng/ml TBT compounds was very toxic to the cells and caused immediate cell death within 24 h, while 200 ng/ml was found to cause apoptosis of the cells. Treatment of the KGN cells for more than 48 h with 20 ng/ml TBT or TPT, which is a concentration level reported to cause imposex in marine species, did not affect cell proliferation but significantly suppressed the aromatase activity determined by a [(3)H]H(2)O release assay. Treatment with 20 ng/ml TBT compounds for 7 days also resulted in a reduction of the E2 production from Delta 4-androstenedione stimulated by db-cAMP. The changes in the aromatase activity by TBT compounds were associated with comparable changes in P450arom mRNA assessed by RT-PCR. The luciferase activity of the P450arom promoter II (1 kb) decreased after the addition of 20 ng/ml TBT compounds in transfected KGN cells either in a basic state or in states stimulated by db-cAMP. The Ad4BP-dependent increase in the luciferase activity of P450arom promoter II was also downregulated by such treatments. These results indicate that TBT compounds inhibited the aromatase activity and also decreased the P450arom mRNA level at the transcriptional level in KGN cells. The direct inhibitory effect of TBT compounds on the aromatase activity may therefore partly explain the induction

  5. Comparative analysis of gene expression in normal and cancer human prostate cell lines

    Directory of Open Access Journals (Sweden)

    E. E. Rosenberg

    2014-04-01

    Full Text Available Prostate cancer is one of the main causes of mortality in men with malignant tumors. The urgent problem was a search for biomarkers of prostate cancer, which would allow distinguishing between aggressive metastatic and latent tumors. The aim of this work was to search for differentially expressed genes in normal epithelial cells PNT2 and prostate cancer cell lines LNCaP, DU145 and PC3, produced from tumors with different aggressiveness and metas­tatic ability. Such genes might be used to create a panel of prognostic markers for aggressiveness and metastasis. Relative gene expression of 65 cancer-related genes was determined by the quantitative polymerase chain reaction (Q-PCR. Expression of 29 genes was changed in LNCaP cells, 20 genes in DU145 and 16 genes in PC3 cell lines, compared with normal line PNT2. The obtained data make it possible to conclude that the epithelial-mesenchymal cell transition took place, which involved the loss of epithelial markers, reduced cell adhesion and increased migration. We have also found few differentially expressed genes among 3 prostate cancer cell lines. We have found that genes, involved in cell adhesion (CDH1, invasiveness and metastasis (IL8, CXCL2 and cell cycle control (P16, CCNE1 underwent most changes. These genes might be used for diagnosis and prognosis of invasive metastatic prostate tumors.

  6. Study of the cell cycle control for human malignant mesothelioma lines. Interferon and radiations effect

    International Nuclear Information System (INIS)

    Vivo, C.

    1999-01-01

    In order to better understand the inhibition mechanisms of the IFN-R-HU on tumoral development, the IFN-R-U effect on MM lines has been studied. Three groups of lines has been distinguished: eight sensitive lines, two intermediate and three resistant. The sensitive lines showed a triple locking of the cell cycle: in phases S, G1 and G2. The study of the cell cycle control points function, realized by the MM lines radiation exposure showed the points function on G1/S and-or on G2/M and the dependence or non dependence of the cycle stop of the protein P53 and P21 W at F1/CIP1. (A.L.B.)

  7. Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

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    Heema Hewitson

    2016-03-01

    Full Text Available The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG. Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. 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. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  8. Generation of KCL018 research grade human embryonic stem cell line carrying a mutation in the DMPK gene

    Directory of Open Access Journals (Sweden)

    Cristian Miere

    2016-03-01

    Full Text Available The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 trinucleotide repeats; 14 for the normal allele. 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. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  9. Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

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    Laureen Jacquet

    2016-03-01

    Full Text Available The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele. 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. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  10. FMSP-Nanoparticles Induced Cell Death on Human Breast Adenocarcinoma Cell Line (MCF-7 Cells: Morphometric Analysis

    Directory of Open Access Journals (Sweden)

    Firdos Alam Khan

    2018-05-01

    Full Text Available Currently, breast cancer treatment mostly revolves around radiation therapy and surgical interventions, but often these treatments do not provide satisfactory relief to the patients and cause unmanageable side-effects. Nanomaterials show promising results in treating cancer cells and have many advantages such as high biocompatibility, bioavailability and effective therapeutic capabilities. Interestingly, fluorescent magnetic nanoparticles have been used in many biological and diagnostic applications, but there is no report of use of fluorescent magnetic submicronic polymer nanoparticles (FMSP-nanoparticles in the treatment of human breast cancer cells. In the present study, we tested the effect of FMSP-nanoparticles on human breast cancer cells (MCF-7. We tested different concentrations (1.25, 12.5 and 50 µg/mL of FMSP-nanoparticles in MCF-7 cells and evaluated the nanoparticles response morphometrically. Our results revealed that FMSP-nanoparticles produced a concentration dependent effect on the cancer cells, a dose of 1.25 µg/mL produced no significant effect on the cancer cell morphology and cell death, whereas dosages of 12.5 and 50 µg/mL resulted in significant nuclear augmentation, disintegration, chromatic condensation followed by dose dependent cell death. Our results demonstrate that FMSP-nanoparticles induce cell death in MCF-7 cells and may be a potential anti-cancer agent for breast cancer treatment.

  11. Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Bodvarsdottir, Sigridur K., E-mail: skb@hi.is [Cancer Research Laboratory, BioMedical Centre, Faculty of Medicine, University of Iceland, Vatnsmyrarvegi 16, 101 Reykjavik (Iceland); Steinarsdottir, Margret [Chromosome Laboratory, Department of Genetics and Molecular Medicine, Landspitali University Hospital, Reykjavik (Iceland); Bjarnason, Hordur; Eyfjord, Jorunn E. [Cancer Research Laboratory, BioMedical Centre, Faculty of Medicine, University of Iceland, Vatnsmyrarvegi 16, 101 Reykjavik (Iceland)

    2012-01-03

    In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and {gamma}-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.

  12. Dysfunctional telomeres in human BRCA2 mutated breast tumors and cell lines

    International Nuclear Information System (INIS)

    Bodvarsdottir, Sigridur K.; Steinarsdottir, Margret; Bjarnason, Hordur; Eyfjord, Jorunn E.

    2012-01-01

    In the present study the possible involvement of telomeres in chromosomal instability of breast tumors and cell lines from BRCA2 mutation carriers was examined. Breast tumors from BRCA2 mutation carriers showed significantly higher frequency of chromosome end-to-end fusions (CEFs) than tumors from non-carriers despite normal telomere DNA content. Frequent CEFs were also found in four different BRCA2 heterozygous breast epithelial cell lines, occasionally with telomere signal at the fusion point, indicating telomere capping defects. Extrachromosomal telomeric repeat (ECTR) DNA was frequently found scattered around metaphase chromosomes and interstitial telomere sequences (ITSs) were also common. Telomere sister chromatid exchanges (T-SCEs), characteristic of cells using alternative lengthening of telomeres (ALT), were frequently detected in all heterozygous BRCA2 cell lines as well as the two ALT positive cell lines tested. Even though T-SCE frequency was similar in BRCA2 heterozygous and ALT positive cell lines they differed in single telomere signal loss and ITSs. Chromatid type alterations were more prominent in the BRCA2 heterozygous cell lines that may have propensity for telomere based chromosome healing. Telomere dysfunction-induced foci (TIFs) formation, identified by co-localization of telomeres and γ-H2AX, supported telomere associated DNA damage response in BRCA2 heterozygous cell lines. TIFs were found in interphase nuclei, at chromosome ends, ITSs and ECTR DNA. In conclusion, our results suggest that BRCA2 has an important role in telomere stabilization by repressing CEFs through telomere capping and the prevention of telomere loss by replication stabilization.

  13. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

    International Nuclear Information System (INIS)

    Zhang, Zhiyu; Ma, Fang; Cai, Zhengdong; Zhang, Lijun; Hua, Yingqi; Jia, Xiaofang; Li, Jian; Hu, Shuo; Peng, Xia; Yang, Pengyuan; Sun, Mengxiong

    2010-01-01

    Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

  14. Enhancement of Radiation Effects by Ursolic Acid in BGC-823 Human Adenocarcinoma Gastric Cancer Cell Line.

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    Yang Yang

    Full Text Available Recent research has suggested that certain plant-derived polyphenols, i.e., ursolic acid (UA, which are reported to have antitumor activities, might be used to sensitize tumor cells to radiation therapy by inhibiting pathways leading to radiation therapy resistance. This experiment was designed to investigate the effects and possible mechanism of radiosensitization by UA in BGC-823 cell line from human adenocarcinoma gastric cancer in vitro. UA caused cytotoxicity in a dose-dependent manner, and we used a sub-cytotoxicity concentration of UA to test radioenhancement efficacy with UA in gastric cancer. Radiosensitivity was determined by clonogenic survival assay. Surviving fraction of the combined group with irradiation and sub-cytotoxicity UA significantly decreased compared with the irradiation group. The improved radiosensitization efficacy was associated with enhanced G2/M arrest, increased reactive oxygen species (ROS, down-regulated Ki-67 level and improved apoptosis. In conclusion, as UA demonstrated potent antiproliferation effect and synergistic effect, it could be used as a potential drug sensitizer for the application of radiotherapy.

  15. Cytotoxicity and morphological effects induced by carvacrol and thymol on the human cell line Caco-2.

    Science.gov (United States)

    Llana-Ruiz-Cabello, María; Gutiérrez-Praena, Daniel; Pichardo, Silvia; Moreno, F Javier; Bermúdez, José María; Aucejo, Susana; Cameán, Ana María

    2014-02-01

    Essential oils used as additives in the food industry due to its flavour, antimicrobial and antioxidant properties. Therefore, human can be exposed orally to these compounds through the ingestion of foods. In this sense, the present work aims to assess toxicological effects of oregano essential oil on the digestive tract. In concrete, the cytotoxic effects of two components of the oregano essential oils, carvacrol and thymol, and their mixture, on the intestinal cells line Caco-2 after 24 and 48 h of exposure are studied. The basal cytotoxicity endpoints assayed (total protein content, neutral red uptake and the tetrazolium salt reduction) and the annexin/propidium iodide staining indicated that carvacrol and the mixture carvacrol/thymol induced toxic effects. Moreover, a morphological study was performed in order to determine the ultrastructural cellular damages caused by these substances. The main morphological alterations were vacuolated cytoplasm, altered organelles and finally cell death. In addition, although no cytotoxic effects were recorded for thymol at any concentration and time of exposure, ultrastructural changes evidenced cellular damage such as lipid degeneration, mitochondrial damage, nucleolar segregation and apoptosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Performance comparison of digital microRNA profiling technologies applied on human breast cancer cell lines.

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    Erik Knutsen

    Full Text Available MicroRNA profiling represents an important first-step in deducting individual RNA-based regulatory function in a cell, tissue, or at a specific developmental stage. Currently there are several different platforms to choose from in order to make the initial miRNA profiles. In this study we investigate recently developed digital microRNA high-throughput technologies. Four different platforms were compared including next generation SOLiD ligation sequencing and Illumina HiSeq sequencing, hybridization-based NanoString nCounter, and miRCURY locked nucleic acid RT-qPCR. For all four technologies, full microRNA profiles were generated from human cell lines that represent noninvasive and invasive tumorigenic breast cancer. This study reports the correlation between platforms, as well as a more extensive analysis of the accuracy and sensitivity of data generated when using different platforms and important consideration when verifying results by the use of additional technologies. We found all the platforms to be highly capable for microRNA analysis. Furthermore, the two NGS platforms and RT-qPCR all have equally high sensitivity, and the fold change accuracy is independent of individual miRNA concentration for NGS and RT-qPCR. Based on these findings we propose new guidelines and considerations when performing microRNA profiling.

  17. Effects of oridonin nanosuspension on cell proliferation and apoptosis of human prostatic carcinoma PC-3 cell line

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2010-10-01

    Full Text Available Zhen Zhang, Xiumei Zhang, Wei Xue, Yuna YangYang, Derong Xu, Yunxue Zhao, Haiyan LouSchool of Medicine, Shandong University, Jinan, Republic of ChinaAbstract: This study aims to investigate the inhibitory effects of oridonin nanosuspension on human prostatic carcinoma PC-3 cell line in vitro. The PC-3 cells were incubated with increasing concentrations of oridonin solution and nanosuspensions for 12 hours, 24 hours, and 36 hours. MTT [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] assay was performed to measure cellular viability and investigate the effect of oridonin on cell growth of PC-3. Annexin V-FITC/PI staining method was used to determine the effect of oridonin by fluorescence microscope and flow cytometry, respectively. Nanosuspension on early apoptosis of PC-3 cells was also evaluated. Oridonin significantly inhibited the growth of PC-3 cells after 12 hours, 24 hours, and 36 hours of treatment in a dose-dependent manner (P < 0.05. Compared with the same concentration of oridonin solution, oridonin nanosuspension enhanced the inhibition ratio of proliferation. The observation of propidium iodide fluorescence staining confirmed the MTT assay results. The cell proportion of PC-3 at the G2/M phase in the nanosuspension treatment group was upregulated compared with that of the control and oridonin solution groups. Both oridonin solution and nanosuspension promoted the early apoptosis of PC-3 cells. Furthermore, while improving the ratio of early apoptosis, oridonin nanosuspensions also enhanced growth suppression, and induced apoptosis of PC-3 cells. This shows great potential in the treatment of androgen-independent carcinoma of prostate by oridonin nanosuspensions.Keywords: oridonin, nanosuspension, carcinoma of prostate, PC-3 cells, cell cycle, apoptosis

  18. [Different effects of anticancer drugs on two human thyroid cell lines with different stages of differentiation].

    Science.gov (United States)

    Yamanaka, T; Hishinuma, A

    1995-01-20

    We established two human thyroid tumor cell lines. One cell line (hPTC) was established from the tissue of a papillary thyroid carcinoma surgically excised from a 27-year-old female patient. The other cell line (hAG) was established from the tissue of an adenomatous goiter excised from a 59-year old female patient. Synthesis of cAMP by hPTC and hAG increased when they were stimulated by TSH. hPTC and hAG continued to divide as a monolayer in a tissue culture for three years and two years, respectively. We assessed the efficacy of anticancer drugs (doxorubicin:ADR, cisplatin:CDDP, nimustine:ACNU, bleomycin:BLM, cyclophosphamide:CPA, aclarubicin:ACR) with resard to hPTC. The hPTC cells were cultured in 24-well plates in the presence of the anticancer drugs for 48 hours, and the cellular DNA of the live cells was measured with diaminobenzoic acid. ADR had the lowest ED50 (0.029 mu g/ml) and the clinical blood concentration was 13.8 times that of the ED50. The clinical blood concentration divided by ED50 for the other anticancer drugs are, in order of higher values, 2.3 for CPA, 1.7 for BLM, 1.2 for CDDP, 0.5 for ACR, and less than 0.1 for ACNU. ADR showed time-independent effects since a 2-hour exposure of ADR to the hPTC cells resulted in the significant reduction of the cellular DNA content of the live cells even after 48 hours. The effects of the other anticancer drugs were time-dependent. We then studied the difference of the effects of ADR on hPTC and hAG. ED50 for hPTC was significantly low (0.035 mu g/ml) compared to that for hAG (0.460 mu g/ml). Since free radical formation is one of the major anticancer mechanisms of ADR the effects of free radicals on ED50's for hPTC and hAG were measured by adding glutathione (GSH), N-acetylcystein (NAC), buthionine sulfoximine (BSO), and alpha-tocopherol (alpha-toco) into the culture media. GSH catches up with free radicals in the extracellular fluid. NAC promotes production of GSH in the cytoplasm, but BSO interferes with

  19. Widespread molecular patterns associated with drug sensitivity in breast cancer cell lines, with implications for human tumors.

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    Chad J Creighton

    Full Text Available BACKGROUND: Recent landmark studies have profiled cancer cell lines for molecular features, along with measuring the corresponding growth inhibitory effects for specific drug compounds. These data present a tool for determining which subsets of human cancer might be more responsive to particular drugs. To this end, the NCI-DREAM-sponsored DREAM7: Drug Sensitivity Prediction Challenge (sub-challenge 1 set out to predict the sensitivities of 18 breast cancer cell lines to 31 previously untested compounds, on the basis of molecular profiling data and a training subset of cell lines. METHODS AND RESULTS: With 47 teams submitting blinded predictions, team Creighton scored third in terms of overall accuracy. Team Creighton's method was simple and straightforward, incorporated multiple expression data types (RNA-seq, gene array, RPPA, and incorporated all profiled features (not only the "best" predictive ones. As an extension of the approach, cell line data, from public datasets of expression profiling coupled with drug sensitivities (Barretina, Garnett, Heiser were used to "predict" the drug sensitivities in human breast tumors (using data from The Cancer Genome Atlas. Drug sensitivity correlations within human breast tumors showed differences by expression-based subtype, with many associations in line with the expected (e.g. Lapatinib sensitivity in HER2-enriched cancers and others inviting further study (e.g. relative resistance to PI3K inhibitors in basal-like cancers. CONCLUSIONS: Molecular patterns associated with drug sensitivity are widespread, with potentially hundreds of genes that could be incorporated into making predictions, as well as offering biological clues as to the mechanisms involved. Applying the cell line patterns to human tumor data may help generate hypotheses on what tumor subsets might be more responsive to therapies, where multiple cell line datasets representing various drugs may be used, in order to assess consistency of

  20. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

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    Clementina Sansone

    Full Text Available Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD, 2-trans,4-trans-octadienal (OD and 2-trans,4-trans-heptadienal (HD on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1 and Fas Associated Death Domain (FADD leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP. The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.

  1. δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    De Siervi, Adriana; Vazquez, Elba S; Rezaval, Carolina; Rossetti, María V; Batlle, Alcira M del [Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), Argentine National Research Council (CONICET), Department of Biological Chemistry, FCEN, University of Buenos Aires (Argentina)

    2002-01-01

    Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

  2. δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

    Directory of Open Access Journals (Sweden)

    del Batlle Alcira M

    2002-03-01

    Full Text Available Abstract Background Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA and porphobilinogen (PBG. ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. Results We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

  3. δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

    International Nuclear Information System (INIS)

    De Siervi, Adriana; Vazquez, Elba S; Rezaval, Carolina; Rossetti, María V; Batlle, Alcira M del

    2002-01-01

    Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations

  4. Efficacy of the HSP90 inhibitor 17-AAG in human glioma cell lines and tumorigenic glioma stem cells.

    Science.gov (United States)

    Sauvageot, Claire Marie-Elisabeth; Weatherbee, Jessica Leigh; Kesari, Santosh; Winters, Susan Elizabeth; Barnes, Jessica; Dellagatta, Jamie; Ramakrishna, Naren Raj; Stiles, Charles Dean; Kung, Andrew Li-Jen; Kieran, Mark W; Wen, Patrick Yung Chih

    2009-04-01

    Glioblastoma multiforme (GBM) arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Studies to date with single-agent targeted molecular therapy have revealed only modest effects in attenuating the growth of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat-shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a defined group of client proteins, many of which are deregulated in GBM. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well-characterized HSP90 inhibitor that should be able to target many of the aberrant signal transduction pathways in GBM. We assessed the ability of 17-AAG to inhibit the growth of glioma cell lines and glioma stem cells both in vitro and in vivo and assessed its ability to synergize with radiation and/or temozolomide, the standard therapies for GBM. Our results reveal that 17-AAG is able to inhibit the growth of both human glioma cell lines and glioma stem cells in vitro and is able to target the appropriate proteins within these cells. In addition, 17-AAG can inhibit the growth of intracranial tumors and can synergize with radiation both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide in any of our models of gliomas. Our results suggest that HSP90 inhibitors like 17-AAG may have therapeutic potential in GBM, either as a single agent or in combination with radiation.

  5. Transfection of wild type ADVP53 gene into human brain tumor cell lines has a radiosensitizing effect independent of apoptosis

    International Nuclear Information System (INIS)

    Geng, L.; Walter, S; Vaughan, A.T.M.

    1997-01-01

    Purpose: Despite attempts with a variety of therapeutic approaches there has been little impact on the survival of patients with Glioblastoma multiforme, with median survivals reported of approximately 12 months. In this study a replication restricted adenovirus vector is used to transfer the wild type p53 gene into two cell lines derived from a human astrocytoma U87MG or glioblastoma T98G, to determine its ability to act as a radiosensitizer in conjunction with conventional radiotherapy. Methods: An adenovirus vector containing the human wild type p53 (Advp53) gene was used in addition to a control vector containing the β-galactosidase (Advγgal) reporter gene. To achieve cellular incorporation both vectors were incubated with cells for 30 minutes - washed and returned to culture. The successful incorporation of each vector was determined by either a p53 assay using either a western blotting or flow cytometry techniques, or specific staining for β-galactosidase activity. The presence of each vector was assayed until the constructs were eliminated from the cell. To determine the effects of these vectors on cell survival sufficient vector was added to produce a measurable reduction in clonogenic survival and this value was used in subsequent irradiation experiments. To determine the ability of wild type p53 to induce apoptosis the cells were examined from 1 to 5 days after irradiation by H and E staining for the characteristic morphology indicating an apoptotic process. Results: Both the Advp53 and Advβgal vectors were successfully incorporated into each cell line. Expression of each gene was reduced to approximately half by 5 days and virtually eliminated by 15 days after transfection in both lines. At the doses used the wild type Advp53 adenovirus was toxic to both cell lines giving surviving fractions between 39-74%. When this toxicity was taken into account the presence of the Advp53 gene had a radiosensitizing effect in each cell line. To determine the

  6. Heterogeneity in 2-deoxy-D-glucose-induced modifications in energetics and radiation responses of human tumor cell lines

    International Nuclear Information System (INIS)

    Dwarkanath, Bilikere S.; Zolzer, Frido; Chandana, Sudhir; Bauch, Thomas; Adhikari, Jawahar S.; Muller, Wolfgang U.; Streffer, Christian; Jain, Viney

    2001-01-01

    Purpose: The glucose analog and glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), has been shown to differentially enhance the radiation damage in tumor cells by inhibiting the postirradiation repair processes. The present study was undertaken to examine the relationship between 2-DG-induced modification of energy metabolism and cellular radioresponses and to identify the most relevant parameter(s) for predicting the tumor response to the combined treatment of radiation + 2-DG. Methods and Materials: Six human tumor cell lines (glioma: BMG-1 and U-87, squamous cell carcinoma: 4451 and 4197, and melanoma: MeWo and Be-11) were investigated. Cells were exposed to 2 Gy of Co-60 γ-rays or 250 kVP X-rays and maintained under liquid-holding conditions 2-4 h to facilitate repair. 2-DG (5 mM, equimolar with glucose) that was added at the time of irradiation was present during the liquid holding. Glucose utilization, lactate production (enzymatic assays), and adenine nucleotides (high performance liquid chromatography and capillary isotachophoresis) were investigated as parameters of energy metabolism. Induction and repair of DNA damage (comet assay), cytogenetic damage (micronuclei formation), and cell death (macrocolony assay) were analyzed as parameters of radiation response. Results: The glucose consumption and lactate production of glioma cell lines (BMG-1 and U-87) were nearly 2-fold higher than the squamous carcinoma cell lines (4197 and 4451). The ATP content varied from 3.0 to 6.5 femto moles/cell among these lines, whereas the energy charge (0.86-0.90) did not show much variation. Presence of 2-DG inhibited the rate of glucose usage and glycolysis by 30-40% in glioma cell lines and by 15-20% in squamous carcinoma lines, while ATP levels reduced by nearly 40% in all the four cell lines. ATP:ADP ratios decreased to a greater extent (∼40%) in glioma cells than in squamous carcinoma 4451 and MeWo cells; in contrast, presence of 2-DG reduced ADP:AMP ratios by 3-fold in

  7. Apoptosis induced by GanoPoly in human gastric cancer cell line ...

    African Journals Online (AJOL)

    In order to investigate polysaccharide effect on the cultured human gastric cancer cells (SGC7901), DNA ladder, flow cytometry and western blot were used to examine the morpholog, proliferation and apoptosis of human gastric cancer SGC-7901 cells when they were affected by polysaccharide. Results show that ...

  8. Sensitivity to ionizing radiation and chemotherapeutic agents in gemcitabine-resistant human tumor cell lines

    NARCIS (Netherlands)

    van Bree, Chris; Castro Kreder, Natasja; Loves, Willem J. P.; Franken, Nicolaas A. P.; Peters, Godefridus J.; Haveman, Jaap

    2002-01-01

    Purpose: To determine cross-resistance to anti-tumor treatments in 2',2'difluorodeoxycytidine (dFdC, gemcitabine)-resistant human tumor cells. Methods and Materials: Human lung carcinoma cells SW-1573 (SWp) were made resistant to dFdC (SWg). Sensitivity to cisplatin (cDDP), paclitaxel,

  9. In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

    Science.gov (United States)

    Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

    2005-06-01

    Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the

  10. Generation of a Nrf2 homozygous knockout human embryonic stem cell line using CRISPR/Cas9

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    So-Jung Kim

    2017-03-01

    Full Text Available Nuclear factor erythroid 2-related factor 2 (NFE2L2 or Nrf2 is a well-known transcription factor that regulates the expression of a large number of anti-oxidant genes in mammalian cells (J.H. Kim et al., 2014. Here, we generated a homozygous Nrf2 knockout human embryonic stem cell (hESC line, H9Nrf2KO-A13, using the CRISPR/Cas9 genome editing method. The Nrf2 homozygous knockout H9 cell line maintains pluripotency, differentiation potential into three germ layers, and a normal karyotype.

  11. Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

    Science.gov (United States)

    Zapata-Linares, Natalia; Rodriguez, Saray; Mazo, Manuel; Abizanda, Gloria; Andreu, Enrique J; Barajas, Miguel; Prosper, Felipe; Rodriguez-Madoz, Juan R

    2016-01-01

    In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Characterization of the response of a human breast carcinoma cell line (T-47D) to radiation and chemotherapeutic agents

    International Nuclear Information System (INIS)

    Prager, A.; Ben-Hur, E.; Riklis, E.

    1981-01-01

    The response of a human breast carcinoma cell line (T-47D) to various antitumour agents, gamma irradiation, UV light and heat was studied, using the colony-forming ability technique. Combinations of radiation with drugs and heat were also tested. The resulting survival curves corresponded to one of five patterns: simple exponential, biphasic exponential, threshold exponential, exponential plateau and ineffectual. Whereas the cells were particularly sensitive to gamma irradiation, the response to UV light was normal. The patient from whom this cell line originated did not respond to METHOTREXATEsup(R) therapy. The in vitro results correlated with this observation. (author)

  13. Regulation of cytochrome P4501A1 expression by hyperoxia in human lung cell lines: Implications for hyperoxic lung injury

    International Nuclear Information System (INIS)

    Bhakta, Kushal Y.; Jiang, Weiwu; Couroucli, Xanthi I.; Fazili, Inayat S.; Muthiah, Kathirvel; Moorthy, Bhagavatula

    2008-01-01

    Supplemental oxygen, used to treat pulmonary insufficiency in newborns, contributes to the development of bronchopulmonary dysplasia (BPD). Cytochrome P4501A enzymes are induced by hyperoxia in animal models, but their role in human systems is unknown. Here we investigated the molecular mechanisms of induction of CYP1A1 by hyperoxia in human lung cell lines. Three human lung cell lines were exposed to hyperoxia (95% O2) for 0-72 h, and CYP1A1 activities, apoprotein contents, and mRNA levels were determined. Hyperoxia significantly induced CYP1A1 activity and protein contents (2-4 fold), and mRNA levels (30-40 fold) over control in each cell line. Transfection of a CYP1A1 promoter/luciferase reporter construct, followed by hyperoxia (4-72 h), showed marked (2-6 fold) induction of luciferase expression. EMSA and siRNA experiments strongly suggest that the Ah receptor (AHR) is involved in the hyperoxic induction of CYP1A1. MTT reduction assays showed attenuation of cell injury with the CYP1A1 inducer beta-naphthoflavone (BNF). Our results strongly suggest that hyperoxia transcriptionally activates CYP1A1 expression in human lung cell lines by AHR-dependent mechanisms, and that CYP1A1 induction is associated with decreased toxicity. This novel finding of induction of CYP1A1 in the absence of exogenous AHR ligands could lead to novel interventions in the treatment of BPD

  14. The human vascular endothelial cell line HUV-EC-C harbors the integrated HHV-6B genome which remains stable in long term culture

    OpenAIRE

    Shioda, Setsuko; Kasai, Fumio; Ozawa, Midori; Hirayama, Noriko; Satoh, Motonobu; Kameoka, Yousuke; Watanabe, Ken; Shimizu, Norio; Tang, Huamin; Mori, Yasuko; Kohara, Arihiro

    2017-01-01

    Human herpes virus 6 (HHV-6) is a common human pathogen that is most often detected in hematopoietic cells. Although human cells harboring chromosomally integrated HHV-6 can be generated in vitro, the availability of such cell lines originating from in vivo tissues is limited. In this study, chromosomally integrated HHV-6B has been identified in a human vascular endothelial cell line, HUV-EC-C (IFO50271), derived from normal umbilical cord tissue. Sequence analysis revealed that the viral gen...

  15. Identification and characterization of cells with cancer stem cell properties in human primary lung cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Ping Wang

    Full Text Available Lung cancer (LC with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC, large cell carcinoma (LCC, squamous cell carcinoma (SCC and adenocarcinoma (AC. We identified a small population of cells strongly positive for CD44 (CD44(high and a main population which was either weakly positive or negative for CD44 (CD44(low/-. Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44(highCD90(+ sub-population. Moreover, these CD44(highCD90(+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44(highCD90(+ population a good candidate for the lung CSCs. Both CD44(highCD90(+ and CD44(highCD90(- cells in the PLCCL derived from SCC formed spheroids, whereas the CD44(low/- cells were lacking this potential. These results indicate that CD44(highCD90(+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44(high sub-population.

  16. Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

    Science.gov (United States)

    Suo, Zhenhe; Munthe, Else; Solberg, Steinar; Ma, Liwei; Wang, Mengyu; Westerdaal, Nomdo Anton Christiaan; Kvalheim, Gunnar; Gaudernack, Gustav

    2013-01-01

    Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44high) and a main population which was either weakly positive or negative for CD44 (CD44low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44highCD90+ sub-population. Moreover, these CD44highCD90+ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44highCD90+ population a good candidate for the lung CSCs. Both CD44highCD90+ and CD44highCD90− cells in the PLCCL derived from SCC formed spheroids, whereas the CD44low/− cells were lacking this potential. These results indicate that CD44highCD90+ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44high sub-population. PMID:23469181

  17. Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

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    Felix Hart

    2017-05-01

    Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

  18. 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.

  19. Differential response of human and rodent cell lines to chemical inhibition of the repair of potentially lethal damage

    Energy Technology Data Exchange (ETDEWEB)

    Little, J.B.; Ueno, A.M.; Dahlberg, W.K.

    1989-07-01

    We have examined the effects of several classes of metabolic inhibitors on the repair of potentially lethal damage in density-inhibited cultures of two rodent and two human cell systems which differ in their growth characteristics. Aphidicolin, 1-..beta..-D-arabinofuranosylcytosine (ara-C) and hydroxyurea showed no effect on PLD repair, whereas the effects of 9-..beta..-D-arabinofuranosyladenine (ara-A) and 3-aminobenzamide (3-AB) were cell line dependent. For example, 3-AB suppressed PLD repair almost completely in CHO cells, but showed no inhibitory effects in human diploid fibroblasts. These results indicate that inhibitors of DNA replication and poly(ADP-ribose) synthesis are not efficient inhibitors of cellular recovery in irradiated cells and, moreover, that such effects may be cell line dependent.

  20. Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines.

    Science.gov (United States)

    Muñoz, Jorge; Lázcoz, Paula; Inda, María Mar; Nistal, Manuel; Pestaña, Angel; Encío, Ignacio J; Castresana, Javier S

    2004-05-01

    Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.31 and 10q25.3-26.1, respectively, which have been found frequently altered in other kinds of neoplasms. We screened both genes for homozygous deletions in 45 primary neuroblastic tumors and 12 neuroblastoma cell lines. Expression of these genes in cell lines was assessed by RT-PCR analysis. We could detect 2 of 41 (5%) primary tumors harboring PTEN homozygous deletions. Three of 41 (7%) primary tumors and 2 of 12 cell lines presented homozygous losses at the g14 STS on the DMBT1 locus. All cell lines analyzed expressed PTEN, but lack of DMBT1 mRNA expression was detected in 2 of them. We tried to see whether epigenetic mechanisms, such as aberrant promoter hypermethylation, had any role in DMBT1 silencing. The 2 cell lines lacking DMBT1 expression were treated with 5-aza-2'-deoxycytidine; DMBT1 expression was restored in only one of them (MC-IXC). From our work, we can conclude that PTEN and DMBT1 seem to contribute to the development of a small fraction of neuroblastomas, and that promoter hypermethylation might have a role in DMBT1 gene silencing. Copyright 2004 Wiley-Liss, Inc.

  1. Cytotoxicity effect of Zataria multiflora Boiss. on two human colon carcinoma cell lines

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    F. Sharififar

    2017-10-01

    Full Text Available Background and objectives: Natural products are one of the major sources for investigations of novel medicines. Zataria multiflora Boiss (ZM has shown pharmacological activities especially in gastrointestinal tract; however, there are limited studies about its cytotoxicity effects. In this study, the effect of Zataria multiflora was examined on two colon cancer cell lines (SW-48 and HT-29. Methods: Hydro-alcoholic extract of ZM and its fractions including chloroform, petroleum ether and methanol extract were prepared by warm maceration method. Different concentrations were prepared and examined on SW-48 and HT-29 cell lines using 2-(4, 5-dimethylthiazol-2-yl 2, 5-diphenyltetrazolium bromide (MTT assay. Results: The results of the present study have shown the cytotoxic effect of some fractions of ZM. The most considerable cytotoxic effect was shown against HT-29 cell line. Also, total ZM extract and the petroleum ether fraction demonstrated cytotoxic effects with IC50 values of 44.22 and 33.42 µg/ml on SW-48 and HT-29 cell lines, respectively. Conclusion: Zataria multiflora was cytotoxic to against colon cancer cell lines HT-29 and SW-48.

  2. Genetic analysis of human traits in vitro: drug response and gene expression in lymphoblastoid cell lines.

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    Edwin Choy

    2008-11-01

    Full Text Available Lymphoblastoid cell lines (LCLs, originally collected as renewable sources of DNA, are now being used as a model system to study genotype-phenotype relationships in human cells, including searches for QTLs influencing levels of individual mRNAs and responses to drugs and radiation. In the course of attempting to map genes for drug response using 269 LCLs from the International HapMap Project, we evaluated the extent to which biological noise and non-genetic confounders contribute to trait variability in LCLs. While drug responses could be technically well measured on a given day, we observed significant day-to-day variability and substantial correlation to non-genetic confounders, such as baseline growth rates and metabolic state in culture. After correcting for these confounders, we were unable to detect any QTLs with genome-wide significance for drug response. A much higher proportion of variance in mRNA levels may be attributed to non-genetic factors (intra-individual variance--i.e., biological noise, levels of the EBV virus used to transform the cells, ATP levels than to detectable eQTLs. Finally, in an attempt to improve power, we focused analysis on those genes that had both detectable eQTLs and correlation to drug response; we were unable to detect evidence that eQTL SNPs are convincingly associated with drug response in the model. While LCLs are a promising model for pharmacogenetic experiments, biological noise and in vitro artifacts may reduce power and have the potential to create spurious association due to confounding.

  3. Estrogenic activity of lambda-cyhalothrin in the MCF-7 human breast carcinoma cell line.

    Science.gov (United States)

    Zhao, Meirong; Zhang, Ying; Liu, Weiping; Xu, Chao; Wang, Lumei; Gan, Jianying

    2008-05-01

    Synthetic pyrethroids are widely used in both agricultural and urban environments for insect control. Lambda-cyhalothrin (LCT) is one of the most common pyrethroids and is used mainly for controlling mosquitoes, fleas, cockroaches, flies, and ants around households. Previous studies have addressed the environmental behaviors and acute toxicities of LCT, but little is known about its chronic toxicity, such as estrogen-like activity. In the present study, the estrogenic potential of LCT was evaluated using the MCF-7 human breast carcinoma cell line. The in vitro E-screen assay showed that 10(-7) M LCT could significantly promote MCF-7 cell proliferation, with a relative proliferative effect ratio of 45%. The cell proliferation induced by LCT could be blocked completely, however, by the addition of 10(-9) M of the estrogen receptor (ER)-antagonist ICI 182,780. The semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) results showed that the Trefoil factor 1 (pS2) and progesterone receptor gene expression were up-regulated by 10(-7) M LCT for 2- and 1.5-fold, respectively. On the other hand, RT-PCR, Western blot analysis, and immunofluorescent assay demonstrated that LCT significantly repressed the mRNA and protein expression levels of ERalpha and ERbeta. These observations indicate that LCT possesses estrogenic properties and may function as a xenoestrogen, likely via a mechanism similar to that of 17beta-estradiol. The endocrine-disruption potential of LCT should be considered when assessing the safety of this compound in sensitive environmental compartments.

  4. Isolation and characterization of a neoplastic epithelial cell line derived from irradiated human submaxillary gland

    International Nuclear Information System (INIS)

    Shirasuna, Kanemitsu; Sato, Mitsunobu; Yura, Yoshiaki; Yanagawa, Tetuo; Kubo, Kazuko

    1979-01-01

    Submaxillary tissues taken from a patient whose oral base was irradiated for squamous cell carcinoma were cultured in order to isolate transformed epithelial cells in vitro. The cells showed a fine structure similar to an intermediate duct cell. When they were transplanted in nude mice, salivary tumors developed. It is epidemiologically known that irradiation induces salivary tumors. In this study, the risk of inducement was revealed and a salivary epithelial cell line was used as a model for the analysis of salivary tumors. (Ichikawa, K.)

  5. In vitro response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells exposed to 60Co at single fraction

    International Nuclear Information System (INIS)

    Andrade, Lidia Maria; Campos, Tarcisio Passos Ribeiro de; Leite, M.F.; Goes, A.M.

    2005-01-01

    Radiotherapy using gamma rays is a common modality of breast cancer treatment. The aim of this research is to investigate the biological response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells (PBMC) exposed in vitro to 60 Co irradiation at a single fraction of 10 Gy, 25 Gy and 50 Gy doses at 136,4 cGy.min -1 rate. Cells were irradiated at room temperature by the Theratron 80 radiotherapy system. Biological response was evaluated through cellular viability using MTT assay and nucleus damages visualized by Propidium Iodide assay and electrophoresis agarose gel after gamma irradiation. Nucleus damages induced by 60 Co irradiation were compared to damage caused by cell exposure to 10% methanol. The 50 Gy dose of irradiation did not stimulate nucleus damages at the same level as that affected by 10% methanol induction in the MDAMB-231. Further studies are necessary to understand these mechanisms in the MDAMB-231 human breast carcinoma cell line.(author)

  6. In vitro response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells exposed to {sup 60}Co at single fraction

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Lidia Maria; Campos, Tarcisio Passos Ribeiro de [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Engenharia Nuclear]. E-mail: lidia.andrade@unifenas.br; Leite, M.F. [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Fisiologia e Biofisica; Goes, A.M. [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia

    2005-10-15

    Radiotherapy using gamma rays is a common modality of breast cancer treatment. The aim of this research is to investigate the biological response of the human breast cancer cell line MDAMB-231 and human peripheral blood mononuclear cells (PBMC) exposed in vitro to {sup 60} Co irradiation at a single fraction of 10 Gy, 25 Gy and 50 Gy doses at 136,4 cGy.min{sup -1} rate. Cells were irradiated at room temperature by the Theratron 80 radiotherapy system. Biological response was evaluated through cellular viability using MTT assay and nucleus damages visualized by Propidium Iodide assay and electrophoresis agarose gel after gamma irradiation. Nucleus damages induced by {sup 60} Co irradiation were compared to damage caused by cell exposure to 10% methanol. The 50 Gy dose of irradiation did not stimulate nucleus damages at the same level as that affected by 10% methanol induction in the MDAMB-231. Further studies are necessary to understand these mechanisms in the MDAMB-231 human breast carcinoma cell line.(author)

  7. Development and Characterisation of a Human Chronic Skin Wound Cell Line-Towards an Alternative for Animal Experimentation.

    Science.gov (United States)

    Caley, Matthew; Wall, Ivan B; Peake, Matthew; Kipling, David; Giles, Peter; Thomas, David W; Stephens, Phil

    2018-03-27

    Background : Chronic skin wounds are a growing financial burden for healthcare providers, causing discomfort/immobility to patients. Whilst animal chronic wound models have been developed to allow for mechanistic studies and to develop/test potential therapies, such systems are not good representations of the human chronic wound state. As an alternative, human chronic wound fibroblasts (CWFs) have permitted an insight into the dysfunctional cellular mechanisms that are associated with these wounds. However, such cells strains have a limited replicative lifespan and therefore a limited reproducibility/usefulness. Objectives : To develop/characterise immortalised cell lines of CWF and patient-matched normal fibroblasts (NFs). Methods and Results : Immortalisation with human telomerase resulted in both CWF and NF proliferating well beyond their replicative senescence end-point (respective cell strains senesced as normal). Gene expression analysis demonstrated that, whilst proliferation-associated genes were up-regulated in the cell lines (as would be expected), the immortalisation process did not significantly affect the disease-specific genotype. Immortalised CWF (as compared to NF) also retained a distinct impairment in their wound repopulation potential (in line with CWF cell strains). Conclusions : These novel CWF cell lines are a credible animal alternative and could be a valuable research tool for understanding both the aetiology of chronic skin wounds and for therapeutic pre-screening.

  8. Radiosensitivity of mesothelioma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

    1996-10-01

    The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

  9. Comparison of the multi-drug resistant human hepatocellular carcinoma cell line Bel-7402/ADM model established by three methods

    Directory of Open Access Journals (Sweden)

    Zhong Xingguo

    2010-08-01

    Full Text Available Abstract Background To compare the biological characteristics of three types of human hepatocellular carcinoma multi-drug resistant cell sub-lines Bel-7402/ADM models established by three methods. Methods Established human hepatocellular carcinoma adriamycin (ADM multi-drug resistant cell sub-lines models Bel-7402/ADMV, Bel-7402/ADML and Bel-7402/ADMS by three methods of in vitro concentration gradient increased induction, nude mice liver-implanted induction and subcutaneous-implanted induction respectively. Phase contrast microscopy was used to observe the cells and the MTT (methyl thiazolyl tetrazolium method was used to detect drug resistance of the three different sub-lines of cells. Results The three groups of drug resistant cells, Bel-7402/ADMV, Bel-7402/ADML and Bel-7402/ADMS generated cross-resistance to ADM and CDDP (cis-Diaminedichloroplatinum, but showed a significant difference in resistance to Bel-7402 IC50 value (P V, 46 h (Bel-7402/ADML, and 45 h (Bel-7402/ADMS. The excretion rates of ADM were significantly increased compared with the parent cell (34.14% line and were 81.06% (Bel-7402/ADMV, 66.56% (Bel-7402/ADML and 61.56% (Bel-7402/ADMS. Expression of P-gp and MRP in the three groups of resistant cells was significantly enhanced (P P > 0.05. Conclusions Stable resistance was involved in the resistant cell line model established by the above three methods. Liver implantation was a good simulation of human hepatocellular and proved to be an ideal model with characteristics similar to human hepatocellular biology and the pharmacokinetics of anticancer drugs.

  10. Effects of currently used pesticides in the AhR-CALUX assay: comparison between the human TV101L and the rat H4IIE cell line

    DEFF Research Database (Denmark)

    Long, M.; Laier, Peter; Vinggaard, Anne

    2003-01-01

    TV101L hepatoma cell lines. In comparison the results indicated that the rat H4IIE cell line is more sensitive than the human TV101L for detection of TCDD inducing AhR-CALUX activity. The pesticides iprodione, chlorpyrifos and prochloraz showed dose-dependent AhR agonistic effects in both cell lines...

  11. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

    Energy Technology Data Exchange (ETDEWEB)

    Katika, Madhumohan R. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Hendriksen, Peter J.M. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Shao, Jia [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Loveren, Henk van [Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Peijnenburg, Ad, E-mail: ad.peijnenburg@wur.nl [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands)

    2012-10-01

    Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

  12. Down-regulation of HSP40 gene family following OCT4B1 suppression in human tumor cell lines

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Mirzaei

    2016-02-01

    Full Text Available Objective(s: The OCT4B1, as one of OCT4 variants, is expressed in cancer cell lines and tissues more than other variants and plays an important role in apoptosis and stress (heat shock protein pathways. The present study was designed to determine the effects of OCT4B1 silencing on expressional profile of HSP40 gene family expression in three different human tumor cell lines. Materials and Methods: The OCT4B1 expression was suppressed by specific siRNA transfection in AGS (gastric adenocarcinoma, 5637 (bladder tumor and U-87MG (brain tumor cell lines employing Lipofectamine reagent. Real-time PCR array technique was employed for RNA qualification. The fold changes were calculated using RT2 Profiler PCR array data analysis software version 3.5. Results: Our results indicated that fifteen genes (from 36 studied genes were down-regulated and two genes (DNAJC11 and DNAJC5B were up-regulated in all three studied tumor cell lines by approximately more than two folds. The result of other studied genes (19 genes showed different expressional pattern (up or down-expression based on tumor cell lines. Conclusion: According to the findings of the present study, we may suggest that there is a direct correlation between OCT4B1 expression in tumor cell lines (and tissues and HSP40 family gene expressions to escape from apoptosis and cancer expansion.

  13. Anti-Cancer Activity of Methanol Extracts of Cichorium Intybus on Human Breast Cancer SKBR3 Cell Line

    Directory of Open Access Journals (Sweden)

    Reza Mehrandish

    2016-11-01

    Full Text Available Background Breast cancer is the most prevalent cancer and the second cause of death among women around the world. In many cancers, including breast cancer, Fatty acid synthase (FASN gene expression is increased significantly. In breast cancer cell lines, expression of FASN is higher in HER2 positive cell line like SKBR3 than the others. FASN is the key enzyme for fatty acid synthesis de novo pathway and it is producing palmitate which is necessary for cell membrane formation. Cichorium intybus is a medicinal plant that effectively leads to inhibition of fatty acid synthase and thus reduces the percentage of survival of cancer cell lines. Objectives The aim of this study was to evaluate the effect of methanol extract of Chicorium intybus root on percentage of survival in SKBR3 cell line. Methods Human breast cancer SKBR3 cell line was cultured in DMEM medium. Methanol extract of Cichorium intybus root was extracted and different dilutions (200, 300, 400, 500 and 600µg/mL were added to cell culture. Cell viability was quantitated by MTT assay after 24, 48 and 72 hours. Results Cichorium intybus could decrease cell viability. The effects of extract on cell viability were observed after 24, 48 and 72 hours on SKBR3 cell line and IC50 was 800, 400 and 300 after 24, 48 and 72 hours of treatment, respectively. Conclusions Our study shows that methanol extract of Cichorium intybus has cytotoxic effects on tumor cells. This is a pilot work for further evaluation in the future.

  14. Production of coagulation factor VII in human cell lines Sk-Hep-1 and HKB-11.

    Science.gov (United States)

    Corrêa de Freitas, Marcela Cristina; Bomfim, Aline de Sousa; Mizukami, Amanda; Picanço-Castro, Virgínia; Swiech, Kamilla; Covas, Dimas Tadeu

    2017-09-01

    Recombinant factor VII (rFVII) is the main therapeutic choice for hemophilia patients who have developed inhibitory antibodies against conventional treatments (FVIII and FIX). Because of the post-translational modifications, rFVII needs to be produced in mammalian cell lines. In this study, for the first time, we have shown efficient rFVII production in HepG2, Sk-Hep-1, and HKB-11 cell lines. Experiments in static conditions for a period of 96 h showed that HepG2-FVII produced the highest amounts of rhFVII, with an average of 1843 ng/mL. Sk-hep-1-FVII cells reached a maximum protein production of 1432 ng/mL and HKB-11-FVII cells reached 1468 ng/mL. Sk-Hep-1-rFVII and HKB-11-rFVII were selected for the first step of scale-up. Over 10 days of spinner flask culture, HKB-11 and SK-Hep-1 cells showed a cumulative production of rFVII of 152 μg and 202.6 μg in 50 mL, respectively. Thus, these human cell lines can be used for an efficient production of recombinant FVII. With more investment in basic research, human cell lines can be optimized for the commercial production of different bio therapeutic proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Structure-cytotoxicity relationships of a series of natural and semi-synthetic simple coumarins as assessed in two human tumour cell lines

    NARCIS (Netherlands)

    Kolodziej, H; Kayser, O; Woerdenbag, HJ; vanUden, W; Pras, N

    1997-01-01

    The cytotoxicity of 22 natural and semi-synthetic simple coumarins 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 > 100 mu M, following a continuous (96h)

  16. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...

  17. Decreased helenalin-induced cytotoxicity by flavonoids from Arnica as studied in a human lung carcinoma cell line

    NARCIS (Netherlands)

    Woerdenbag, HJ; Merfort, [No Value; Schmidt, TJ; Passreiter, CM; Willuhn, G; vanUden, W; Pras, N; Konings, AWT

    1995-01-01

    The effect of the flavones apigenin, luteolin, hispidulin and eupafolin, and of the flavonols kaempferol, quercetin, 6-methoxykaempferol and patuletin from Amica spp, on the cytotoxicity of the sesquiterpene lactone helenalin was studied in the human lung carcinoma cell line GLC(4) using the

  18. Cellular gene expression upon human immunodeficiency virus type 1 infection of CD4(+)-T-cell lines

    NARCIS (Netherlands)

    van 't Wout, Angélique B.; Lehrman, Ginger K.; Mikheeva, Svetlana A.; O'Keeffe, Gemma C.; Katze, Michael G.; Bumgarner, Roger E.; Geiss, Gary K.; Mullins, James I.

    2003-01-01

    The expression levels of approximately 4,600 cellular RNA transcripts were assessed in CD4(+)-T-cell lines at different times after infection with human immunodeficiency virus type 1 strain BRU (HIV-1(BRU)) using DNA microarrays. We found that several classes of genes were inhibited by HIV-1(BRU)

  19. The neonicotinoid insecticide Clothianidin adversely affects immune signaling in a human cell line.

    Science.gov (United States)

    Di Prisco, Gennaro; Iannaccone, Marco; Ianniello, Flora; Ferrara, Rosalba; Caprio, Emilio; Pennacchio, Francesco; Capparelli, Rosanna

    2017-10-18

    Clothianidin is a widely used neonicotinoid insecticide, which is a potent agonist of the nicotinic acetylcholine receptor in insects. This neurotoxic compound has a negative impact on insect immunity, as it down-regulates the activation of the transcription factor NF-κB. Given the evolutionary conserved role of NF-κB in the modulation of the immune response in the animal kingdom, here we want to assess any effect of Clothianidin on vertebrate defense barriers. In presence of this neonicotinoid insecticide, a pro-inflammatory challenge with LPS on the human monocytic cell line THP-1 results both in a reduced production of the cytokine TNF-α and in a down-regulation of a reporter gene under control of NF-κB promoter. This finding is corroborated by a significant impact of Clothianidin on the transcription levels of different immune genes, characterized by a core disruption of TRAF4 and TRAF6 that negatively influences NF-κB signaling. Moreover, exposure to Clothianidin concurrently induces a remarkable up-regulation of NGFR, which supports the occurrence of functional ties between the immune and nervous systems. These results suggest a potential risk of immunotoxicity that neonicotinoids may have on vertebrates, which needs to be carefully assessed at the organism level.

  20. Correlations between RNA and protein expression profiles in 23 human cell lines

    Directory of Open Access Journals (Sweden)

    Pontén Fredrik

    2009-08-01

    Full Text Available Abstract Background The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays and protein profiles (determined immunohistochemically in tissue microarrays of 1066 gene products in 23 human cell lines. Results A high mean correlation coefficient (0.52 was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Conclusion Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.

  1. Tumorigenicity and Validity of Fluorescence Labelled Mesenchymal and Epithelial Human Oral Cancer Cell Lines in Nude Mice

    Directory of Open Access Journals (Sweden)

    Wei Xin Cai

    2016-01-01

    Full Text Available Tumorigenicity and metastatic activity can be visually monitored in cancer cells that were labelled with stable fluorescence. The aim was to establish and validate local and distant spread of subcutaneously previously injected fluorescence transduced human tongue cancer cell lines of epithelial and mesenchymal phenotype in nude mice. A total of 32 four-week-old male athymic Balb/c nude mice were randomly allocated into 4 groups (n=8. A single dose of 0.3 mL PBS containing 1 × 107 of four different cancer cell-lines (UM1, UM1-GFP, UM2, and UM2-RFP was injected subcutaneously into the right side of their posterolateral back. Validity assessment of the labelled cancer cells’ tumorigenicity was assessed by physical examination, imaging, and histology four weeks after the injection. The tumor take rate of cancer cells was similar in animals injected with either parental or transduced cancer cells. Transduced cancer cells in mice were easily detectable in vivo and after cryosection using fluorescent imaging. UM1 cells showed increased tumor take rate and mean tumor volume, presenting with disorganized histopathological patterns. Fluorescence labelled epithelial and mesenchymal human tongue cancer cell lines do not change in tumorigenicity or cell phenotype after injection in vivo.

  2. Derivation of the human induced pluripotent stem cell line MUi017-A from a patient with homozygous Hemoglobin Constant Spring

    Directory of Open Access Journals (Sweden)

    Wasinee Wongkummool

    2017-04-01

    Full Text Available Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52 year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling.

  3. Establishment of a high production system for AIDS retroviruses with a human T-leukemic cell line Molt-4

    International Nuclear Information System (INIS)

    Koyanagi, Yoshio; Harada, Shinji; Yamamoto, Naoki

    1986-01-01

    A cell culture system was developed for the continuous and efficient production of acquired immune deficiency syndrome (AIDS) retrovirus. After infection of a human T-cell line Molt-4 with HTLV-III and LAV the cells grow permanently and produce large amounts of virus continuously. The yields of production of virus were assessed either with reverse transcriptase activity or a newly established biological quantitation assay of active virus. The amounts of virus with this cell system were much higher than those of the H9 cell system. This procedure enabled us first to compare the two viral isolates HTLV-III and LAV directly in the same cell lines. Establishment of the culture system, allowing efficient production of AIDS retroviruses, provides a useful tool for the isolation of the virus from patients with AIDS and for more basic research, such as the mechanisms of immune destruction caused by the virus leading to the occurence of various malignancies (author)

  4. Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ibtissam Youlyouz

    2002-01-01

    Full Text Available Platelet-activating factor receptor (PAF-R transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells, the pancreas (Miapaca cells and the bladder (5637 cells in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.

  5. Effects of treatment with Maraviroc a CCR5 inhibitor on a human hepatic stellate cell line.

    Science.gov (United States)

    Coppola, Nicola; Perna, Angelica; Lucariello, Angela; Martini, Salvatore; Macera, Margherita; Carleo, Maria A; Guerra, Germano; Esposito, Vincenzo; De Luca, Antonio

    2018-08-01

    After an acute liver damage, tissue regeneration repairs lesions with degradation of deposed fibrotic material, while mechanisms of tissue restoration are persistently activated following several repeated injuries, inducing deposition of extracellular matrix. (ECM). Factors responsible for ECM remodeling have been identified in a pathway involving a family of zinc-dependent enzyme matrix metalloproteinases (MMPs), together with tissue inhibitor of metalloproteinases (TIMPs). Recent experimental models suggested a role of CCR5 receptor in the genesis of liver fibrosis. Drawing from these background we decided to evaluate the effects of the treatment with the CCR5 inhibitor Maraviroc on LX-2, a human hepatic stellate cell line (HSC). Treatment with Maraviroc resulted in a block in S phase of LX-2 cells with increased expression levels of cyclin D1 and p21 while the expression of p53 was reduced. Treatment with Maraviroc was also able to block the accumulation of fibrillar collagens and extracellular matrix proteins (ECM), as demonstrated by the decrease of specific markers as Collagen type I, α-SMA, and TGF-β1. In addition we observed a down regulation of both metalloproteins (MMP-2, MMP-9), used for the degradation of the extracellular matrix and their inhibitors (TIMP-1, TIMP-2). The identification of a compound that may modulate the dynamic of liver fibrosis could be crucial in all chronic liver diseases. Maraviroc could play an important role because, in addition to its own anti-HIV activity, it could reduce the release of pro-inflammatory citokynes implicated in liver fibrogenesis. © 2018 Wiley Periodicals, Inc.

  6. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative

    Czech Academy of Sciences Publication Activity Database

    Adewumi, O.; Aflatoonian, B.; Ahrlund-Richter, L.; Amit, M.; Andrews, P.W.; Beighton, G.; Bello, P.A.; Benvenisty, N.; Berry, L.S.; Bevan, S.; Blum, B.; Brooking, J.; Chen, K.G.; Choo, A.B.H.; Churchill, G.A.; Corbel, M.; Damjanov, I.; Draper, J.S.; Dvořák, Petr; Emanuelsson, K.; Fleck, R.A.; Ford, A.; Gertow, K.; Gertsenstein, M.; Gokhale, P.J.; Hamilton, R.S.; Hampl, Aleš; Healy, L.E.; Hovatta, O.; Hyllner, J.; Imreh, M.P.; Itskovitz-Eldor, J.; Jackson, J.; Johnson, J.L.; Jones, M.; Kee, K.; King, B.L.; Knowles, B.B.; Lako, M.; Lebrin, F.; Mallon, B.S.; Manning, D.; Mayshar, Y.; Mckay, D.G.; Michalska, A.E.; Mikkola, M.; Mileikovsky, M.; Minger, S.L.; Moore, H.D.; Mummery, Ch.L.; Nagy, A.; Nakutsuji, N.; O´Brien, C.M.; Oh, S.K.W.; Olsson, C.; Otonkoski, T.; Park, K.Y.; Passier, R.; Patel, H.; Patel, M.; Pedersen, R.; Pera, M.F.; Piekarczyk, M.S.; Pera, R.A.P.; Reubinoff, B.E.; Robins, A.J.; Rossant, J.; Rugg-Gunn, P.; Schulz, T.C.; Semb, H.; Sherrer, E.S.; Siemen, H.; Stacey, G.N.; Stojkovic, M.; Suemori, H.; Szatkiewicz, J.; Turetsky, T.; Tuuri, T.; Van den Brink, S.; Vintersten, K.; Vuoristo, S.; Ward, D.; Weaver, T.A.; Young, L.A.; Zhang, W.

    2007-01-01

    Roč. 25, č. 7 (2007), s. 803-816 ISSN 1087-0156 R&D Projects: GA MŠk 1M0538; GA ČR GA301/05/0463; GA ČR GA305/05/0434 Institutional research plan: CEZ:AV0Z50390512 Keywords : International Stem Cell Initiative Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 22.848, year: 2007

  7. Anti-Cancer Effects of Imperata cylindrica Leaf Extract on Human Oral Squamous Carcinoma Cell Line SCC-9 in Vitro.

    Science.gov (United States)

    Keshava, Rohini; Muniyappa, Nagesh; Gope, Rajalakshmi; Ramaswamaiah, Ananthanarayana Saligrama

    2016-01-01

    Imperata cylindrica, a tall tufted grass which has multiple pharmacological applications is one of the key ingredients in various traditional medicinal formula used in India. Previous reports have shown that I. cylindrica plant extract inhibited cell proliferation and induced apoptosis in various cancer cell lines. To our knowledge, no studies have been published on the effect of I. cylindrica leaf extract on human oral cancers. The present study was undertaken in order to evaluate the anticancer properties of the leaf extract of I. cylindrica using an oral squamous cell carcinoma cell line SCC-9 as an in vitro model system. A methanol extract from dried leaves of I. cylindrica (ICL) was prepared by standard procedures. Effects of the ICL extract on the morphology of SCC-9 cells was visualized by microscopy. Cytotoxicity was determined by MTT assay. Effects of the ICL extract on colony forming ability of SCC-9 cells was evaluated using clonogenic assay. Cell cycle analysis was performed by flow cytometry and induction of apoptosis was determined by DNA fragmentation assay. The ICL extract treatment caused cytotoxicity and induced cell death in vitro in SCC-9 cells in a dose-dependent manner. This treatment also significantly reduced the clonogenic potential and inhibited cell proliferation by arresting the cell cycle in the G2/M phase. Furthermore, DNA fragmentation assays showed that the observed cell death was caused by apoptosis. This is the first report showing the anticancer activity of the methanol extracts from the leaves of I. cylindrica in human oral cancer cell line. Our data indicates that ICL extract could be considered as one of the lead compounds for the formulation of anticancer therapeutic agents to treat/manage human oral cancers. The natural abundance of I. cylindrica and its wide geographic distribution could render it one of the primary resource materials for preparation of anticancer therapeutic agents.

  8. Specific receptors for epidermal growth factor in human bone tumour cells and its effect on synthesis of prostaglandin E2 by cultured osteosarcoma cell line

    International Nuclear Information System (INIS)

    Hirata, Y.; Uchihashi, M.; Nakashima, H.; Fujita, T.; Matsukura, S.; Matsui, K.

    1984-01-01

    Using tumour cell lines derived from human bone tumours, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator in many tissues, and its effect on synthesis of prostaglandin (PG) E 2 , a potent bone-resorbing factor, by cultured osteosarcoma cell line were studied. Three tumour cell lines, one osteosarcoma (HOSO) and two giant cell tumours of the bone (G-1 and G-2), all possessed specific binding sites for 125 I-labelled EGF: the apparent dissociation constant was approximately 4-10 x 10 -10 M and the maximal binding capacity was 50 000-80 000 sites/cell. EGF had no mitogenic effect in these cell lines. However, these cell lines did not have specific binding sites for 125 I-labelled parathyroid hormone (PTH) or calcitonin. HOSO line produced and secreted PGE 2 into medium, while no significant amount of PGE 2 was demonstrated in G-1 or G-2 line. EGF significantly stimulated PGE 2 production in HOSO line in a dose-dependent manner (0.5-50 ng/ml); its stimulatory effect was completely abolished by indomethacin, an inhibitor of PG biosynthesis. Exogenous PGE 1 significantly stimulated cyclic AMP formation in HOSO line, whereas PGFsub(2α) PTH, calcitonin, or EGF had no effect. None of these calcium-regulating hormones affected cyclic AMP generation in either G-1 of G-2 line. These data indicate that human bone tumour cells have specific EGF receptors unrelated to cell growth, and suggest that EGF may be involved in bone resorption through a PGE 2 -mediated process in human osseous tissues. (author)

  9. Fourier analysis of the cell shape of paired human urothelial cell lines of the same origin but of different grades of transformation.

    Science.gov (United States)

    Ostrowski, K; Dziedzic-Goclawska, A; Strojny, P; Grzesik, W; Kieler, J; Christensen, B; Mareel, M

    1986-01-01

    The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I = nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II = nonmalignant cell lines with an infinite life span. TGr III = malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs--each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)--were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by "spontaneous" transformation during propagation in vitro of the respective maternal TGr II-cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Characterization of human glioblastoma cell lines in vitro and their xenografts in nude mice by DNA fingerprinting

    DEFF Research Database (Denmark)

    Türeci, O; Fischer, H; Lagoda, P

    1990-01-01

    Human gliomas were grown as permanent tissue cultures and xenografts in nude mice. DNA fingerprint patterns from two human gliomas were established using two different hypervariable multilocus probes [( GTG]5 and 33.15). In general the cell lines investigated showed an overall stability in the DNA...... fingerprint pattern. However, differences in the DNA fingerprint patterns were shown to occur depending upon the above mentioned parameters....

  11. Anti-cancer effects of bioactive compounds from rose hip fruit in human breast cancer cell lines

    OpenAIRE

    Zhong, Lijie

    2017-01-01

    Rose hips have long been used in human diets as a food ingredient and supplement. Their multiple medical properties, which have been attributed to their abundant carotenoid composition, have attracted widespread scientific attention. This thesis examined the carotenoid composition in rose hips from five rose species. The anti-cancer effect of different carotenoid fractions from rose hips was investigated in human breast cancer cell lines, using the natural variation in carotenoid content in h...

  12. Apoptosis induced by GanoPoly in human gastric cancer cell line ...

    African Journals Online (AJOL)

    use

    2011-12-12

    Dec 12, 2011 ... ... cancer's active therapy. Key words: Apoptosis, polysaccharide, human gastric cancer cells. ... The active components of polysaccharides are all glucans, which have a ... for the treatment of alleviated fatigue, night sweating,.

  13. Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

    Science.gov (United States)

    Wang, Z-M; Kang, Y-H; Yang, X; Wang, J-F; Zhang, Q; Yang, B-X; Zhao, K-L; Xu, L-P; Yang, L-P; Ma, J-X; Huang, G-H; Cai, J; Sun, X-C

    2016-01-01

    To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. © 2014 International Society for Diseases of the Esophagus.

  14. Single-molecule optical genome mapping of a human HapMap and a colorectal cancer cell line.

    Science.gov (United States)

    Teo, Audrey S M; Verzotto, Davide; Yao, Fei; Nagarajan, Niranjan; Hillmer, Axel M

    2015-01-01

    Next-generation sequencing (NGS) technologies have changed our understanding of the variability of the human genome. However, the identification of genome structural variations based on NGS approaches with read lengths of 35-300 bases remains a challenge. Single-molecule optical mapping technologies allow the analysis of DNA molecules of up to 2 Mb and as such are suitable for the identification of large-scale genome structural variations, and for de novo genome assemblies when combined with short-read NGS data. Here we present optical mapping data for two human genomes: the HapMap cell line GM12878 and the colorectal cancer cell line HCT116. High molecular weight DNA was obtained by embedding GM12878 and HCT116 cells, respectively, in agarose plugs, followed by DNA extraction under mild conditions. Genomic DNA was digested with KpnI and 310,000 and 296,000 DNA molecules (≥ 150 kb and 10 restriction fragments), respectively, were analyzed per cell line using the Argus optical mapping system. Maps were aligned to the human reference by OPTIMA, a new glocal alignment method. Genome coverage of 6.8× and 5.7× was obtained, respectively; 2.9× and 1.7× more than the coverage obtained with previously available software. Optical mapping allows the resolution of large-scale structural variations of the genome, and the scaffold extension of NGS-based de novo assemblies. OPTIMA is an efficient new alignment method; our optical mapping data provide a resource for genome structure analyses of the human HapMap reference cell line GM12878, and the colorectal cancer cell line HCT116.

  15. [Linezolid-induced Apoptosis through Mitochondrial Damage and Role of Superoxide Dismutase-1 in Human Monocytic Cell Line U937].

    Science.gov (United States)

    Fujii, Satoshi; Muraoka, Sanae; Miyamoto, Atsushi; Sakurai, Koichi

    2018-01-01

     Cytopenia is a major adverse event associated with linezolid therapy. The objective of this study was to examine whether the cytotoxicity of linezolid to eukaryotic cells was associated with mitochondrial dysfunction and apoptosis-like cell death in human leukemic monocyte lymphoma cell line U937. Apoptosis-like cell death was clearly observed when cells were incubated with linezolid, depending on the duration and linezolid concentration. Mitochondrial membrane potential of cells treated with linezolid collapsed in a short period of time, but the number of mitochondria did not decrease. Cytotoxicity of linezolid was relieved by the knockdown of superoxide dismutase-1 in U937 cells. On the other hand, no autophagy was observed in cells treated with linezolid. These results suggest that mitochondrial damages would be linked to the induction of apoptosis in U937 cells treated with linezolid and that its mechanism does not involve autophagy.

  16. Adaptive Response to ionizing Radiation Induced by Low Doses of Gamma Rays in Human Lymphoblastoid Cell Lines

    International Nuclear Information System (INIS)

    Seong, Jin Sil; Suh, Chang Ok; Kim, Gwi Eon

    1994-01-01

    When cells are exposed to low doses of a mutagenic or clastogenic agents, they often become less sensitive to the effects of a higher does administered subsequently. Such adaptive responses were first described in Escherichia coli and mammalian cells to low doses of an alkylating agent. Since most of the studies have been carried out with human lymphocytes, it is urgently necessary to study this effect in different cellular systems. Its relation with inherent cellular radiosensitivity and underlying mechanism also remain to be answered. In this study, adaptive response by 1 cGy of gamma rays was investigated in three human lymphoblastoid cell lines which were derived from ataxia telangiectasia homozygote, ataxia telangiectasia heterozygote, and normal individual. Experiments were carried out by delivering 1 cGy followed by 50 cGy of gamma radiation and chromatid breaks were scored as an endpoint. The results indicate that prior exposure to 1 cGy of gamma rays reduces the number of chromatid breaks induced by subsequent higher does (50 cGy). The expression of this adaptive response was similar among three cell lines despite of their different radiosensitivity. When 3-aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase, was added after 50 cGy, adaptive responses were abolished in all the tested cell lines. Therefore it is suggested that the adaptive response can be observed in human lymphoblastoid cell lines. Which was first documented through this study. The expression of adaptive response was similar among the cell lines regardless of their radiosensitivity. The elimination of the adaptive response by 3-aminobenzamide is consistent with the proposal that this adaptive response is the result of the induction of a certain chromosomal repair mechanism

  17. Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line

    Directory of Open Access Journals (Sweden)

    Binhai Ren

    2016-04-01

    Full Text Available Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone, H4IIE/ND (NeuroD1 gene alone, and H4IIEins/ND (insulin and NeuroD1 genes. The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ± 171.8 pmol/insulin/5 × 106 cells, respectively. Additionally, several β cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0–20 mmol/L was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes.

  18. The effect of β-ionone on telomerase activity in the human leukemia cell line K562

    Directory of Open Access Journals (Sweden)

    Zohreh Faezizadeh

    2015-06-01

    Full Text Available Background: Telomerase is highly activated in most human cancer cells, therefore, its inhibition has been proposed as a novel and promising strategy for cancer therapy. Many plant-derived anticancer agents act through inhibition of telomerase activity and induction of apoptosis. β-ionone, a carotenoid compound isolated from Roseaceae, has been reported to possess anticancer properties. The present study was undertaken to examine the mechanism of β-ionone-induced apoptosis in human leukemia cell line K562 with special emphasis on its role in telomerase inhibition. Method: In this study the anti-proliferation effect of β-ionone on K562 cells was evaluated by MTT assay. Apoptosis rate was detected by Hoechst staining and flow cytometry analysis. Telomerase activity was measured by (TRAP ELISA assay. Results: Exposure of K562 cells to β-ionone caused a dose-dependent decrease in proliferation. Flow cytometry analysis and Hoechst staining showed that percentage of apoptotic cells markedly increased with an increase in β-ionone concentration. Compared to control cells, treatment of K562 cells with β-ionone resulted in a significant decrease of telomerase activity. Moreover, a positive correlation was detected between telomerase inhibition and apoptosis induction in the treated K562 cells. Conclusion: Based on these results, β-ionone is an appropriate candidate for inhibiting telomerase activity in K562 cells. Therefore, it may be utilized as a novel drug against some leukemia cell lines.

  19. Radiosensitivity of a monoclonal human lung adenocarcinoma cell line with MDR phenotype induced by CDDP: an in vitro study

    International Nuclear Information System (INIS)

    Zhang Junxiang; Kong Zhaolu; Shen Zhifen; Tong Shungao; Jin Yizun

    2006-01-01

    The study was to evaluate radiosensitivity of a monoclonal human lung adenocarcinoma cell line SPC-A-1/CDDP-4 with MDR phenotype induced by cisplatin (CDDP) compared with its parental cell SPC-A-1 in vitro. The glutathione (GSH) content and the radiosensitivity of SPC-A-1/CDDP-4 and SPC-A-1 cells were investigated in aerobic and under hypoxia, respectively. The radiosensitization effect of buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, to SPC-A-1/CDDP-4 and SPC-A-1 cells was observed. The results indicated that the monoclonal human lung adenocarcinoma cell line SPC-A-1/CDDP-4 showed, to some extent, a cross-resistance to 137 Cs γ-ray, in addition to its resistance to anticancer drugs (CDDP, ADM, MTX and VCR). The GSH content of SPC-A-1/CDDP-4 cells was higher than that of SPC-A-1 cells both in aerobic and under hypoxia which might account for it. BSO had radiosensitization effect to SPC-A-1/CDDP-4 and SPC-A-1 cells both in aerobic and under hypoxia, but it was stronger under hypoxia than in aerobic and it was stronger to SPC-A-1/CDDP-4 cells than to SPC-A-1 cells. (authors)

  20. Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines

    International Nuclear Information System (INIS)

    Maneckjee, R.; Minna, J.D.

    1990-01-01

    Using specific radioactively-labeled ligands, the authors find that lung cancer cell lines of diverse histologic types express multiple, high-affinity membrane receptors for μ, δ, and κ opioid agonists and for nicotine and α-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas μ, δ, and κ opioid agonists at low concentrations inhibited lung cancer growth in vitro. They also found that lung cancer cells expressed various combinations of immunoreactive opioid peptides (β-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, they tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a tumor suppressor system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer

  1. Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Maneckjee, R.; Minna, J.D. (National Cancer Institute-Navy Medical Oncology Branch, Bethesda, MD (USA) Uniformed Services Univ. of the Health Sciences, Bethesda, MD (USA))

    1990-05-01

    Using specific radioactively-labeled ligands, the authors find that lung cancer cell lines of diverse histologic types express multiple, high-affinity membrane receptors for {mu}, {delta}, and {kappa} opioid agonists and for nicotine and {alpha}-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas {mu}, {delta}, and {kappa} opioid agonists at low concentrations inhibited lung cancer growth in vitro. They also found that lung cancer cells expressed various combinations of immunoreactive opioid peptides ({beta}-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, they tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a tumor suppressor system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer.

  2. Anti-proliferative and apoptosis-inducing activity of lycopene against three subtypes of human breast cancer cell lines

    Science.gov (United States)

    Takeshima, Mikako; Ono, Misaki; Higuchi, Takako; Chen, Chen; Hara, Takayuki; Nakano, Shuji

    2014-01-01

    Although lycopene, a major carotenoid component of tomatoes, has been suggested to attenuate the risk of breast cancer, the underlying preventive mechanism remains to be determined. Moreover, it is not known whether there are any differences in lycopene activity among different subtypes of human breast cancer cells. Using ER/PR positive MCF-7, HER2-positive SK-BR-3 and triple-negative MDA-MB-468 cell lines, we investigated the cellular and molecular mechanism of the anticancer activity of lycopene. Lycopene treatment for 168 consecutive hours exhibited a time-dependent and dose-dependent anti-proliferative activity against these cell lines by arresting the cell cycle at the G0/G1 phase at physiologically achievable concentrations found in human plasma. The greatest growth inhibition was observed in MDA-MB-468 where the sub-G0/G1 apoptotic population was significantly increased, with demonstrable cleavage of PARP. Lycopene induced strong and sustained activation of the ERK1/2, with concomitant cyclin D1 suppression and p21 upregulation in these three cell lines. In triple negative cells, lycopene inhibited the phosphorylation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of proapoptotic Bax without affecting anti-apoptotic Bcl-xL. Taken together, these data indicate that the predominant anticancer activity of lycopene in MDA-MB-468 cells suggests a potential role of lycopene for the prevention of triple negative breast cancer. PMID:24397737

  3. Fucoidan cytotoxicity against human breast cancer T47D cell line increases with higher level of sulfate ester group

    Science.gov (United States)

    Saepudin, Endang; Alfita Qosthalani, Fildzah; Sinurat, Ellya

    2018-01-01

    The anticancer activity of different sulfate ester group content in different molecular weight was examined. The anticancer activity was achieved in vitro on human breast cancer T47D cell line. Fucoidan with lower molecular weight (5.79 kDa) tends to have lower sulfate ester group content (8.69%) and resulted in higher IC50 value (184.22 μg/mL). While fucoidan with higher molecular weight (785.12 kDa) tends to have higher sulfate level (18.63%) and achieved lower IC50 value (75.69 μg/mL). The result showed that in order to maintain fucoidan cytotoxic activity against human breast cancer T47D cell line, the sulfate content should be remain high. Keywords: fucoidan, sulfate ester group, human breast cancer

  4. A Recombinant Fragment of Human Surfactant Protein D induces Apoptosis in Pancreatic Cancer Cell Lines via Fas-Mediated Pathway.

    Science.gov (United States)

    Kaur, Anuvinder; Riaz, Muhammad Suleman; Murugaiah, Valarmathy; Varghese, Praveen Mathews; Singh, Shiv K; Kishore, Uday

    2018-01-01

    Human surfactant protein D (SP-D) is a potent innate immune molecule, which is emerging as a key molecule in the recognition and clearance of altered and non-self targets. Previous studies have shown that a recombinant fragment of human SP-D (rfhSP-D) induced apoptosis via p53-mediated apoptosis pathway in an eosinophilic leukemic cell line, AML14.3D10. Here, we report the ability of rfhSP-D to induce apoptosis via TNF-α/Fas-mediated pathway regardless of the p53 status in human pancreatic adenocarcinoma using Panc-1 (p53 mt ), MiaPaCa-2 (p53 mt ), and Capan-2 (p53 wt ) cell lines. Treatment of these cell lines with rfhSP-D for 24 h caused growth arrest in G1 cell cycle phase and triggered transcriptional upregulation of pro-apoptotic factors such as TNF-α and NF-κB. Translocation of NF-κB from the cytoplasm into the nucleus of pancreatic cancer cell lines was observed via immunofluorescence microscopy following treatment with rfhSP-D as compared to the untreated cells. The rfhSP-D treatment caused upregulation of pro-apoptotic marker Fas, as analyzed via qPCR and western blot, which then triggered caspase cascade, as evident from cleavage of caspase 8 and 3 analyzed via western blot at 48 h. The cell number following the rfhSP-D treatment was reduced in the order of Panc-1 (~67%) > MiaPaCa-2 (~60%) > Capan-2 (~35%). This study appears to suggest that rfhSP-D can potentially be used to therapeutically target pancreatic cancer cells irrespective of their p53 phenotype.

  5. Differential responses to radiation and hyperthermia of cloned cell lines derived from a single human melanoma xenograft

    International Nuclear Information System (INIS)

    Rofstad, E.K.; Brustad, T.

    1984-01-01

    One uncloned and five cloned cell lines were derived from a single human melanoma xenograft. Cells from passages 7-12 were exposed to either radiation or hyperthermia (42.5 0 C, pH = 7.4) under aerobic conditions and the colony forming ability of the cells was assayed in soft agar. The five cloned lines showed individual and characteristic responses to radiation as well as to hyperthermia. The variation in the response to radiation was mainly reflected in the size of the shoulders of the survival curves rather than in the D 0 -values. The variation in the response to hyperthermia was mainly reflected in the terminal slopes of the survival curves. The survival curve of cells from the uncloned line, both when exposed to radiation and hyperthermia, was positioned in the midst of those of the cloned lines. The response of the cloned lines to radiation did not correlate with the response to hyperthermia, indicating that tumor cell subpopulations which are resistant to radiation may respond well to hyperthermia

  6. SYNTHESIS AND CYTOTOXIC ACTIVITY OF CHALCONE DERIVATIVES ON HUMAN BREAST CANCER CELL LINES

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    Nuraini Harmastuti

    2012-12-01

    Full Text Available Chalcone, an α,β-unsaturated ketone, has been shown have many biological activities such as anticancer and antifungi. This research was conducted to synthesize the chalcone derivatives and to obtain their cytotoxic activity on human cervix cancer cell lines. Synthesis of chalcone and its derivatives, 4II-methylchalcone, 4II-methoxychalcone, and 3II,4II-dichlorochalcone was carried out using starting materials of benzaldehide and acetofenon, p-methylacetophenone, p-methoxyacetophenone, as well as m,p-dichloroacetophenone through Claisen Schmidt condensation catalized by NaOH in ethanol at 15 °C. The purity of synthesized compounds were analyzed by thin layer chromatography, melting range, and gas chromatography. Structure elucidations were conducted by UV spectrophotometer, IR spectrometer, 1H-NMR spectrometer, as well as mass spectrometer. Cytotoxic activities were determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT microculture tetrazolium viability assay. The results showed that chalcone and derivatives compounds have been able to be synthesized and purified and had the same structure as a predicted structure. Chalcone had highest cytotoxic activity compared to that of its derivatives, with the IC50 values of chalcone, 4II-methylchalcone, 4II-methoxychalcone, and 3II,4II-dichlorochalcone were 9.49, 14.79, 11.48, and 24.26 µg/mL respectively. It was concluded that methyl, methoxy as well as chlorine substitution at 3 II and 4II position decrease the cytotoxic activity of chalcone.

  7. Identification of pyrogallol as an antiproliferative compound present in extracts from the medicinal plant Emblica officinalis: effects on in vitro cell growth of human tumor cell lines.

    Science.gov (United States)

    Khan, Mahmud Tareq Hassan; Lampronti, Ilaria; Martello, Dino; Bianchi, Nicoletta; Jabbar, Shaila; Choudhuri, Mohammad Shahabuddin Kabir; Datta, Bidduyt Kanti; Gambari, Roberto

    2002-07-01

    In this study we compared the in vitro antiproliferative activity of extracts from medicinal plants toward human tumor cell lines, including human erythromyeloid K562, B-lymphoid Raji, T-lymphoid Jurkat, erythroleukemic HEL cell lines. Extracts from Emblica officinalis were the most active in inhibiting in vitro cell proliferation, after comparison to those from Terminalia arjuna, Aphanamixis polystachya, Oroxylum indicum, Cuscuta reflexa, Aegle marmelos, Saraca asoka, Rumex maritimus, Lagerstroemia speciosa, Red Sandalwood. Emblica officinalis extracts have been studied previously, due to their hepatoprotective, antioxidant, antifungal, antimicrobial and anti-inflammatory medicinal activities. Gas chromatography/mass spectrometry analyses allowed to identify pyrogallol as the common compound present both in unfractionated and n-butanol fraction of Emblica officinalis extracts. Antiproliferative effects of pyrogallol were therefore determined on human tumor cell lines thus identifying pyrogallol as an active component of Emblica officinalis extracts.

  8. Immunocapture of CD133-positive cells from human cancer cell lines by using monodisperse magnetic poly(glycidyl methacrylate) microspheres containing amino groups

    Energy Technology Data Exchange (ETDEWEB)

    Kuan, Wei-Chih [Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Minhisung 621, Taiwan (China); Horák, Daniel, E-mail: horak@imc.cas.cz [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6 (Czech Republic); Plichta, Zdeněk [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6 (Czech Republic); Lee, Wen-Chien [Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Minhisung 621, Taiwan (China)

    2014-01-01

    Magnetic poly(glycidyl methacrylate)-based macroporous microspheres with an average particle size of 4.2 μm were prepared using a modified multi-step swelling polymerization method and by introducing amino functionality on their surfaces. Antibody molecules were oxidized on their carbohydrate moieties and bound to the amino-containing magnetic microspheres via a site-directed procedure. CD133-positive cells could be effectively captured from human cancer cell lines (HepG2, HCT116, MCF7, and IMR-32) by using magnetic microspheres conjugated to an anti-human CD133 antibody. After further culture, the immunocaptured CD133-expressing cells from IMR-32 proliferated and gradually detached from the magnetic microspheres. Flow-cytometric analysis confirmed the enrichment of CD133-expressing cells by using the antibody-bound magnetic microspheres. Such microspheres suitable for immunocapture are very promising for cancer diagnosis because the CD133-expressing cells in cancer cell lines have been suggested to be cancer stem cells. - Highlights: • Multi-step swelling polymerization produced poly(glycidyl methacrylate) microspheres. • Anti-human CD133 antibodies were bound to the amino-containing magnetic microspheres. • CD133-positive cells were effectively captured from human cancer cell lines. • Immunocaptured CD133-expressing cells proliferated and were detached from microspheres. • Enrichment of CD133-expressing cells was confirmed by flow-cytometric analysis.

  9. Polarity of fatty acid uptake and metabolism in a human intestinal cell line (CACO-2)

    International Nuclear Information System (INIS)

    Trotter, P.J.; Storch, J.

    1990-01-01

    Free fatty acids (ffa) can enter the intestinal cell via the apical (AP) or basolateral (BL) membrane. The authors are using the Caco-2 intestinal cell line to examine the polarity of ffa uptake and metabolism in the enterocyte. Cells are grown on permeable polycarbonate Transwell filters in order to obtain access to both AP and BL compartments. Differentiated Caco-2 cells form tight polarized monolayers which express small intestine-specific enzymes and are impermeable to the fluid phase marker Lucifer Yellow. Submicellar concentrations of 3 H-palmitic acid (2uM) were added to AP or BL sides of Caco-2 monolayers at 37 degrees C and cells were incubated for various times between 2 and 120 minutes. Total AP and BL uptake is similar; however, when relative membrane surface areas are accounted for, AP uptake is about 2-fold higher. The metabolism of AP and BL ffa is not significantly different: triacylglycerol and phosphatidylcholine account for most of the metabolites (32±4 and 24±2% respectively at 5 minutes). Little ffa oxidation is observed. Preincubation with albumin-bound 2-monoolein (100uM) and palmitate (50uM) increases the level of TG metabolites. The results suggest that in this cell line the uptake of AP ffa may be greater than BL ffa, but that AP (dietary) ffa and BL (plasma) ffa are metabolized similarly

  10. Investigation of hTERT gene expression levels in two cell lines infected by high-risk human papilloma virus

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    Maryam Akhtari

    2016-07-01

    Full Text Available Background: Human papilloma virus (HPV is one of the most important factors in cervical cancer. Viral sequences are integrated into the host cell genome. In mild cases the virus causes skin damages, in severe cases it leads to cancer. Like many other cancers, telomerase gene expression was increased in cervical cancer. This enzyme is a reverse transcriptase that contains two common subunits: i catalytic protein called human telomerase reverse transcriptase (hTERT and, ii RNA sequence called hTR. hTERT expression is hardly found in any somatic tissues. Detection of high telomerase activity in human cells, lead to tumor genesis. So hTERT can be used as a diagnostic tool in cancer detection. Methods: This experimental study was carried out from May 2013 to April 2014 in Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences in Tehran, Iran. Caski and Hela cancer cell lines were used which contain HPV16 and HPV18 respectively. Cell lines were cultured and total RNA was extracted. Following normalization agent glyceraldehyde-3-phosphate dehydrogenase (GADPH, hTERT expression level was determining by real-time PCR method. For each sample, the expression level of hTERT and GAPDH were quantified as copy numbers (per reaction using the standard curve. Finally, hTERT levels in Hela and Caski cell lines were compared quantitatively by t-test using GraphPad statistic software version 5 (San Diego, CA, USA. Results: According to the charts real-time PCR, hTERT gene expression in Hela and Caski cancer cell lines is significantly different (t=0.0319. Conclusion: All results confirm that hTERT expression levels in Hela and Caski cell lines are significantly different and the level of hTERT expression in the Caski cell line was slightly higher than that of Hela cell line. The significant difference between hTERT mRNA expression levels reported here could be used as a tumor marker for HPV16 and HPV18 in cervical cancer.

  11. Anti-Proliferative and Apoptotic Effects of Beta-Ionone in Human Leukemia Cell Line K562

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    Zohreh Faezizadeh

    2016-06-01

    Full Text Available Background Beta-ionone is an aroma compound found in the Rosaceae family. Some evidence supported that beta-ionone has a great potential for cancer prevention. To date, the anti-proliferative and apoptotic effects of beta-ionone in human leukemia cell line K562 were not studied. Objectives Hence, we investigated whether beta-ionone could inhibit cell growth and induce apoptosis in the K562 cells. Materials and Methods In this experimental study, human leukemia cell line K562 was cultured and anti-proliferation effect of beta-ionone with different doses (25 - 400 µm at different times (24 - 96 hours on treated cells was evaluated by the MTT assay. To determine apoptosis rate, the Hoechst 33342 staining and flow cytometry was performed. Results The MTT assay showed that beta-ionone inhibited proliferation of K562 cells in a dose-dependent manner significantly (P = 0.0008. Moreover, the increased apoptotic rate was found after incubation of K562 cells with 200 µm beta-ionone. The Hoechst staining and flow cytometry analysis indicated that beta-ionone could increase apoptosis of K562 cells in a dose-dependent manner. Conclusions The results demonstrated that beta-ionone has anti-proliferative and apoptotic effects on K562 cells, and in the future may be used in the treatment of some leukemia sub-types.

  12. TRIM32 promotes retinoic acid receptor α-mediated differentiation in human promyelogenous leukemic cell line HL60

    International Nuclear Information System (INIS)

    Sato, Tomonobu; Okumura, Fumihiko; Iguchi, Akihiro; Ariga, Tadashi; Hatakeyama, Shigetsugu

    2012-01-01

    Highlights: ► TRIM32 enhanced RARα-mediated transcriptional activity even in the absence of RA. ► TRIM32 stabilized RARα in the human promyelogenous leukemic cell line HL60. ► Overexpression of TRIM32 in HL60 cells induced granulocytic differentiation. ► TRIM32 may function as a coactivator for RARα-mediated transcription in APL cells. -- Abstract: Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor α (RARα). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RARα and enhances transcriptional activity of RARα in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RARα, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RARα-mediated transcriptional activity even in the absence of RA and stabilizes RARα in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RARα-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL.

  13. Differential effects of arsenic trioxide on chemosensitization in human hepatic tumor and stellate cell lines

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    Rangwala Fatima

    2012-09-01

    Full Text Available Abstract Background Crosstalk between malignant hepatocytes and the surrounding peritumoral stroma is a key modulator of hepatocarcinogenesis and therapeutic resistance. To examine the chemotherapy resistance of these two cellular compartments in vitro, we evaluated a well-established hepatic tumor cell line, HepG2, and an adult hepatic stellate cell line, LX2. The aim was to compare the chemosensitization potential of arsenic trioxide (ATO in combination with sorafenib or fluorouracil (5-FU, in both hepatic tumor cells and stromal cells. Methods Cytotoxicity of ATO, 5-FU, and sorafenib, alone and in combination against HepG2 cells and LX2 cells was measured by an automated high throughput cell-based proliferation assay. Changes in survival and apoptotic signaling pathways were analyzed by flow cytometry and western blot. Gene expression of the 5-FU metabolic enzyme, thymidylate synthase, was analyzed by real time PCR. Results Both HepG2 and LX2 cell lines were susceptible to single agent sorafenib and ATO at 24 hr (ATO IC50: 5.3 μM in LX2; 32.7 μM in HepG2; Sorafenib IC50: 11.8 μM in LX2; 9.9 μM in HepG2. In contrast, 5-FU cytotoxicity required higher concentrations and prolonged (48–72 hr drug exposure. Concurrent ATO and 5-FU treatment of HepG2 cells was synergistic, leading to increased cytotoxicity due in part to modulation of thymidylate synthase levels by ATO. Concurrent ATO and sorafenib treatment showed a trend towards increased HepG2 cytotoxicity, possibly due to a significant decrease in MAPK activation in comparison to treatment with ATO alone. Conclusions ATO differentially sensitizes hepatic tumor cells and adult hepatic stellate cells to 5-FU and sorafenib. Given the importance of both of these cell types in hepatocarcinogenesis, these data have implications for the rational development of anti-cancer therapy combinations for the treatment of hepatocellular carcinoma (HCC.

  14. Differential effects of arsenic trioxide on chemosensitization in human hepatic tumor and stellate cell lines

    International Nuclear Information System (INIS)

    Rangwala, Fatima; Williams, Kevin P; Smith, Ginger R; Thomas, Zainab; Allensworth, Jennifer L; Lyerly, H Kim; Diehl, Anna Mae; Morse, Michael A; Devi, Gayathri R

    2012-01-01

    Crosstalk between malignant hepatocytes and the surrounding peritumoral stroma is a key modulator of hepatocarcinogenesis and therapeutic resistance. To examine the chemotherapy resistance of these two cellular compartments in vitro, we evaluated a well-established hepatic tumor cell line, HepG2, and an adult hepatic stellate cell line, LX2. The aim was to compare the chemosensitization potential of arsenic trioxide (ATO) in combination with sorafenib or fluorouracil (5-FU), in both hepatic tumor cells and stromal cells. Cytotoxicity of ATO, 5-FU, and sorafenib, alone and in combination against HepG2 cells and LX2 cells was measured by an automated high throughput cell-based proliferation assay. Changes in survival and apoptotic signaling pathways were analyzed by flow cytometry and western blot. Gene expression of the 5-FU metabolic enzyme, thymidylate synthase, was analyzed by real time PCR. Both HepG2 and LX2 cell lines were susceptible to single agent sorafenib and ATO at 24 hr (ATO IC 50 : 5.3 μM in LX2; 32.7 μM in HepG2; Sorafenib IC 50 : 11.8 μM in LX2; 9.9 μM in HepG2). In contrast, 5-FU cytotoxicity required higher concentrations and prolonged (48–72 hr) drug exposure. Concurrent ATO and 5-FU treatment of HepG2 cells was synergistic, leading to increased cytotoxicity due in part to modulation of thymidylate synthase levels by ATO. Concurrent ATO and sorafenib treatment showed a trend towards increased HepG2 cytotoxicity, possibly due to a significant decrease in MAPK activation in comparison to treatment with ATO alone. ATO differentially sensitizes hepatic tumor cells and adult hepatic stellate cells to 5-FU and sorafenib. Given the importance of both of these cell types in hepatocarcinogenesis, these data have implications for the rational development of anti-cancer therapy combinations for the treatment of hepatocellular carcinoma (HCC)

  15. Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480.

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    Akari Takaya

    Full Text Available Human cancer stem-like cells (CSCs/cancer-initiating cells (CICs can be isolated as side population (SP cells, aldehyde dehydrogenase high (ALDHhigh cells or cell surface marker-positive cells includin