WorldWideScience

Sample records for cell line expressing

  1. Isolation of Oct4-expressing extraembryonic endoderm precursor cell lines.

    Directory of Open Access Journals (Sweden)

    Bisrat G Debeb

    Full Text Available BACKGROUND: The extraembryonic endoderm (ExEn defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines ("XEN-P cell lines", which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. CONCLUSIONS/SIGNIFICANCE: Our findings (i suggest strongly that the ExEn precursor is a self-renewable entity, (ii indicate that active Oct4 gene expression (transcription plus translation is part of its molecular identity, and (iii provide an in vitro model of early ExEn differentiation.

  2. Nestin expression in the cell lines derived from glioblastoma multiforme

    International Nuclear Information System (INIS)

    Veselska, Renata; Kuglik, Petr; Cejpek, Pavel; Svachova, Hana; Neradil, Jakub; Loja, Tomas; Relichova, Jirina

    2006-01-01

    Nestin is a protein belonging to class VI of intermediate filaments that is produced in stem/progenitor cells in the mammalian CNS during development and is consecutively replaced by other intermediate filament proteins (neurofilaments, GFAP). Down-regulated nestin may be re-expressed in the adult organism under certain pathological conditions (brain injury, ischemia, inflammation, neoplastic transformation). Our work focused on a detailed study of the nestin cytoskeleton in cell lines derived from glioblastoma multiforme, because re-expression of nestin together with down-regulation of GFAP has been previously reported in this type of brain tumor. Two cell lines were derived from the tumor tissue of patients treated for glioblastoma multiforme. Nestin and other cytoskeletal proteins were visualized using imunocytochemical methods: indirect immunofluorescence and immunogold-labelling. Using epifluorescence and confocal microscopy, we described the morphology of nestin-positive intermediate filaments in glioblastoma cells of both primary cultures and the derived cell lines, as well as the reorganization of nestin during mitosis. Our most important result came through transmission electron microscopy and provided clear evidence that nestin is present in the cell nucleus. Detailed information concerning the pattern of the nestin cytoskeleton in glioblastoma cell lines and especially the demonstration of nestin in the nucleus represent an important background for further studies of nestin re-expression in relationship to tumor malignancy and invasive potential

  3. TKTL1 expression in human malign and benign cell lines.

    Science.gov (United States)

    Kämmerer, Ulrike; Gires, Olivier; Pfetzer, Nadja; Wiegering, Armin; Klement, Rainer Johannes; Otto, Christoph

    2015-06-10

    Overexpression of transketolase-like 1 protein TKTL1 in cancer cells has been reported to correlate with enhanced glycolysis and lactic acid production. Furthermore, enhanced TKTL1 expression was put into context with resistance to chemotherapy and ionizing radiation. Here, a panel of human malign and benign cells, which cover a broad range of chemotherapy and radiation resistance as well as reliance on glucose metabolism, was analyzed in vitro for TKTL1 expression. 17 malign and three benign cell lines were characterized according to their expression of TKTL1 on the protein level with three commercially available anti-TKTL1 antibodies utilizing immunohistochemistry and Western blot, as well as on mRNA level with three published primer pairs for RT-qPCR. Furthermore, sensitivities to paclitaxel, cisplatin and ionizing radiation were assessed in cell survival assays. Glucose consumption and lactate production were quantified as surrogates for the "Warburg effect". Considerable amounts of tktl1 mRNA and TKTL1 protein were detected only upon stable transfection of the human embryonic kidney cell line HEK293 with an expression plasmid for human TKTL1. Beyond that, weak expression of endogenous tktl1 mRNA was measured in the cell lines JAR and U251. Western blot analysis of JAR and U251 cells did not detect TKTL1 at the expected size of 65 kDa with all three antibodies specific for TKTL1 protein and immunohistochemical staining was observed with antibody JFC12T10 only. All other cell lines tested here revealed expression of tktl1 mRNA below detection limits and were negative for TKTL1 protein. However, in all cell lines including TKTL1-negative HEK293-control cells, antibody JFC12T10 detected multiple proteins with different molecular weights. Importantly, JAR and U251 did neither demonstrate an outstanding production of lactic acid nor increased resistance against chemotherapeutics or to ionizing radiation, respectively. Using RT-qPCR and three different antibodies we

  4. TKTL1 expression in human malign and benign cell lines

    International Nuclear Information System (INIS)

    Kämmerer, Ulrike; Gires, Olivier; Pfetzer, Nadja; Wiegering, Armin; Klement, Rainer Johannes; Otto, Christoph

    2015-01-01

    Overexpression of transketolase-like 1 protein TKTL1 in cancer cells has been reported to correlate with enhanced glycolysis and lactic acid production. Furthermore, enhanced TKTL1 expression was put into context with resistance to chemotherapy and ionizing radiation. Here, a panel of human malign and benign cells, which cover a broad range of chemotherapy and radiation resistance as well as reliance on glucose metabolism, was analyzed in vitro for TKTL1 expression. 17 malign and three benign cell lines were characterized according to their expression of TKTL1 on the protein level with three commercially available anti-TKTL1 antibodies utilizing immunohistochemistry and Western blot, as well as on mRNA level with three published primer pairs for RT-qPCR. Furthermore, sensitivities to paclitaxel, cisplatin and ionizing radiation were assessed in cell survival assays. Glucose consumption and lactate production were quantified as surrogates for the “Warburg effect”. Considerable amounts of tktl1 mRNA and TKTL1 protein were detected only upon stable transfection of the human embryonic kidney cell line HEK293 with an expression plasmid for human TKTL1. Beyond that, weak expression of endogenous tktl1 mRNA was measured in the cell lines JAR and U251. Western blot analysis of JAR and U251 cells did not detect TKTL1 at the expected size of 65 kDa with all three antibodies specific for TKTL1 protein and immunohistochemical staining was observed with antibody JFC12T10 only. All other cell lines tested here revealed expression of tktl1 mRNA below detection limits and were negative for TKTL1 protein. However, in all cell lines including TKTL1-negative HEK293-control cells, antibody JFC12T10 detected multiple proteins with different molecular weights. Importantly, JAR and U251 did neither demonstrate an outstanding production of lactic acid nor increased resistance against chemotherapeutics or to ionizing radiation, respectively. Using RT-qPCR and three different antibodies

  5. CellMinerHCC: a microarray-based expression database for hepatocellular carcinoma cell lines.

    Science.gov (United States)

    Staib, Frank; Krupp, Markus; Maass, Thorsten; Itzel, Timo; Weinmann, Arndt; Lee, Ju-Seog; Schmidt, Bertil; Müller, Martina; Thorgeirsson, Snorri S; Galle, Peter R; Teufel, Andreas

    2014-04-01

    Therapeutic options for hepatocellular carcinoma (HCC) still remain limited. Development of gene targeted therapies is a promising option. A better understanding of the underlying molecular biology is gained in in vitro experiments. However, even with targeted manipulation of gene expression varying treatment responses were observed in diverse HCC cell lines. Therefore, information on gene expression profiles of various HCC cell lines may be crucial to experimental designs. To generate a publicly available database containing microarray expression profiles of diverse HCC cell lines. Microarray data were analyzed using an individually scripted R program package. Data were stored in a PostgreSQL database with a PHP written web interface. Evaluation and comparison of individual cell line expression profiles are supported via public web interface. This database allows evaluation of gene expression profiles of 18 HCC cell lines and comparison of differential gene expression between multiple cell lines. Analysis of commonly regulated genes for signaling pathway enrichment and interactions demonstrates a liver tumor phenotype with enrichment of major cancer related KEGG signatures like 'cancer' and 'inflammatory response'. Further molecular associations of strong scientific interest, e.g. 'lipid metabolism', were also identified. We have generated CellMinerHCC (http://www.medicalgenomics.org/cellminerhcc), a publicly available database containing gene expression data of 18 HCC cell lines. This database will aid in the design of in vitro experiments in HCC research, because the genetic specificities of various HCC cell lines will be considered. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Systemic alteration of cell-surface and secreted glycoprotein expression in malignant breast cancer cell lines

    OpenAIRE

    Timpe, Leslie C; Yen, Roger; Haste, Nicole V; Litsakos-Cheung, Christina; Yen, Ten-Yang; Macher, Bruce A

    2013-01-01

    Breast cancer cell lines express fewer transmembrane and secreted glycoproteins than nonmalignant ones. The objective of these experiments was to characterize the changes in the expression of several hundred glycoproteins quantitatively. Secreted and cell-surface glycoproteins were isolated using a glycoprotein capture protocol and then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared with th...

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

    DEFF Research Database (Denmark)

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

    1992-01-01

    Epidermal growth factor (EGF) receptor expression was evaluated in a panel of 21 small cell lung cancer cell lines with radioreceptor assay, affinity labeling, and Northern blotting. We found high-affinity receptors to be expressed in 10 cell lines. Scatchard analysis of the binding data...... lung cancer cell lines express the EGF receptor....... 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...

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

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

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

  9. 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....

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

    DEFF Research Database (Denmark)

    Rygaard, K; Møller, C; Bock, E

    1992-01-01

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

  11. Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

    Science.gov (United States)

    Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

    2016-06-01

    Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

  12. Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

    DEFF Research Database (Denmark)

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

    1993-01-01

    A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse....... WE examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc......-myc. In general, the level of expression of c-myc and N-myc was similar at the mRNA and the protein level. Expression of c-myc was positively correlated with the proliferative index (sum of S and G2+M phases) of cell lines, but not with the population doubling time. In general, L-myc-expressing cell lines had...

  13. [Expression of human Jagged-1 protein on eukaryotic cells and establishment of stable transfectant cell line].

    Science.gov (United States)

    Gan, Zhi-Hua; Chen, Yu; Yan, Hua; Wang, Kan-Kan

    2010-08-01

    Jagged-1 protein is one of the ligands belonging to Notch signaling pathway. Notch signaling pathway is one of the major signaling pathways mediated by contact between cells and plays an important role to regulate the process of proliferation and differentiation of hematopoietic cells in the hematopoietic microenvironment. To study the biological effect after the combination of receptor and ligand in Notch signaling pathway and the mechanism of Notch signaling pathway in bone marrow stromal cells mediated-drug resistance, a NIH-3T3 cell line over-expressing Jagged-1 protein was constructed for further research purposes. A full coding region of Jagged-1 gene was cloned and inserted into eukaryotic expression plasmid to construct pEGFP-IRES2-Jagged-1 eukaryotic expression vector, then transfected into NIH-3T3 cell line, a mammalian cells. As a result Western blot analysis confirmed that the transfectant NIH-3T3 cells highly expressed Jagged-1 protein and flow cytometry analysis confirmed that the NIH-3T3-pEGFP-IRES2-Jagged-1 cell line over-expressed Jagged-1 protein was monoclonal after screened by selective medium and limiting dilution analysis. It is concluded that the pEGFP-IRES2-Jagged-1 eukaryotic expression vector and a stable transfectant monoclonal NIH-3T3 cell line are successfully established. The construction of the stable transfectant monoclonal NIH-3T3 cell line which overexpressed Jagged-1 protein, provides the conditions to further study the mechanism of the bone marrow stromal cell-mediated drug resistance and to discover the new drug targets.

  14. 'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

    International Nuclear Information System (INIS)

    Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ulleras, Erik; Dastych, JarosIaw

    2005-01-01

    The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals

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

    DEFF Research Database (Denmark)

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

    1998-01-01

    receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot...... analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...

  16. Assessment of tumor characteristic gene expression in cell lines using a tissue similarity index (TSI).

    Science.gov (United States)

    Sandberg, Rickard; Ernberg, Ingemar

    2005-02-08

    The gene expression profiles of 60 cell lines, derived from nine different tissues, were compared with their corresponding in vivo tumors and tissues. Cell lines expressed few tissue-specific (2%) or tumor-specific (5%) genes when analyzed group-wise. A tissue similarity index (TSI) was designed based upon singular value decomposition that measured in vivo tumor characteristic gene expression in each cell line independently. Only 34 of the 60 cell lines received the highest TSI toward its tumor of origin. In addition, we identified the most appropriate cell lines to be used as model systems for different in vivo tumors. Seven cell lines were identified as being of another origin than the originally presumed one. The proposed TSI will likely become an important tool for the selection of the most appropriate cell lines in pharmaceutical screening programs and experimental and biomedical research.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1997-07-01

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

  18. Differential CCR4 Expression And Function in Cutaneous T-Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Chieh-Shan Wu

    2008-11-01

    Full Text Available Cutaneous T cell lymphoma (CTCL is a clonal epidermotropic malignancy of memory T cells primarily involving the skin. However, the mechanisms governing migration of CTCL cells have not been fully clarified. It has been shown that certain chemokine receptors are upregulated in CTCL cells, but it remains unanswered whether these chemokine receptors play a critical role in the migration dynamics of CTCL. Using cell lines originally derived from patients with different subtypes of CTCL, we have shown higher CCR4 expression in the line derived from the mycosis fungoides (MJ, compared with the line derived from Sézary syndrome (Hut78. In specific responses to CCL22 (a CCR4 ligand treatments, MJ cells showed significant chemotactic migration, enhanced activation and adhesion of certain integrins (CD49d and CD29 in vitro, while the control cells (Hut78, CD4+CD45RO+ memory T cells, and Jurkat cells did not. Furthermore, compared with Hut78 cells, MJ cells manifested significantly more transendothelial migration in responses to treatments with either CCL22 or conditioned medium from dendritic cells in vitro. These results provide further dynamic evidence, in line with the multistep cascade paradigm for leukocyte transendothelial migration, to support a critical role for CCR4 in CTCL migration.

  19. Antitumor effects of celecoxib in COX-2 expressing and non-expressing canine melanoma cell lines.

    Science.gov (United States)

    Seo, Kyoung-Won; Coh, Ye-Rin; Rebhun, Robert B; Ahn, Jin-Ok; Han, Sei-Myung; Lee, Hee-Woo; Youn, Hwa-Young

    2014-06-01

    Cyclooxygenase-2 (COX-2) is a potential target for chemoprevention and cancer therapy. Celecoxib, a selective COX-2 inhibitor, inhibits cell growth of various types of human cancer including malignant melanoma. In dogs, oral malignant melanoma represents the most common oral tumor and is often a fatal disease. Therefore, there is a desperate need to develop additional therapeutic strategies. The purpose of this study was to investigate the anticancer effects of celecoxib on canine malignant melanoma cell lines that express varying levels of COX-2. Celecoxib induced a significant anti-proliferative effect in both LMeC and CMeC-1 cells. In the CMeC cells, treatment of 50 μM celecoxib caused an increase in cells in the G0/G1 and a decreased proportion of cells in G-2 phase. In the LMeC cells, 50 μM of celecoxib led to an increase in the percentage of cells in the sub-G1 phase and a significant activation of caspase-3 when compared to CMeC-1 cells. In conclusion, these results demonstrate that celecoxib exhibits antitumor effects on canine melanoma LMeC and CMeC-1 cells by induction of G1-S cell cycle arrest and apoptosis. Our data suggest that celecoxib might be effective as a chemotherapeutic agent against canine malignant melanoma. Copyright © 2014. Published by Elsevier Ltd.

  20. Systemic alteration of cell-surface and secreted glycoprotein expression in malignant breast cancer cell lines.

    Science.gov (United States)

    Timpe, Leslie C; Yen, Roger; Haste, Nicole V; Litsakos-Cheung, Christina; Yen, Ten-Yang; Macher, Bruce A

    2013-11-01

    Breast cancer cell lines express fewer transmembrane and secreted glycoproteins than nonmalignant ones. The objective of these experiments was to characterize the changes in the expression of several hundred glycoproteins quantitatively. Secreted and cell-surface glycoproteins were isolated using a glycoprotein capture protocol and then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared with those expressed by a nonmalignant set. The average number of spectral counts (proportional to relative protein abundance) and the total number of glycopeptides in the malignant samples were reduced to about two-thirds of the level in the nonmalignant samples. Most glycoproteins were expressed at a different level in the malignant samples, with nearly as many increasing as decreasing. The glycoproteins with reduced expression accounted for a larger change in spectral counts, and hence for the net loss of spectral counts in the malignant lines. Similar results were found when the glycoproteins were studied via identified glycosylation sites only, or through identified sites together with non-glycopeptides. The overall reduction is largely due to the loss of integrins, laminins and other proteins that form or interact with the basement membrane.

  1. 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

  2. S100 protein expression in human melanoma cells: Comparison of levels of expression among different cell lines and individual cells in different phases of the cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Marks, A.; O' Hanlon, D.; Dunn, R. (Univ. of Toronto, Ontario (Canada)); Petsche, D.; Baumal, R. (Hospital for Sick Children, Toronto, Ontario (Canada)); Kwong, P.C.; Stead, R. (McMaster Univ., Hamilton, Ontario (Canada)); Liao, S.K. (McMaster Univ., Hamilton, Ontario (Canada) Biotherapeutics, Inc., Franklin, TN (United States))

    1990-03-01

    The synthesis of S100 protein in cultured human melanoma cells was examined using metabolic labeling with ({sup 35}S)methionine, immunoprecipitation with anti-S100 protein antiserum, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Six of seven cell lines derived from melanomas synthesized relatively large amounts of S100 protein, whereas three cell lines derived from normal melanocytes synthesized lesser amounts. Synthesis of S100 protein was not detected in 10 human cell lines of non-neuroectodermal origin. Analysis of poly(A{sup +}) RNA form one melanoma cell line by Northern blot hybridization with a probe specific for the {beta} subunit of rat S100 protein revealed a single mRNA species of 1.0 kb coding for the human protein. Flow cytometric analysis of individual cells of two melanoma cell lines and the rat glioma cell line C6 indicated that G0/G1 cells were heterogeneous with respect to S100 protein expression, while almost all the cells in S+G2+M expressed S100 protein. These results suggest that expression of S100 protein in G0/G1 could be a prerequisite for progression of the cells through the cell cycle.

  3. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E.; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based do......We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...... transfer. PFV cells supported Flp mediated cassette exchange for transgene substitution of eGFP with dsRED, and the dsRED transgenic PFV cells generated blastocysts with transgene expression. Hence, the PFV cell line constitutes a valuable pig equivalent to transformed cell lines from other mammalian...

  4. Gene expression profiling in chemoresistant variants of three cell lines of different origin

    DEFF Research Database (Denmark)

    Johnsson, Anders; Vallon-Christensson, Johan; Strand, Carina

    2005-01-01

    . Several genes encoding ABC transporters were highly up-regulated, most notably ABCB1 (MDR1) and ABCB4 in several cell lines and ABCG2 (MXR) specifically in MX-resistant cell lines. A pronounced down-regulation of several histones was noted in the MCF-7-derived resistant sublines. Altered expression...

  5. 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.

  6. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

    DEFF Research Database (Denmark)

    Efrat, S; Linde, S; Kofod, Hans

    1988-01-01

    Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture...... by glucose, although with a lower threshold for maximal stimulation than that for normal beta cells. beta TC lines can be repeatedly derived from primary beta-cell tumors that heritably arise in the transgenic mice. Thus, targeted expression of an oncogene with a cell-specific regulatory element can be used...

  7. 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)

  8. 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...

  9. Construction of a high-EGFR expression cell line and its biological ...

    African Journals Online (AJOL)

    Targeted screening of EGFR compounds has become one of the medical research focuses for tumor therapy. A431, which naturally expresses high levels of EGFR, was compared with the stably high expressing EGFR cell line HEK293. Flow cytometry was used to analyze cell growth and Western blot was used to ...

  10. Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

    Directory of Open Access Journals (Sweden)

    Zahra Moinfar

    Full Text Available Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43 and estrogen receptors (ERs. Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2 on Cx43 expression in two glioma cell lines with variable native expression of Cx43.F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

  11. Comprehensive sampling of gene expression in human cell lines with massively parallel signature sequencing.

    Science.gov (United States)

    Jongeneel, C Victor; Iseli, Christian; Stevenson, Brian J; Riggins, Gregory J; Lal, Anita; Mackay, Alan; Harris, Robert A; O'Hare, Michael J; Neville, A Munro; Simpson, Andrew J G; Strausberg, Robert L

    2003-04-15

    Whereas information is rapidly accumulating about the structure and position of genes encoded in the human genome, less is known about the complexity and relative abundance of their expression in individual human cells and tissues. Here, we describe the characteristics of the transcriptomes of two cultured cell lines, HB4a (normal breast epithelium) and HCT-116 (colon adenocarcinoma), using massively parallel signature sequencing (MPSS). We generated in excess of 10(7) short signature sequences per cell line, thus providing a comprehensive snapshot of gene expression, within the technical limitations of the method. The number of genes expressed at one copy per cell or more in either of the lines was estimated to be between 10,000 and 15,000. The vast majority of the transcripts found in these cells can be mapped to known genes and their polyadenylation variants. Among the genes that could be identified from their signature sequences, approximately 8,500 were expressed by both cell lines, whereas 6,000 showed cellular specificity. Taking into account sequence tags that map uniquely to the genome but not to known transcripts, overall the data are consistent with an upper limit of 17,000 for the total number of genes expressed at more than one copy per cell in one or both of the two cell lines examined.

  12. Evaluation of procoagulant tissue factor expression in canine hemangiosarcoma cell lines.

    Science.gov (United States)

    Witter, Lauren E; Gruber, Erika J; Lean, Fabian Z X; Stokol, Tracy

    2017-01-01

    OBJECTIVE To evaluate expression of procoagulant tissue factor (TF) by canine hemangiosarcoma cells in vitro. SAMPLES 4 canine hemangiosarcoma cell lines (SB-HSA [mouse-passaged cutaneous tumor], Emma [primary metastatic brain tumor], and Frog and Dal-1 [primary splenic tumors]) and 1 nonneoplastic canine endothelial cell line (CnAoEC). PROCEDURES TF mRNA and TF antigen expression were evaluated by quantitative real-time PCR assay and flow cytometry, respectively. Thrombin generation was measured in canine plasma and in coagulation factor-replete or specific coagulation factor-deficient human plasma by calibrated automated thrombography. Corn trypsin inhibitor and annexin V were used to examine contributions of contact activation and membrane-bound phosphatidylserine, respectively, to thrombin generation. RESULTS All cell lines expressed TF mRNA and antigen, with significantly greater expression of both products in SB-HSA and Emma cells than in CnAoEC. A greater percentage of SB-HSA cells expressed TF antigen, compared with other hemangiosarcoma cell lines. All hemangiosarcoma cell lines generated significantly more thrombin than did CnAoEC in canine or factor-replete human plasma. Thrombin generation induced by SB-HSA cells was significantly lower in factor VII-deficient plasma than in factor-replete plasma and was abolished in factor X-deficient plasma; residual thrombin generation in factor VII-deficient plasma was abolished by incubation of cells with annexin V. Thrombin generation by SB-HSA cells was unaffected by the addition of corn trypsin inhibitor. CONCLUSIONS AND CLINICAL RELEVANCE Hemangiosarcoma cell lines expressed procoagulant TF in vitro. Further research is needed to determine whether TF can be used as a biomarker for hemostatic dysfunction in dogs with hemangiosarcoma.

  13. Augmenter of liver regeneration gene expression in human colon cancer cell lines and clinical tissue samples.

    Science.gov (United States)

    Gatzidou, Elisavet; Mantzourani, Marina; Giaginis, Constantinos; Giagini, Athina; Patsouris, Efstratios; Kouraklis, Gregory; Theocharis, Stamatios

    2015-01-01

    Augmenter of liver regeneration (ALR) is an hepatotrophic factor responsible for the increased regenerative capacity of mammalian liver and ALR gene expression has been well-documented in liver cirrhosis and hepatocellular carcinoma tissue samples. The present study aimed to quantify and evaluate ALR gene expression in human colon cancer cell lines and tissue samples. Total RNA was isolated from 6 colorectal cancer cell lines and 23 primary colorectal tumors, cDNA was prepared and ALR mRNA expression analysis was performed using quantitative real-time PCR. ALR mRNA expression was confirmed in all 6 colorectal cancer cell lines (SW480, SW620, DLD-1, RKO, COLO-205 and HTC-116) and an epithelial one (WISH). DLD-1 cell line showed the highest ALR mRNA levels, followed by RKO, COLO-205, HCT-116, SW480, SW620 and WISH cell lines. ALR gene expression levels were detected in all cancer tissue samples (N=23), being significantly increased in well/moderately compared to poorly differentiated tumors (p=0.0208). ALR gene expression levels were increased in Dukes' stage A/B compared to stage C tumors, at a non significant level (p=0.2842). ALR mRNA levels were slightly higher in colon cancer tissues compared to adjacent non-neoplastic ones (N=19), at a non significant level (p=0.2122). The present study verified for the first time the ALR gene expression in both human colon cancer cell lines and clinical samples. Enhanced ALR gene expression was negatively correlated with advanced histopathological grade and stage in both colon cancer cell lines and human tissue samples, implicating ALR participation at the early stage of colon malignant progression.

  14. Functional importance of GLP-1 receptor species and expression levels in cell lines.

    Science.gov (United States)

    Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

    2012-04-10

    Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Functional analysis of synthetic insectatachykinin analogs on recombinant neurokinin receptor expressing cell lines

    NARCIS (Netherlands)

    Torfs, H.; Akerman, K.E.; Nachman, R.J.; Oonk, H.B.; Detheux, M.; Poels, J.; Loy, van T.; Loof, A.; Meloen, R.H.; Vassart, G.; Parmentier, M.; Broeck, van den J.

    2002-01-01

    The activity of a series of synthetic tachykinin-like peptide analogs was studied by means of microscopic calcium imaging on recombinant neurokinin receptor expressing cell lines. A C-terminal pentapeptide (FTGMRa) is sufficient for activation of the stomoxytachykinin receptor (STKR) expressed in

  16. [Influence of hepatocyte cell adhesion molecule on gene expression profile of human bladder transitional cell carcinoma cell line].

    Science.gov (United States)

    Wang, Qiu-ju; Lv, Chang-kun; Tao, Jia; Du, Hong-fei; Fan, Yan-ru; Song, Xue-dong; Luo, Chun-li

    2013-04-01

    To investigate the changes of gene expression file in transitional cell carcinoma of bladder after hepatocyte cell adhesion molecule(hepaCAM) overexpression. Affymetrix Human Genome U133 Plus 2.0 Array was used to investigate the changes of gene expression profile between adenovirus-green fluorescent protein(GFP) -hepaCAM group and GFP group in transitional cell carcinoma of bladder EJ cells.Significant Analysis of Microarray(SAM) was used to screen the differentially expressed genes, DAVID software was used to conduct gene ontology analysis and wikiPathway analysis based on the differentially expressed genes. Reverse transcription-polymerase chain reaction and Western blot were applied to verify microarray data. Compared with the GFP group, a total of 2469 genes were up-regulated or down-regulated by more than 2 times in the GFP-hepaCAM group. Among these genes, 1602 genes were up-regulated and 867 were down-regulated.Most of the differentially expressed genes were involved in the function of cell proliferation and cell cycle regulation. The mRNA expressions of nibrin, liver kinase B1, and cyclin D1 detected by reverse transcription-polymerase chain reaction in three different bladder cancer cell lines were consistent with the microarray data.The protein expressions of nibrin and liver kinase B1 in these three cell lines measured by Western blot were consistent with the mRNA expression. HepaCAM can alter the gene expression profile of bladder cancer EJ cells. The well-known anti-tumor effect of hepaCAM may be mediated by regulating the gene expression via multiple pathways.

  17. Mammosphere formation in breast carcinoma cell lines depends upon expression of E-cadherin.

    Directory of Open Access Journals (Sweden)

    Juan Manuel Iglesias

    Full Text Available Tumors are heterogeneous at the cellular level where the ability to maintain tumor growth resides in discrete cell populations. Floating sphere-forming assays are broadly used to test stem cell activity in tissues, tumors and cell lines. Spheroids are originated from a small population of cells with stem cell features able to grow in suspension culture and behaving as tumorigenic in mice. We tested the ability of eleven common breast cancer cell lines representing the major breast cancer subtypes to grow as mammospheres, measuring the ability to maintain cell viability upon serial non-adherent passage. Only MCF7, T47D, BT474, MDA-MB-436 and JIMT1 were successfully propagated as long-term mammosphere cultures, measured as the increase in the number of viable cells upon serial non-adherent passages. Other cell lines tested (SKBR3, MDA-MB-231, MDA-MB-468 and MDA-MB-435 formed cell clumps that can be disaggregated mechanically, but cell viability drops dramatically on their second passage. HCC1937 and HCC1569 cells formed typical mammospheres, although they could not be propagated as long-term mammosphere cultures. All the sphere forming lines but MDA-MB-436 express E-cadherin on their surface. Knock down of E-cadherin expression in MCF-7 cells abrogated its ability to grow as mammospheres, while re-expression of E-cadherin in SKBR3 cells allow them to form mammospheres. Therefore, the mammosphere assay is suitable to reveal stem like features in breast cancer cell lines that express E-cadherin.

  18. Expression of three reporter genes in four cell lines developed from Papilio demoleus Linnaeus (Lepidoptera: Papilionidae).

    Science.gov (United States)

    Ding, Wei-Feng; Zhang, Xin; Li, Xian; Liu, Zhi-Gang; Xie, Shi-Cong; Feng, Ying

    2018-03-01

    This paper used recombinant baculoviruses that carried three reporter genes, green fluorescent protein (GFP), β-galactosidase, and secreted alkaline phosphatase (SEAP), to infect four new cell lines from Papilio demoleus Linnaeus larvae (named RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3, and RIRI-PaDe-4). The expression levels of the three recombinant proteins were detected at 24, 48, 72, 96, 120, and 144 h after infection and compared with Sf9 and High Five cells to evaluate the characteristics of these four cell lines as host cells. The inoculation densities of the tested cell lines were 2 × 10 4 cells/well (96-well plate) and 1 × 10 5 cells/well (24-well plate), and adding a volume of virus stock resulted in an MOI of 5.0. The results showed that the four cell lines could be infected by recombinant baculovirus and that cell lysis occurred 96 h after infection. In the four tested cell lines, only a small number of RIRI-PaDe-1 and RIRI-PaDe-3 cells expressed recombinant GFP and showed green fluorescence. The expression was much lower than that of Sf9 and High Five. Comparing the intracellular and extracellular activity of β-galactosidase indicated that the P. demoleus cell system was more suitable for the expression of secreted proteins, and its extracellular β-galactosidase level was close to that of Sf9, but the expression level of SEAP was far lower than those of Sf9 and High Five.

  19. 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.

  20. Expression of Ku correlates with radiation sensitivities in the head and neck cancer cell lines

    International Nuclear Information System (INIS)

    Lee, Sang Wook; Yu, Eun Sil; Yi, So Lyoung; Son, Se Hee; Kim, Jong Hoon; Ahn, Seung Do; Shin, Seong Soo; Choi, Eun Kyung

    2004-01-01

    DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase consisting of a 470 kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex, called Ku, which is composed of 70 kDa (Ku 70) and 86 kDa (Ku 80) proteins. The DNA-PK has been shown to play a pivotal role in rejoining DNA double-strand-breaks (dsb) in mammalian cells. The purpose of this study is to examine the relationship between the level of Ku expression and radiation sensitivity. Nine head and neck, cancer cell lines showed various intrinsic radiation sensitivities. Among the nine, AMC-HN-3 cell was the most sensitive for X-ray irradiation and AMC-HN-9 cell was the most resistance. The most sensitive and resistant cell lines were selected and the test sensitivity of radiation and expression of Ku were measured. Radiation sensitivity was obtained by colony forming assay and Ku protein expression using Western blot analysis. Ku80 increased expression by radiation, wheras Ku70 did not. Overexpression of Ku80 protein increased radiation resistance in AMC-HN9 cell line. There was a correlation between Ku80 expression and radiation resistance. Ku80 was shown to play an important role in radiation damage response. Induction of Ku80 expression had an important role in DNA damage repair by radiation. Ku80 expression may be an effective predictive assay of radiosensitivity on head and neck cancer

  1. Successful Reconstruction of Tooth Germ with Cell Lines Requires Coordinated Gene Expressions from the Initiation Stage

    Directory of Open Access Journals (Sweden)

    Yasuhiro Tomooka

    2012-10-01

    Full Text Available Tooth morphogenesis is carried out by a series of reciprocal interactions between the epithelium and mesenchyme in embryonic germs. Previously clonal dental epithelial cell (epithelium of molar tooth germ (emtg lines were established from an embryonic germ. They were odontogenic when combined with a dental mesenchymal tissue, although the odontogenesis was quantitatively imperfect. To improve the microenvironment in the germs, freshly isolated dental epithelial cells were mixed with cells of lines, and germs were reconstructed in various combinations. The results demonstrated that successful tooth construction depends on the mixing ratio, the age of dental epithelial cells and the combination with cell lines. Analyses of gene expression in these germs suggest that some signal(s from dental epithelial cells makes emtg cells competent to communicate with mesenchymal cells and the epithelial and mesenchymal compartments are able to progress  odontogenesis from the initiation stage.

  2. Construction of a high-EGFR expression cell line and its biological ...

    African Journals Online (AJOL)

    USER

    2010-07-26

    Jul 26, 2010 ... eukaryotic vector and obtained cell lines with stable and high-expression of EGFR. Results demonstrated ... dilution was transferred to new plates. Two days after infection,. G418 (200, 400 ... RNA from 1 × 107 cells was isolated using TRIzol reagent (Invitrogen,. Carlsbad, CA, USA) and reverse-transcribed ...

  3. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    International Nuclear Information System (INIS)

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing

    2006-01-01

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression

  4. The Expression and Biological Significance of PD-L1 on Lung Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Cheng CHEN

    2009-08-01

    Full Text Available Background and objective Tumor-associated PD-L1 expression was recently shown to promote T-cell apoptosis and proposed as a potential mechanism of immune evasion by tumors. On the basis of the ability of tumor-associated PD-L1 to mediate activated T-cell death, it is likely that manipulation of the PD-L1 pathway at defined time points during the development of the T-cell antitumor immune response can enhance the efficacy of T-cell-based immunotherapy. Here, the levels of expression of PD-L1 on lung cancer cell lines and its role in interaction of CTL and target cells was investigated. Methods Human PBMC derived DCs were loaded with apoptotic tumor cells and stimulated by CD40 mAb (5C11. Tumor specific CTL was generated in vitro by autologous T cells co-cultured with mature DCs. Expression of PD-L1 on lung cancer cell lines H1299 and A549 were analyzed by FCM. JAM assay was used to detect the cytolytic activity of CTL with or without blocking PD-L1 by PD-L1 mAb respectively. The concentrations of IFN-γ in supernatants from distinct groups were analyzed by ELISA. Results Tumor cells-loaded mature DCs could induce the generation of the tumor specific CTL. Expression of PD-L1 was low on A549 cell, but high on H1299 cell. Blockade of PD-L1 on A549 could not improve cytolytic effect of CTL on target cells and IFN-γ production, but fragmentation of H1299 cells and IFN-γ production were significantly enhanced by the combination of PD-L1 mAb and CTL. Conclusion Expression of PD-L1 on lung cancer cell line can decrease the cytolytic effect of CTL on target cells.

  5. Induction of expression of two phenotypic markers of pulmonary type II cells in a cultured cell line

    International Nuclear Information System (INIS)

    Henderson, R.F.; Waide, J.J.; Scott, G.G.

    1994-01-01

    The functions of pulmonary type II cells, such as synthesis of pulmonary surfactant and metabolism of inhaled xenobiotics, can be studied in primary isolates of lung cells. However, isolated type II cells, when cultured, quickly lose the phenotypic expressions characteristics of type II cells, including surfactant lipid and protein synthesis and alkaline phosphatase (AP) activity. A cultured cell line that maintained expression of type II cell markers of differentiation would be advantageous for the study of such functions as surfactant synthesis and secretion. Such a cell line would allow generation of a large number of homogeneous cells for study. The purpose of the current study was to induce markers of differentiated type II cells in a cultured cell line to facilitate studies of factors that control surfactant synthesis and secretion

  6. Altered expression of LINC-ROR in cancer cell lines and tissues.

    Science.gov (United States)

    Rezaei, Majdaddin; Emadi-Baygi, Modjtaba; Hoffmann, Michèle J; Schulz, Wolfgang A; Nikpour, Parvaneh

    2016-02-01

    According to GLOBOCAN 2012, the worldwide burden of cancer increased and is expected to worsen within the next decades. Therefore, universal combat against cancer will not succeed with treatment solely; effective prevention and early detection are urgently needed to tackle the cancer crisis. Emerging data demonstrate that long non-coding RNAs are involved in numerous biological and pathological processes like development and differentiation and in a variety of human diseases including cancer. Located at 18q21, LINC-ROR (regulator of reprogramming) is a modulator of ESCs maintenance and hypoxia-signaling pathways in hepatocellular cancer cells. The aim of this study was to examine the expression of LINC-ROR in various cell lines and representative samples of human cancers by quantitative real-time RT-PCR to provide a snapshot on how LINC-ROR expression may be deregulated in cancer. More than 30 cell lines and 112 patient specimens from various tissues were assessed for relative expression of LINC-ROR. Our results revealed that the expression of LINC-ROR was lower in all somatic cancer cell lines compared to stem cells or cells with stem cell-like capabilities, like the embryonic carcinoma cell line, NTERA-2. In tissues, expression patterns vary, but some cancerous tissues displayed increased LINC-ROR expression compared to corresponding normal tissues. Thus, we hypothesize that LINC-ROR may have a key function in a subpopulation of cells from the tumor bulk, i.e., the cancer stem cells associated with specific properties including resistance to adverse environmental conditions.

  7. Matrigel Basement Membrane Matrix influences expression of microRNAs in cancer cell lines

    International Nuclear Information System (INIS)

    Price, Karina J.; Tsykin, Anna; Giles, Keith M.; Sladic, Rosemary T.; Epis, Michael R.; Ganss, Ruth; Goodall, Gregory J.; Leedman, Peter J.

    2012-01-01

    Highlights: ► Matrigel alters cancer cell line miRNA expression relative to culture on plastic. ► Many identified Matrigel-regulated miRNAs are implicated in cancer. ► miR-1290, -210, -32 and -29b represent a Matrigel-induced miRNA signature. ► miR-32 down-regulates Integrin alpha 5 (ITGA5) mRNA. -- Abstract: Matrigel is a medium rich in extracellular matrix (ECM) components used for three-dimensional cell culture and is known to alter cellular phenotypes and gene expression. microRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and have roles in cancer. While miRNA profiles of numerous cell lines cultured on plastic have been reported, the influence of Matrigel-based culture on cancer cell miRNA expression is largely unknown. This study investigated the influence of Matrigel on the expression of miRNAs that might facilitate ECM-associated cancer cell growth. We performed miRNA profiling by microarray using two colon cancer cell lines (SW480 and SW620), identifying significant differential expression of miRNAs between cells cultured in Matrigel and on plastic. Many of these miRNAs have previously been implicated in cancer-related processes. A common Matrigel-induced miRNA signature comprised of up-regulated miR-1290 and miR-210 and down-regulated miR-29b and miR-32 was identified using RT-qPCR across five epithelial cancer cell lines (SW480, SW620, HT-29, A549 and MDA-MB-231). Experimental modulation of these miRNAs altered expression of their known target mRNAs involved in cell adhesion, proliferation and invasion, in colon cancer cell lines. Furthermore, ITGA5 was identified as a novel putative target of miR-32 that may facilitate cancer cell interactions with the ECM. We propose that culture of cancer cell lines in Matrigel more accurately recapitulates miRNA expression and function in cancer than culture on plastic and thus is a valuable approach to the in vitro study of miRNAs.

  8. Wild-type p53 gene expression sensitizes radioresistant esophageal cancer cell lines

    International Nuclear Information System (INIS)

    Gao Xianshu; Lu Fuhe; Zhou Zhiguo; Song Yonghui; Qiao Xueying; Wan Jun

    2002-01-01

    Objective: To define the radiosensitizing effect of wild-type p3 (Wt-p53) on human radioresistant esophageal cancer cell lines and the application of p53 gene therapy combined with radiotherapy. Methods: The human esophageal cancer cell lines TE-13 and its radioresistant variant TE-13R50 derived from repeated irradiation were initially transfected with Ad5CMV-p53, a recombined adenovirus vector containing human Wt-p53 cDNA and cytomegalovirus (CMV) promoter. The impact of Ad5CMV-p53 expression on radiation sensitivity was observed and analyzed both after transfected cell lines (in vitro) and their transplanted tumors (in vivo) had been irradiated. Results: Significant difference in radiosensitivity between the TE-13 (D 0 = 1.38 Gy) and TE-13R50 (D 0 = 2.48 Gy) cell lines was confirmed. When Ad5CMV-p53 had been transfected and expressed in there cells, their sensitivity to irradiation was enhanced obviously, with declined D 0 values of 0.97 Gy and 1.14 Gy, respectively. On the other hand, the growth rate of transplanted tumors in nude mice was more suppressed by combined radiation and injection of Ad5CMV-p53, as compared with irradiation alone, especially for TE-13R50. Conclusion: The potentiation of adenovirus-mediated wt-p53 gene expression has a significant impact on improving the radiosensitivity of esophageal cancer cell lines

  9. Cloning and Expression of Luteinizing Hormone Subunits in Chinese Hamster Ovary Cell Line

    Directory of Open Access Journals (Sweden)

    Zeinab Soleimanifar

    2016-10-01

    Full Text Available Background: Luteinizing hormone (LH was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively.Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary (CHO eukaryotic system.Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR.  Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody.Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly.

  10. Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

    International Nuclear Information System (INIS)

    Xu, Ling; Hausmann, Martin; Dietmaier, Wolfgang; Kellermeier, Silvia; Pesch, Theresa; Stieber-Gunckel, Manuela; Lippert, Elisabeth; Klebl, Frank; Rogler, Gerhard

    2010-01-01

    Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab

  11. 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.

  12. Protein disulfide isomerases: Impact of thapsigargin treatment on their expression in melanoma cell lines.

    Science.gov (United States)

    Silva, Zélia; Veríssimo, Teresa; Videira, Paula A; Novo, Carlos

    2015-08-01

    Anti-cancer treatments usually elevate the content of unfolded or misfolded proteins in the endoplasmic reticulum (ER). Here we aimed to get insights into the relation between sensitivity of melanoma cell lines to the ER stress inducer thapsigargin (THG) and the genetic expression of protein disulfide isomerase family members (PDIs). The expression of PDIs was analysed by flow cytometry and real-time PCR. The results showed that SK-MEL-30, the less THG sensitive cell line, displays higher basal PDIs' expression levels and the sensitivity is increased by the PDIs inhibitor bacitracin. While SK-MEL-30 PDIs' expression is not THG dose-dependent, an increase in glucose related protein 78 (GRP78), PDIA5, PDIA6, and thioredoxin-related-transmembrane proteins' (TMX3 and TMX4) expression, in response to higher drug concentrations, was observed in MNT-1. The differences in PDIs' gene expression in MNT-1 suggest a different response to ER stress compared to the other cell lines and highlight the importance of understanding the diversity among cancer cells. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

    Science.gov (United States)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Ye, C.; Vassilev, P. M.; Sanders, J. L.; Brown, E. M.

    2001-01-01

    We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.

  14. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

  15. Human hepatocellular carcinoma cell lines exhibit multidrug resistance unrelated to MRD1 gene expression.

    Science.gov (United States)

    Shen, D W; Lu, Y G; Chin, K V; Pastan, I; Gottesman, M M

    1991-03-01

    Multidrug resistance of human cancer cells may result from expression of P-glycoprotein, the product of the MRD1 gene, acting as an energy-dependent drug efflux pump. However, direct evidence that expression of the MDR1 gene contributes to the multidrug resistance of human liver carcinomas has not been established. In this study, we tested five cell lines derived from human hepatocellular carcinomas for sensitivity to a variety of drugs used widely as anticancer agents; these included vinblastine, doxorubicin, actinomycin D, mitomycin C, 5-fluorouracil, 6-mercaptopurine, melphalan, methotrexate, cis-platinum and etoposide (VP-16). All five hepatoma cell lines were resistant at different levels to these chemicals compared to human KB cells. Although it has been demonstrated that resistance to vinblastine, colchicine, doxorubicin and actinomycin D in human multidrug-resistant cells is associated with overexpression of P-glycoprotein, very little expression of P-glycoprotein was found in these human hepatoma cells. Neither verapamil nor quinidine, inhibitors of the drug efflux pump, were able to overcome multidrug resistance in hepatoma cells. These results indicate that the multidrug resistance phenotype in human hepatocellular carcinoma cells cannot be attributed to expression of the MDR1 gene, but that novel mechanisms may account for the resistance of these cancer cells.

  16. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Schilling, D.; Multhoff, G. [Klinikum rechts der Isar der Technischen Univ. Muenchen (Germany). Dept. of Radiation Oncology; Helmholtz Center Munich, CCG - Innate Immunity in Tumor Biology, Munich (Germany). German Research Center for Environmental Health - Inst. of Pathology; Bayer, C.; Emmerich, K.; Molls, M.; Vaupel, P. [Klinikum rechts der Isar der Technischen Univ. Muenchen (Germany). Dept. of Radiation Oncology; Huber, R.M. [Klinikum der Univ. Muenchen (Germany). Dept. of Pneumology

    2012-04-15

    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.)

  18. Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters.

    NARCIS (Netherlands)

    Wilmer, M.J.G.; Saleem, M.A.; Masereeuw, R.; Ni, L.; Velden, T.J.A.M. van der; Russel, F.G.M.; Mathieson, P.W.; Monnens, L.A.H.; Heuvel, L.P.W.J. van den; Levtchenko, E.N.

    2010-01-01

    Reabsorption of filtered solutes from the glomerular filtrate and excretion of waste products and xenobiotics are the main functions of the renal proximal tubular (PT) epithelium. A human PT cell line expressing a range of functional transporters would help to augment current knowledge in renal

  19. Gene expression profile of colon cancer cell lines treated with SN-38

    DEFF Research Database (Denmark)

    Wallin, A; Francis, P; Nilbert, M

    2010-01-01

    the incidence in fact has increased. To improve chemotherapy and enable personalised treatment, the need of biomarkers is of great significance. In this study, we evaluated the gene expression profiles of the colon cancer cell lines treated with SN-38, the active metabolite of topoisomerase-1 inhibitor...

  20. Gene expression profiles of human promyelocytic leukemia cell lines exposed to volatile organic compounds.

    Science.gov (United States)

    Sarma, Sailendra Nath; Kim, Youn-Jung; Ryu, Jae-Chun

    2010-05-27

    Benzene, toluene, o-xylene, ethylbenzene, trichloroethylene and dichloromethane are the most widely used volatile organic compounds (VOCs), and their toxic mechanisms are still undefined. This study analyzed the genome-wide expression profiles of human promyelocytic leukemia HL-60 cells exposed to VOCs using a 35-K whole human genome oligonucleotide microarray to ascertain potential biomarkers. Genes with a significantly increased expression levels (over 1.5-fold and p-values lines to VOC exposure.

  1. Inflammation-induced chemokine expression in uveal melanoma cell lines stimulates monocyte chemotaxis

    DEFF Research Database (Denmark)

    Jehs, Tina; Faber, Carsten; Juel, Helene B

    2014-01-01

    of activated T cells on the expression of chemotactic cytokines in UM cells. Furthermore, we examined the ability of stimulated UM cells to attract monocytes. METHODS: We used an in vitro coculture system in which UM cell lines and T cells were cultured together, but separated by a membrane. Uveal melanoma...... resulted in an upregulation of chemokines such as CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5, VEGF, intracellular adhesion molecule 1 (ICAM1), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The upregulation of these molecules was confirmed at the protein level. This increase of chemokines...

  2. Differential expression of human homeodomain TGIFLX in brain tumor cell lines.

    Directory of Open Access Journals (Sweden)

    Reza Raoofian

    2013-12-01

    Full Text Available Glioblastoma is the most common and the most lethal primary brain cancer. This malignancy is highly locally invasive, rarely metastatic and resistant to current therapies. Little is known about the distinct molecular biology of glioblastoma multiforme (GBM in terms of initiation and progression. So far, several molecular mechanisms have been suggested to implicate in GBM development. Homeodomain (HD transcription factors play central roles in the expression of genomic information in all known eukaryotes. The TGIFX homeobox gene was originally discovered in human adult testes. Our previous study showed implications of TGIFLX in prostate cancer and azoospermia, although the molecular mechanism by which TGIFLX acts is unknown. Moreover, studies reported that HD proteins are involved in normal and abnormal brain developments. We examined the expression pattern of TGIFLX in different human brain tumor cell lines including U87MG, A172, Daoy and 1321N1. Interestingly, real time RT-PCR and western blot analysis revealed a high level of TGIFLX expression in A172 cells but not in the other cell lines. We subsequently cloned the entire coding sequence of TGIFLX gene into the pEGFP-N1 vector, eukaryotic expression vector encoding eGFP, and transfected into the U-87 MG cell line. The TGIFLX-GFP expression was confirmed by real time RT-PCR and UV-microscopic analysis. Upon transfection into U87 cells, fusion protein TGIFLX-GFP was found to locate mainly in the nucleus. This is the first report to determine the nuclear localization of TGIFLX and evaluation of its expression level between different brain tumor cell lines. Our data also suggest that TGIFLX gene dysregulation could be involved in the pathogenesis of some human brain tumors.

  3. Differential Expression Profiles of the Transcriptome in Breast Cancer Cell Lines Revealed by Next Generation Sequencing

    Directory of Open Access Journals (Sweden)

    Yu Shi

    2017-11-01

    Full Text Available Background/Aims: As MCF-7 and MDA-MB-231 cells are the typical cell lines of two clinical breast tumour subtypes, the aim of the present study was to elucidate the transcriptome differences between MCF-7 and MDA-MB-231 breast cancer cell lines. Methods: The mRNA, miRNA (MicroRNA and lncRNA (Long non-coding RNA expression profiles were examined using NGS (next generation sequencing instrument Illumina HiSeq-2500. GO (Gene Ontology and KEGG (Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to identify the biological functions of differentially expressed coding RNAs. Subsequently, we constructed an mRNA-ncRNA (non-coding RNA targeting regulatory network. Finally, we performed RT-qPCR (real-time quantitative PCR to confirm the NGS results. Results: There are sharp distinctions of the coding and non-coding RNA profiles between MCF-7 and MDA-MB-231 cell lines. Among the mRNAs and ncRNAs with the most differential expression, SLPI, SOD2, miR-7, miR-143 and miR-145 were highly expressed in MCF-7 cells, while CD55, KRT17, miR-21, miR-10b, miR-9, NEAT1 and PICSAR were over-expressed in MDA-MB-231 cells. Differentially expressed mRNAs are primarily involved in biological processes of locomotion, biological adhesion, ECM-receptor interaction pathway and focal adhesion. In the targeting regulatory network of differentially expressed RNAs, mRNAs and miRNAs are primarily associated with tumour metastasis, but the functions of lncRNAs remain uncharacterized. Conclusion: These results provide a basis for future studies of breast cancer metastasis and drug resistance.

  4. Comprehensive sampling of gene expression in human cell lines with massively parallel signature sequencing

    OpenAIRE

    Jongeneel, C. Victor; Iseli, Christian; Stevenson, Brian J.; Riggins, Gregory J.; Lal, Anita; Mackay, Alan; Harris, Robert A.; O'Hare, Michael J.; Neville, A. Munro; Simpson, Andrew J. G.; Strausberg, Robert L.

    2003-01-01

    Whereas information is rapidly accumulating about the structure and position of genes encoded in the human genome, less is known about the complexity and relative abundance of their expression in individual human cells and tissues. Here, we describe the characteristics of the transcriptomes of two cultured cell lines, HB4a (normal breast epithelium) and HCT-116 (colon adenocarcinoma), using massively parallel signature sequencing (MPSS). We generated in excess of 107 short signature sequences...

  5. Dual regulation of P-glycoprotein expression by Trichostatin A in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Balaguer Trinidad

    2012-07-01

    Full Text Available Abstract Background It has been reported that the histone deacetylase inhibitor (iHDAc trichostatin A (TSA induces an increase in MDR1 gene transcription (ABCB1. This result would compromise the use of iHDACs in combination with other cytotoxic agents that are substrates of P-glycoprotein (Pgp. It has also been reported the use of alternative promoters by the ABCB1 gene and the existence of a translational control of Pgp protein. Finally, the ABCB1 gene is located in a genetic locus with the nested gene RUNDC3B in the complementary DNA strand, raising the possibility that RUNDC3B expression could interfere with ABCB1 alternative promoter regulation. Methods A combination of RT-PCR, real time RT-PCR, Western blot and drug accumulation assays by flow cytometry has been used in this study. Results The iHDACs-induced increase in MDR1 mRNA levels is not followed by a subsequent increase in Pgp protein levels or activity in several pancreatic and colon carcinoma cell lines, suggesting a translational control of Pgp in these cell lines. In addition, the MDR1 mRNA produced in these cell lines is shorter in its 5′ end that the Pgp mRNA produced in cell lines expressing Pgp protein. The different size of the Pgp mRNA is due to the use of alternative promoters. We also demonstrate that these promoters are differentially regulated by TSA. The translational blockade of Pgp mRNA in the pancreatic carcinoma cell lines could be related to alterations in the 5′ end of the MDR1 mRNA in the Pgp protein expressing cell lines. In addition, we demonstrate that the ABCB1 nested gene RUNDC3B expression although upregulated by TSA is independent of the ABCB1 alternative promoter used. Conclusions The results show that the increase in MDR1 mRNA expression after iHDACs treatment is clinically irrelevant since this mRNA does not render an active Pgp protein, at least in colon and pancreatic cancer cell lines. Furthermore, we demonstrate that TSA in fact, regulates

  6. 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.

  7. Association of differential gene expression with imatinib mesylate and omacetaxine mepesuccinate toxicity in lymphoblastoid cell lines

    Science.gov (United States)

    2012-01-01

    Background Imatinib mesylate is currently the drug of choice to treat chronic myeloid leukemia. However, patient resistance and cytotoxicity make secondary lines of treatment, such as omacetaxine mepesuccinate, a necessity. Given that drug cytotoxicity represents a major problem during treatment, it is essential to understand the biological pathways affected to better predict poor drug response and prioritize a treatment regime. Methods We conducted cell viability and gene expression assays to determine heritability and gene expression changes associated with imatinib and omacetaxine treatment of 55 non-cancerous lymphoblastoid cell lines, derived from 17 pedigrees. In total, 48,803 transcripts derived from Illumina Human WG-6 BeadChips were analyzed for each sample using SOLAR, whilst correcting for kinship structure. Results Cytotoxicity within cell lines was highly heritable following imatinib treatment (h2 = 0.60-0.73), but not omacetaxine treatment. Cell lines treated with an IC20 dose of imatinib or omacetaxine showed differential gene expression for 956 (1.96%) and 3,892 transcripts (7.97%), respectively; 395 of these (0.8%) were significantly influenced by both imatinib and omacetaxine treatment. k-means clustering and DAVID functional annotation showed expression changes in genes related to kinase binding and vacuole-related functions following imatinib treatment, whilst expression changes in genes related to cell division and apoptosis were evident following treatment with omacetaxine. The enrichment scores for these ontologies were very high (mostly >10). Conclusions Induction of gene expression changes related to different pathways following imatinib and omacetaxine treatment suggests that the cytotoxicity of such drugs may be differentially tolerated by individuals based on their genetic background. PMID:22917222

  8. Roles of histamine on the expression of aldehyde dehydrogenase 1 in endometrioid adenocarcinoma cell line

    Science.gov (United States)

    Wang, Yi; Jiang, Yang; Ikeda, Jun-ichiro; Tian, Tian; Sato, Atsushi; Ohtsu, Hiroshi; Morii, Eiichi

    2014-01-01

    Cancer-initiating cells (CICs) are a limited number of cells that are essential for maintenance, recurrence, and metastasis of tumors. Aldehyde dehydrogenase 1 (ALDH1) has been recognized as a marker of CICs. We previously reported that ALDH1-high cases of uterine endometrioid adenocarcinoma showed poor prognosis, and that ALDH1 high population was more tumorigenic, invasive, and resistant to apoptosis than ALDH1 low population. Histamine plays a critical role in cancer cell proliferation, migration, and invasion. Here, we examined the effect of histamine on ALDH1 expression in endometrioid adenocarcinoma cell line. The addition of histamine increased ALDH1 high population, which was consistent with the result that histamine enhanced the invasive ability and the resistance to anticancer drug. Among 4 types of histamine receptors, histamine H1 and H2 receptor (H1R and H2R) were expressed in endometrioid adenocarcinoma cell line. The addition of H1R agonist but not H2R agonist increased ALDH1. The antagonist H1R but not H2R inhibited the effect of histamine on ALDH1 expression. These results indicated that histamine increased the expression of ALDH1 via H1R but not H2R. These findings may provide the evidence for exploring a new strategy to suppress CICs by inhibiting ALDH1 expression with histamine. PMID:25045085

  9. 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.

  10. Roles of histamine on the expression of aldehyde dehydrogenase 1 in endometrioid adenocarcinoma cell line.

    Science.gov (United States)

    Wang, Yi; Jiang, Yang; Ikeda, Jun-Ichiro; Tian, Tian; Sato, Atsushi; Ohtsu, Hiroshi; Morii, Eiichi

    2014-10-01

    Cancer-initiating cells (CICs) are a limited number of cells that are essential for maintenance, recurrence, and metastasis of tumors. Aldehyde dehydrogenase 1 (ALDH1) has been recognized as a marker of CICs. We previously reported that ALDH1-high cases of uterine endometrioid adenocarcinoma showed poor prognosis, and that ALDH1 high population was more tumorigenic, invasive, and resistant to apoptosis than ALDH1 low population. Histamine plays a critical role in cancer cell proliferation, migration, and invasion. Here, we examined the effect of histamine on ALDH1 expression in endometrioid adenocarcinoma cell line. The addition of histamine increased ALDH1 high population, which was consistent with the result that histamine enhanced the invasive ability and the resistance to anticancer drug. Among 4 types of histamine receptors, histamine H1 and H2 receptor (H1R and H2R) were expressed in endometrioid adenocarcinoma cell line. The addition of H1R agonist but not H2R agonist increased ALDH1. The antagonist H1R but not H2R inhibited the effect of histamine on ALDH1 expression. These results indicated that histamine increased the expression of ALDH1 via H1R but not H2R. These findings may provide the evidence for exploring a new strategy to suppress CICs by inhibiting ALDH1 expression with histamine. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  11. Differential expression of the MT-1E gene in estrogen-receptor-positive and -negative human breast cancer cell lines.

    Science.gov (United States)

    Friedline, J A; Garrett, S H; Somji, S; Todd, J H; Sens, D A

    1998-01-01

    The goal of this study was to determine which of the 10 functional metallothionein (MT) genes are expressed in four human breast cancer cell lines and whether expression varies among the cell lines. Using reverse transcription polymerase chain reaction (RT-PCR) technology, it was shown that there was no expression of mRNA for the MT-1A, MT-1B, MT-1F, MT-1G, MT-1H, MT-3, and MT-4 genes in any of the four cell lines. All four cell lines were shown to express mRNA for the MT-2A and MT-1X genes. The expression level of mRNA for the MT-2A gene demonstrated modest differences among the cell lines, whereas expression of the MT-1X gene was consistent. In contrast, mRNA for the MT-1E gene was expressed in only two of the four cell lines and expression correlated to the estrogen receptor status of the cell lines. The two estrogen-receptor-positive cell lines showed no mRNA expression for the MT-1E gene. In the two estrogen-receptor-negative cell lines, mRNA expression for the MT-1E gene was elevated with expression levels similar to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. The cellular content of MT protein was also shown to be elevated in the estrogen-receptor-negative cell lines that express MT-1E mRNA. These results suggest a possible relationship between estrogen receptor status and MT-1E gene expression in human breast cancer.

  12. Alterations in gene expression profiles between radioresistant and radiosensitive cell lines

    International Nuclear Information System (INIS)

    Zhou Fuxiang; Zhou Yunfeng; Xie Conghua; Dai Jing; Cao Zhen; Yu Haijun; Liao Zhengkai; Luo Zhiguo

    2007-01-01

    Objective: To study the-difference of gene expressions by the contrastive model including the cells with same pathological origin and genetic background, but definitely different radioresponse, and to find the main molecular targets related to radiosensitivity. Methods: Human larynx squamous carcinoma cell, Hep -2 was irradiated with dose of 637 cGy repeatedly to establish a radioresistant daughter cell line. The radiobiology characteristics were obtained using clone forming assay. The difference of gene expression between parent and daughter cells was detected by cDNA microarray using two different arrays including 14000 genes respectively. Results: A radioresistant cell strain Hep-2R was isolated from its parental strain Hep-2 cell. The SF 2 , D 0 , α, β for Hep-2R cell line were 0.6798, 3.24, 0.2951 and 0.0363, respectively, while 0.4148, 2.06, 0.1074 and 0.0405 for Hep-2, respectively (for SF 2 , χ 2 =63.957, P<0.001). Compared with Hep-2 cells, the expressions of 41 genes were significantly altered in the radioresistant Hep-2R cells, including 22 genes up-regulated and 19 genes down-regulated, which were involved in DNA repair, regulation of the cell cycle, cell proliferation, cytoskeleton, protein synthesis, cellular metabolism and especially apoptosis which is responsible for the different radiosensitivity between these two larynx cancer cells. The telomere protection protein gene, POT1, was the mostly up-regulated by 3.348 times. Conclusions: There is difference of gene expression between the radioresistant contrastive models. POT1 gene may be the target of radiosensitization. (authors)

  13. Analysis of the regulation of fatty acid binding protein 7 expression in human renal carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Sugiyama Takayuki

    2011-07-01

    Full Text Available Abstract Background Improving the treatment of renal cell carcinoma (RCC will depend on the development of better biomarkers for predicting disease progression and aiding the design of appropriate therapies. One such marker may be fatty acid binding protein 7 (FABP7, also known as B-FABP and BLBP, which is expressed normally in radial glial cells of the developing central nervous system and cells of the mammary gland. Melanomas, glioblastomas, and several types of carcinomas, including RCC, overexpress FABP7. The abundant expression of FABP7 in primary RCCs compared to certain RCC-derived cell lines may allow the definition of the molecular components of FABP7's regulatory system. Results We determined FABP7 mRNA levels in six RCC cell lines. Two were highly expressed, whereas the other and the embryonic kidney cell line (HEK293 were weakly expressed FABP7 transcripts. Western blot analysis of the cell lines detected strong FABP7 expression only in one RCC cell line. Promoter activity in the RCC cell lines was 3- to 21-fold higher than that of HEK293. Deletion analysis demonstrated that three FABP7 promoter regions contributed to upregulated expression in RCC cell lines, but not in the HEK293 cell. Competition analysis of gel shifts indicated that OCT1, OCT6, and nuclear factor I (NFI bound to the FABP7 promoter region. Supershift experiments indicated that BRN2 (POU3F2 and NFI bound to the FABP7 promoter region as well. There was an inverse correlation between FABP7 promoter activity and BRN2 mRNA expression. The FABP7-positive cell line's NFI-DNA complex migrated faster than in other cell lines. Levels of NFIA mRNA were higher in the HEK293 cell line than in any of the six RCC cell lines. In contrast, NFIC mRNA expression was lower in the HEK293 cell line than in the six RCC cell lines. Conclusions Three putative FABP7 promoter regions drive reporter gene expression in RCC cell lines, but not in the HEK293 cell line. BRN2 and NFI may be key

  14. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...... and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

  15. Analysis of P53 mutations and their expression in 56 colorectal cancer cell lines

    DEFF Research Database (Denmark)

    Liu, Ying; Bodmer, Walter F

    2006-01-01

    A comprehensive analysis of the TP53 gene and its protein status was carried out on a panel of 56 colorectal cancer cell lines. This analysis was based on a combination of denaturing HPLC mutation screening of all exons of the p53 gene, sequencing the cDNA, and assessing the function of the p53...... protein by assaying the induced expression of phosphorylated p53 and p21 after exposing cells to gamma-rays. In a few cases where there was no production of p53 message nor evidence of functional p53 protein, all of the p53 exons were sequenced directly. Thirteen of the 56 cell lines had functional p53......, 21 lines had missense mutations (one of which made no detectable protein), 4 lines produced no p53 transcripts, and the remaining 18 lines carried truncating TP53 mutations. Thus, our results showed a relatively high frequency of TP53 mutations (76.8%) in our cell lines, with almost half...

  16. Increased Expression of Serglycin in Specific Carcinomas and Aggressive Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Angeliki Korpetinou

    2015-01-01

    Full Text Available In the present pilot study, we examined the presence of serglycin in lung, breast, prostate, and colon cancer and evaluated its expression in cell lines and tissues. We found that serglycin was expressed and constitutively secreted in culture medium in high levels in more aggressive cancer cells. It is worth noticing that aggressive cancer cells that harbor KRAS or EGFR mutations secreted serglycin constitutively in elevated levels. Furthermore, we detected the transcription of an alternative splice variant of serglycin lacking exon 2 in specific cell lines. In a limited number of tissue samples analyzed, serglycin was detected in normal epithelium but was also expressed in higher levels in advanced grade tumors as shown by immunohistochemistry. Serglycin staining was diffuse, granular, and mainly cytoplasmic. In some cancer cells serglycin also exhibited membrane and/or nuclear immunolocalization. Interestingly, the stromal cells of the reactive tumor stroma were positive for serglycin, suggesting an enhanced biosynthesis for this proteoglycan in activated tumor microenvironment. Our study investigated for first time the distribution of serglycin in normal epithelial and cancerous lesions in most common cancer types. The elevated levels of serglycin in aggressive cancer and stromal cells may suggest a key role for serglycin in disease progression.

  17. Increased Expression of Serglycin in Specific Carcinomas and Aggressive Cancer Cell Lines.

    Science.gov (United States)

    Korpetinou, Angeliki; Papachristou, Dionysios J; Lampropoulou, Angeliki; Bouris, Panagiotis; Labropoulou, Vassiliki T; Noulas, Argyrios; Karamanos, Nikos K; Theocharis, Achilleas D

    2015-01-01

    In the present pilot study, we examined the presence of serglycin in lung, breast, prostate, and colon cancer and evaluated its expression in cell lines and tissues. We found that serglycin was expressed and constitutively secreted in culture medium in high levels in more aggressive cancer cells. It is worth noticing that aggressive cancer cells that harbor KRAS or EGFR mutations secreted serglycin constitutively in elevated levels. Furthermore, we detected the transcription of an alternative splice variant of serglycin lacking exon 2 in specific cell lines. In a limited number of tissue samples analyzed, serglycin was detected in normal epithelium but was also expressed in higher levels in advanced grade tumors as shown by immunohistochemistry. Serglycin staining was diffuse, granular, and mainly cytoplasmic. In some cancer cells serglycin also exhibited membrane and/or nuclear immunolocalization. Interestingly, the stromal cells of the reactive tumor stroma were positive for serglycin, suggesting an enhanced biosynthesis for this proteoglycan in activated tumor microenvironment. Our study investigated for first time the distribution of serglycin in normal epithelial and cancerous lesions in most common cancer types. The elevated levels of serglycin in aggressive cancer and stromal cells may suggest a key role for serglycin in disease progression.

  18. Novel stable HBV producing cell line systems for expression and screening antiviral inhibitor of hepatitis B virus in human hepatoma cell line.

    Science.gov (United States)

    Ogura, Naoki; Ogawa, Kazuya; Watashi, Koichi; Ito, Takayoshi; Wakita, Takaji

    2018-03-25

    Chronic hepatitis B virus (HBV) infection is currently a major public health burden. Therefore, there is an urgent need for the development of novel antiviral inhibitors. The stable HBV-producing cell lines of genotype D are widely used to investigate the HBV life cycle and to evaluate antiviral agents. However, stable HBV-producing cell lines of different genotypes do not exist. To construct more convenient and efficient novel cell systems, stable cell lines of genotypes A, B, and C were established using a full-length HBV genome sequence isolated from chronic HBV patients in human hepatoma HepG2 cells. Novel HBV clones were identified and stable HBV-producing cell lines derived from these clones were constructed. HBV replication activities demonstrated time-dependent expression, and the novel cell lines were susceptible to several antiviral inhibitors with no cytotoxicity. Furthermore, infectious viruses were produced from these cell lines. In conclusion, we have established novel stable HBV-producing cell line systems of genotypes A, B, and C. These systems can provide valuable tools for screening antiviral agents and analyzing viral phenotypes in vitro. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

    International Nuclear Information System (INIS)

    Nemoto, Kiyomitsu; Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki; Fujiwara, Hironori; Yokosuka, Akihito; Mimaki, Yoshihiro; Ohizumi, Yasushi; Degawa, Masakuni

    2013-01-01

    Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

  20. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Kiyomitsu, E-mail: nemoto@u-shizuoka-ken.ac.jp [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)

    2013-02-15

    Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

  1. Cycling CD34 expression in subpopulations of head and neck squamous cell carcinoma cell lines is involved in radioresistance and change in cytokeratin expression profile.

    Science.gov (United States)

    Ettl, Tobias; Hautmann, Matthias; Reichert, Torsten E; Bauer, Richard

    2017-11-01

    The expression of the hair follicle stem cell marker CD34 was analyzed in five different head and neck squamous cell carcinoma (HNSCC) cell lines with different antibodies. All HNSCC cell lines expressed CD34 on their cell surface. After cell cycle synchronization via serum starvation, we observed cyclic CD34 expression in HNSCC cells dependent on cell cycle progression via immunofluorescent staining and flow cytometric analysis. Investigation of the CD34(+) and CD34(-) HNSCC populations revealed most of the cells in S-phase and G2/M-Phase in CD34(+) cells in contrast to CD34(-) cells. Knockdown of CD34 in HNSCC cells led to diminished clonal expansion in a colony forming assay after subjecting the cells to ionizing radiation. Furthermore, knockdown of CD34 after cell cycle synchronization induced high CK1, CK4, and CK5 gene expression and downregulation of CK10 gene expression as shown by Taqman ® quantitative PCR analysis. The expression levels of CK1 and CK10 were verified via western blot analysis. In summary, our study shows that CD34 plays a role during cell cycle progression of head and neck squamous cell carcinoma and additionally is involved in irradiation resistance and differentiation of malignant oral keratinocytes.

  2. Expression of nma, a novel gene, inversely correlates with the metastatic potential of human melanoma cell lines and xenografts

    NARCIS (Netherlands)

    Degen, W. G.; Weterman, M. A.; van Groningen, J. J.; Cornelissen, I. M.; Lemmers, J. P.; Agterbos, M. A.; Geurts van Kessel, A.; Swart, G. W.; Bloemers, H. P.

    1996-01-01

    nma, a novel gene, was isolated by using a subtractive hybridization technique in which the gene expression was compared in a panel of human melanoma cell lines with different metastatic potential. nma mRNA expression (1.5 kb) is high in poorly metastatic human melanoma cell lines and xenografts and

  3. Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR

    DEFF Research Database (Denmark)

    de Bruin, M; Miyake, K; Litman, Thomas

    1999-01-01

    The emergence of several newly identified members of the ABC transporter family has necessitated the development of antagonists that are able to inhibit more than one transporter. We assessed the ability of the chemosensitizer GF120918 to function as a multispecific antagonist using cytotoxicity...... assays, rhodamine and calcein efflux assays, and confocal microscopy in cell lines expressing different multidrug resistance transporters. At a concentration of 1 microM in cytotoxicity assays, GF120918 was able to sensitize both S1-B1-20, a subline expressing P-glycoprotein (Pgp), and S1-M1......-80, a subline expressing a newly identified mitoxantrone transporter, MXR. GF120918 was ineffective in sensitizing MRP-overexpressing MCF-7 VP-16 cells to etoposide as determined by cytotoxicity studies. In flow cytometry experiments, rhodamine 123 efflux in S1-B1-20 cells was decreased at GF120918...

  4. TCLP: an online cancer cell line catalogue integrating HLA type, predicted neo-epitopes, virus and gene expression.

    Science.gov (United States)

    Scholtalbers, Jelle; Boegel, Sebastian; Bukur, Thomas; Byl, Marius; Goerges, Sebastian; Sorn, Patrick; Loewer, Martin; Sahin, Ugur; Castle, John C

    2015-11-20

    Human cancer cell lines are an important resource for research and drug development. However, the available annotations of cell lines are sparse, incomplete, and distributed in multiple repositories. Re-analyzing publicly available raw RNA-Seq data, we determined the human leukocyte antigen (HLA) type and abundance, identified expressed viruses and calculated gene expression of 1,082 cancer cell lines. Using the determined HLA types, public databases of cell line mutations, and existing HLA binding prediction algorithms, we predicted antigenic mutations in each cell line. We integrated the results into a comprehensive knowledgebase. Using the Django web framework, we provide an interactive user interface with advanced search capabilities to find and explore cell lines and an application programming interface to extract cell line information. The portal is available at http://celllines.tron-mainz.de.

  5. Radiation up-regulated the expression of VEGF in a canine oral melanoma cell line

    International Nuclear Information System (INIS)

    Flickinger, I.; Rütgen, B.C.; Gerner, W.; Tichy, A.; Saalmüller, A.; Kleiter, M.; Calice, I.

    2013-01-01

    To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance. (author)

  6. Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

    Science.gov (United States)

    Jiao, Wei; Liu, Fei; Tang, Feng-Zhu; Lan, Jiao; Xiao, Rui-Ping; Chen, Xing-Zhou; Ye, Hui-Lan; Cai, Yong-Lin

    2013-01-01

    To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

  7. 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)

  8. mRNA expression profile in DLD-1 and MOLT-4 cancer cell lines cultured under Microgravity

    Data.gov (United States)

    National Aeronautics and Space Administration — DLD-1 and MOLT-4 cell lines were cultured in a Rotating cell culture system to simulate microgravity and mRNA expression profile was observed in comparison to Static...

  9. Nuclear expression of claudin-3 in human colorectal adenocarcinoma cell lines and tissues.

    Science.gov (United States)

    Tokuhara, Yasunori; Morinishi, Tatsuya; Matsunaga, Toru; Sakai, Manabu; Sakai, Takayoshi; Ohsaki, Hiroyuki; Kadota, Kyuichi; Kushida, Yoshio; Haba, Reiji; Hirakawa, Eiichiro

    2018-01-01

    Claudins are members of a large family of transmembrane proteins, which are essential for the formation of tight junctions and have a significant effect on the biological behavior of tumor progression. Previous studies have demonstrated that several claudins show aberrant expression patterns in numerous types of cancer. The present study investigated the expression and localization of claudin-3 and claudin-7 in human colorectal adenocarcinoma cell lines and tissues. The protein expression levels of claudin-3 and claudin-7 were determined using immunocytochemical and immunohistochemical staining. Claudin-3, but not claudin-7, exhibited nuclear localization in the human colorectal adenocarcinoma Caco-2 and SW620 cell lines. Surgically resected colorectal adenocarcinoma tissue specimens were obtained, and the associations between the expression of claudin-3 or claudin-7 and various clinicopathological parameters were analyzed. The membranous expression rates of claudin-3 and claudin-7 were 58.0 and 50.0%, while their nuclear expression rates were 22.0 and 2.0%, respectively. The membranous expression of claudin-3 and claudin-7 was not associated with any clinicopathological factors, whereas the nuclear expression of claudin-3 was associated with histological type and was significantly increased in colorectal mucinous adenocarcinomas compared with that in well- to moderately-differentiated colorectal adenocarcinomas (P<0.01). However, no associations were observed between the nuclear expression of claudin-7 and any clinicopathological parameter. In conclusion, the nuclear expression of claudin-3 in colorectal mucinous adenocarcinoma may be involved in the biological transformation of tumors. The results from the present study indicated that claudin-3 is an important protein associated with histological type and has potential as a prognostic marker. Although the mechanisms underlying the nuclear localization of claudin-3 in tumorigenesis have not yet been elucidated in

  10. Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line

    International Nuclear Information System (INIS)

    Choi, Hyun-Jeung; Kim, Tae Yong; Ruiz-Llorente, Sergio; Jeon, Min Ji; Han, Ji Min; Kim, Won Gu; Shong, Young Kee; Kim, Won Bae

    2012-01-01

    Introduction: Patients with metastatic thyroid cancers that do not uptake iodine need effective therapeutic option. Differentiation-inducing agents have been tried to restore functional expression of sodium iodide symporter (NIS) without success. Our objective was to assess the effect of alpha-lipoic acid (ALA), known as potential antioxidant, on expression of sodium iodide symporter in thyroid cancer cells. Methods: Human thyroid cancer-derived cell lines, TPC-1, were treated with ALA, and changes in NIS mRNA and protein expression were measured. ALA's effect on NIS gene promoter was evaluated, and functional NIS expression was assessed by iodide uptake assay. Results: Treatment with ALA increased NIS mRNA expression up to ten folds of control dose-dependently after 24 h of exposure. ALA increased NIS promoter activity, and increased iodide uptake by 1.6 fold. ALA induced expression of NIS protein, but had no significant effect on the plasma membrane trafficking. ALA increased phosphorylation of CREB and nuclear translocation of pCREB, and co-treatment of ALA and trichostatin A increased iodide uptake by three folds in TPC-1 cells. Conclusions: ALA is a potential agent to increase NIS transcription in TPC-1. It could be used as an adjunctive agent to increase efficacy of radioiodine therapy if combined with a strategy to increase NIS protein trafficking to cell membrane.

  11. MHC class I-related chain A and B ligands are differentially expressed in human cervical cancer cell lines

    Directory of Open Access Journals (Sweden)

    Bravo-Cuellar Alejandro

    2011-06-01

    Full Text Available Abstract Background Natural killer (NK cells are an important resource of the innate immune system directly involved in the spontaneous recognition and lysis of virus-infected and tumor cells. An exquisite balance of inhibitory and activating receptors tightly controls the NK cell activity. At present, one of the best-characterized activating receptors is NKG2D, which promotes the NK-mediated lysis of target cells by binding to a family of cell surface ligands encoded by the MHC class I chain-related (MIC genes, among others. The goal of this study was to describe the expression pattern of MICA and MICB at the molecular and cellular levels in human cervical cancer cell lines infected or not with human papillomavirus, as well as in a non-tumorigenic keratinocyte cell line. Results Here we show that MICA and MICB exhibit differential expression patterns among HPV-infected (SiHa and HeLa and non-infected cell lines (C33-A, a tumor cell line, and HaCaT, an immortalized keratinocyte cell line. Cell surface expression of MICA was higher than cell surface expression of MICB in the HPV-positive cell lines; in contrast, HPV-negative cells expressed lower levels of MICA. Interestingly, the MICA levels observed in C33-A cells were overcome by significantly higher MICB expression. Also, all cell lines released higher amounts of soluble MICB than of soluble MICA into the cell culture supernatant, although this was most pronounced in C33-A cells. Additionally, Real-Time PCR analysis demonstrated that MICA was strongly upregulated after genotoxic stress. Conclusions This study provides evidence that even when MICA and MICB share a high degree of homology at both genomic and protein levels, differential regulation of their expression and cell surface appearance might be occurring in cervical cancer-derived cells.

  12. The radiosensitivity of glioblastoma cell lines after hypoxia-induced Bax expression

    International Nuclear Information System (INIS)

    Chen, J.K.; Hu, L.J.; Kong, E.L.; Lamborn, K.R.; Deen, D.F.

    2003-01-01

    Full text: Radiation therapy is the most effective treatment after surgery for patients with malignant gliomas. However, the hypoxic cells exclusive to tumor tissue have proven resistant to both radiotherapy and many forms of chemotherapy. In order to specifically target these hypoxic cells, U-251 MG and U-87 MG human glioblastoma cells were stably transfected with constructs containing the suicide gene Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible-factor 1 (HIF-1) binds to HRE and facilitates the transcription of downstream genes. Previously, hypoxia-induced Bax expression in transfected U-251 and U-87 clone cells has been shown to increase cell killing. The benefits of the gene therapy could be further expanded if Bax also acted to increase the sensitivity of these clone cells to radiation. To determine whether this was the case, parent and clone cells were irradiated with graded doses of X-rays under hypoxic conditions. These cells were then left hypoxic for varying durations of time, after which they were incubated for two weeks under aerated conditions to assay for clonogenic cell survival. After less than an hour under hypoxia, both U-251 and U-87 clone cells appeared significantly more sensitive to radiation than their respective parent cells. However, after longer amounts of time under anoxia, higher surviving fractions were found in each clone that were consistent with those of their respective parent cell line, showing that potentially lethal damage repair (PLDR) had occurred in the clone cells. Parent cells did not exhibit PLDR. Results are inconclusive at this point in time. Western blot analyses detailing the amount of Bax expression at each time point as well as further research exploring different durations of hypoxia will be necessary to reveal the nature of the correlation between Bax expression and radiosensitivity. Supported by NS-42927 and CA-85356

  13. Radiostatine and radioiodine uptake characterization in sodium iodine symporter-expressing cell lines

    International Nuclear Information System (INIS)

    Petrich, T.; Helmeke, H.J.; Meyer, G.J.; Knapp, W.H.; Poetter, E.

    2002-01-01

    Full text: The sodium iodide symporter (NIS) has been recognized as an attractive target for cancer gene therapy. Here we investigated NIS-mediated transport of the high LET α-emitter astatine, 211 At, in comparison to radioiodine. A constitutive expression vector harbouring the human NIS cDNA was used in combination with reporter gene vectors for transient transfection of 13 different human cancer cell lines. Radioiodine uptake was measured as well as transfection efficiencies. Six stable NIS-expressing cell lines (3 derived from thyroid carcinomas, 2 colon carcinoma, 1 glioblastoma) were generated by antibiotic selection. NIS expression was monitored by immunohistochemistry and RT-PCR. Subsequently the radioastatine and radioiodine uptake characteristics of genetically modified cells were studied in comparison to the respective control cells. After xenotransplantation in nude mice in vivo tumor imaging by scintigraphy and biodistribution studies following organ removal were performed. Transient transfection of NIS cDNA led to high specific sodium perchlorate-sensitive radioiodine uptake in NIS-expressing cells that roughly correlates to transfection efficiencies. Similarly, stable NIS-expressing cell lines were able to concentrate high levels of radioiodine and in addition showed comparable transport capacity for radioastatine. Accumulation of 211 At was inhibited by sodium perchlorate like iodide uptake and displayed dependency an extracellular Na + - and I - -ions as well. Compared to wash-out experiments in cell culture the effective half life of radioiodine and radioastatine in vivo was significantly prolonged. Preliminary dose calculations by MIRD concepts indicated higher tumor radiation doses for 211 At compared to 131 I. Tumor cells of different origins transfected with the NIS-expression vector specifically and significantly take-up radioiodine and radioastatine in vitro and in vivo. The data provide direct evidence that the NIS efficiently transports

  14. 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.

  15. Heterogeneity of expression of epithelial-mesenchymal transition (EMT markers in melanocytes and melanoma cell lines

    Directory of Open Access Journals (Sweden)

    Ji Eun eKim

    2013-05-01

    Full Text Available The epithelial-mesenchymal transition (EMT describes a reversible switch from an epithelial-like to a mesenchymal-like phenotype. It is essential for the development of the normal epithelium and also contributes to the invasive properties of carcinomas. At the molecular level, the EMT transition is characterised by a series of coordinated changes including downregulation of the junctional protein E-cadherin (CDH1, up-regulation of transcriptional repressors of E-cadherin such as Snail (SNAI1 and Slug (SNAI2, and up-regulation of N-cadherin. We wished to determine whether cultured normal melanocytes and melanoma cell lines, which are derived from the neural crest, showed signs of a similarly coordinated phenotypic switch. We investigated normal melanocytes and 25 cell lines derived from New Zealand patients with metastatic melanoma. Most lines had been previously genotyped for common mutations such as BRAF, NRAS, PIK3CA, TP53 and CDKN2A. Expression of E-cadherin, N-cadherin, MITF, Snail, Slug, Axl, p53 and Hdm2 was compared by western blotting. Normal melanocytes expressed each of these proteins except for Snail, while normal melanocytes and almost every melanoma line expressed Slug. Expression of individual markers among different melanoma lines varied from high to low or undetectable. Quantitation of western blots showed that expression of MITF-M, the melanocyte-specific isoform of MITF, was positively related to that of E-cadherin but inversely related to that of N-cadherin and Axl. There was also no apparent relationship between expression of any particular marker and the presence of BRAF, NRAS, PIK3CA, TP53 or CDKN2A mutations. The results suggest that melanomas do not show the classical epithelial and mesenchymal phenotypes but rather display either high E-cadherin/high MITF-M expression on one hand, or high N-cadherin/high Axl expression on the other. These may correspond to differentiated and invasive phenotypes in vivo.

  16. Radiation induced expression of survivin in Ewing sarcoma cell-lines

    International Nuclear Information System (INIS)

    Sheikh-Mounessi, F.; Willich, N.; Greve, B.

    2009-01-01

    Full text: Introduction: Survivin belongs to the Inhibitor of Apoptosis Protein Family (IAP), is a protein of 16.5 kD and active as a homodimer. It is overexpressed in nearly all human tumors and has a vital function in cell division and apoptotic processes. Beside its role as a relevant prognostic and predictive factor it was described to be a molecular target to improve effectiveness of radiotherapy. We investigated the radiation induced survivin expression in Ewing sarcoma cell-lines. Methods: Ewing sarcoma cells were either irradiated with 10 Gy X-ray and harvested at different time points (0, 2, 4, 6, 10 and 24 h) or irradiated with different doses (0, 2, 5 and 10 Gy) and harvested 24 h later. Protein and mRNA expression was analysed by Westernblot or Real-Time PCR. Results: Directly after irradiation with 10 Gy X-ray survivin mRNA expression was increased in relation to the reference GAPDH. Protein expression was increased in a time dependent manner and reached a maximum after 24h. Three of four investigated cell-lines showed a significant dose dependent increase of survivin protein concentration 24h after irradiation. The same three cell-lines showed a LD50 of >30 Gy. The line with the lowest dose dependent survivin induction was investigated to be most radiosensitive (LD50 = 24 Gy). Discussion: Ewing sarcoma is a childhood tumor with relatively poor prognosis. This tumor often shows significant therapeutic resistance to chemo- and/or radiotherapy. It would be of high interest to find new therapeutic approaches for its treatment. We found a remarkable overexpression of survivin in untreated Ewing sarcoma and a time and dose dependent increase of survivin protein concentration after irradiation with X-ray. The cell-line with the lowest survivin induction showed the highest radiosensitivity. In conclusion, our results show that survivin is an inducible radioresistance factor in Ewing sarcoma. This may open new therapeutic options to treat this aggressive

  17. Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines

    DEFF Research Database (Denmark)

    Litvinov, Ivan V; Cordeiro, Brendan; Fredholm, Simon Mayland

    2014-01-01

    Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype......R-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA....... by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines...

  18. 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.

  19. Enhancing dengue virus maturation using a stable furin over-expressing cell line.

    Science.gov (United States)

    Mukherjee, Swati; Sirohi, Devika; Dowd, Kimberly A; Chen, Zhenguo; Diamond, Michael S; Kuhn, Richard J; Pierson, Theodore C

    2016-10-01

    Flaviviruses are positive-stranded RNA viruses that incorporate envelope (E) and premembrane (prM) proteins into the virion. Furin-mediated cleavage of prM defines a required maturation step in the flavivirus lifecycle. Inefficient prM cleavage results in structurally heterogeneous virions with unique antigenic and functional characteristics. Recent studies with dengue virus suggest that viruses produced in tissue culture cells are less mature than those produced in primary cells. In this study, we describe a Vero cell line that ectopically expresses high levels of human furin (Vero-furin) for use in the production of more homogenous mature flavivirus populations. Laboratory-adapted and clinical dengue virus isolates grow efficiently in Vero-furin cells. Biochemical and structural techniques demonstrate efficient prM cleavage in Vero-furin derived virus preparations. These virions also were less sensitive to neutralization by antibodies that bind efficiently to immature virions. This furin-expressing cell line will be of considerable utility for flavivirus neutralization and structural studies. Published by Elsevier Inc.

  20. 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.

  1. The morphologies of breast cancer cell lines in three-dimensionalassays correlate with their profiles of gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Kenny, Paraic A.; Lee, Genee Y.; Myers, Connie A.; Neve, RichardM.; Semeiks, Jeremy R.; Spellman, Paul T.; Lorenz, Katrin; Lee, Eva H.; Barcellos-Hoff, Mary Helen; Petersen, Ole W.; Gray, Joe W.; Bissell, MinaJ.

    2007-01-31

    3D cell cultures are rapidly becoming the method of choice for the physiologically relevant modeling of many aspects of non-malignant and malignant cell behavior ex vivo. Nevertheless, only a limited number of distinct cell types have been evaluated in this assay to date. Here we report the first large scale comparison of the transcriptional profiles and 3D cell culture phenotypes of a substantial panel of human breast cancer cell lines. Each cell line adopts a colony morphology of one of four main classes in 3D culture. These morphologies reflect, at least in part, the underlying gene expression profile and protein expression patterns of the cell lines, and distinct morphologies were also associated with tumor cell invasiveness and with cell lines originating from metastases. We further demonstrate that consistent differences in genes encoding signal transduction proteins emerge when even tumor cells are cultured in 3D microenvironments.

  2. Combination of FACS and homologous recombination for the generation of stable and high-expression engineered cell lines.

    Directory of Open Access Journals (Sweden)

    Lei Shi

    Full Text Available Traditionally, cell line generation requires several months and involves screening of over several hundred cell clones for high productivity before dozens are selected as candidate cell lines. Here, we have designed a new strategy for the generation of stable and high-expression cell lines by combining homologous recombination (HR and fluorescence-activated cell sorting (FACS. High expression was indicated by the expression of secreted green fluorescent protein (SEGFP. Parental cell lines with the highest expression of SEGFP were then selected by FACS and identified by stability analysis. Consequently, HR vectors were constructed using the cassette for SEGFP as the HR region. After transfecting the HR vector, the cells with negative SEGFP expression were enriched by FACS. The complete exchange between SEGFP and target gene (TNFR-Fc cassettes was demonstrated by DNA analysis. Compared with the traditional method, by integrating the cassette containing the gene of interest into the pre-selected site, the highest producing cells secreted a more than 8-fold higher titer of target protein. Hence, this new strategy can be applied to isolated stable cell lines with desirable expression of any gene of interest. The stable cell lines can rapidly produce proteins for researching protein structure and function and are even applicable in drug discovery.

  3. Paclitaxel and trastuzumab treatment affects insulin growth factor I expression in breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Yu-Xian Qian

    2015-12-01

    Full Text Available Breast cancer is the most common type of cancers and second primary cause of death among women. Insulin-like growth factor I (IGF-1 signaling pathway plays a vital role in cancer cell survival, proliferation, chemotaxis and angiogenesis. In this study, the effect of combination of two drugs, paclitaxel and trastuzumab on IGF signaling and cell cycle arrest in breast cancer cell lines, T47D and Hs0578T were explored. The interaction of paclitaxel and trastuzumab on IGF-1 signaling pathway was studied with IGF-1 and phosphoinositide 3-kinase inhibitor, LY294002. The protein expression of IGF signaling molecules were reduced in the drug treated cancer cells. LY294002 and IGF-1 with paclitaxel and trastuzumab treatment inhibited phosphorylated Akt. During G0/G1 phase, cell cycle arrest and accumulation of apoptotic cells were observed in drug treated cancer cells. The synergistic effect of paclitaxel and trastuzumab decreased the multiplication of breast cancer cells by altering the expression of IGF-I signaling molecules. This combination proves to be one of the useful methods to treat breast cancer.

  4. The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in Breast Cancer Cell Lines

    National Research Council Canada - National Science Library

    Dovichi, Norman J

    2005-01-01

    .... Capillary sieving electrophoresis and capillary micellar electrophoresis were used to characterize proteins in single cells in one-dimensional separations, while the two techniques were combined...

  5. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines.

    Science.gov (United States)

    Boegel, Sebastian; Löwer, Martin; Bukur, Thomas; Sahin, Ugur; Castle, John C

    Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel-opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antigen (HLA) type and HLA expression of tumor cell lines, characterizations necessary for the development of cancer vaccines, have remained largely incomplete and, such information, when available, has been distributed in many publications. Here, we determine the 4-digit HLA type and HLA expression of 167 cancer and 10 non-cancer cell lines from publically available RNA-Seq data. We use standard NGS RNA-Seq short reads from "whole transcriptome" sequencing, map reads to known HLA types, and statistically determine HLA type, heterozygosity, and expression. First, we present previously unreported HLA Class I and II genotypes. Second, we determine HLA expression levels in each cancer cell line, providing insights into HLA downregulation and loss in cancer. Third, using these results, we provide a fundamental cell line "barcode" to track samples and prevent sample annotation swaps and contamination. Fourth, we integrate the cancer cell-line specific HLA types and HLA expression with available cell-line specific mutation information and existing HLA binding prediction algorithms to make a catalog of predicted antigenic mutations in each cell line. The compilation of our results are a fundamental resource for all researchers selecting specific cancer cell lines based on the HLA type and HLA expression, as well as for the development of immunotherapeutic tools for novel cancer treatment modalities.

  6. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines

    Science.gov (United States)

    Boegel, Sebastian; Löwer, Martin; Bukur, Thomas; Sahin, Ugur; Castle, John C

    2014-01-01

    Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel­opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antigen (HLA) type and HLA expression of tumor cell lines, characterizations necessary for the development of cancer vaccines, have remained largely incomplete and, such information, when available, has been distributed in many publications. Here, we determine the 4-digit HLA type and HLA expression of 167 cancer and 10 non-cancer cell lines from publically available RNA-Seq data. We use standard NGS RNA-Seq short reads from “whole transcriptome” sequencing, map reads to known HLA types, and statistically determine HLA type, heterozygosity, and expression. First, we present previously unreported HLA Class I and II genotypes. Second, we determine HLA expression levels in each cancer cell line, providing insights into HLA downregulation and loss in cancer. Third, using these results, we provide a fundamental cell line “barcode” to track samples and prevent sample annotation swaps and contamination. Fourth, we integrate the cancer cell-line specific HLA types and HLA expression with available cell-line specific mutation information and existing HLA binding prediction algorithms to make a catalog of predicted antigenic mutations in each cell line. The compilation of our results are a fundamental resource for all researchers selecting specific cancer cell lines based on the HLA type and HLA expression, as well as for the development of immunotherapeutic tools for novel cancer treatment modalities. PMID:25960936

  7. Comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression.

    Directory of Open Access Journals (Sweden)

    Byungwoo Ryu

    2007-07-01

    Full Text Available Gene expression profiling has revolutionized our ability to molecularly classify primary human tumors and significantly enhanced the development of novel tumor markers and therapies; however, progress in the diagnosis and treatment of melanoma over the past 3 decades has been limited, and there is currently no approved therapy that significantly extends lifespan in patients with advanced disease. Profiling studies of melanoma to date have been inconsistent due to the heterogeneous nature of this malignancy and the limited availability of informative tissue specimens from early stages of disease.In order to gain an improved understanding of the molecular basis of melanoma progression, we have compared gene expression profiles from a series of melanoma cell lines representing discrete stages of malignant progression that recapitulate critical characteristics of the primary lesions from which they were derived. Here we describe the unsupervised hierarchical clustering of profiling data from melanoma cell lines and melanocytes. This clustering identifies two distinctive molecular subclasses of melanoma segregating aggressive metastatic tumor cell lines from less-aggressive primary tumor cell lines. Further analysis of expression signatures associated with melanoma progression using functional annotations categorized these transcripts into three classes of genes: 1 Upregulation of activators of cell cycle progression, DNA replication and repair (CDCA2, NCAPH, NCAPG, NCAPG2, PBK, NUSAP1, BIRC5, ESCO2, HELLS, MELK, GINS1, GINS4, RAD54L, TYMS, and DHFR, 2 Loss of genes associated with cellular adhesion and melanocyte differentiation (CDH3, CDH1, c-KIT, PAX3, CITED1/MSG-1, TYR, MELANA, MC1R, and OCA2, 3 Upregulation of genes associated with resistance to apoptosis (BIRC5/survivin. While these broad classes of transcripts have previously been implicated in the progression of melanoma and other malignancies, the specific genes identified within each class

  8. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

    Directory of Open Access Journals (Sweden)

    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  9. Resveratrol raisesin vitroanticancer effects of paclitaxel in NSCLC cell line A549 through COX-2 expression.

    Science.gov (United States)

    Kong, Fanhua; Zhang, Runqi; Zhao, Xudong; Zheng, Guanlin; Wang, Zhou; Wang, Peng

    2017-09-01

    The aim of this study was to determine the raising anticancer effects of resveratrol (Res) on paclitaxel (PA) in non-small cell lung cancer (NSCLC) cell line A549. The 10 µg/ml of Res had no effect on human fetal lung fibroblast MRC-5 cells or on A549 cancer cells and the 5 or 10 µg/ml of PA also had no effect on MRC-5 normal cells. PA-L (5 µg/ml) and PA-H (10 µg/ml) had the growth inhibitory effects in NSCLC cell line A549, and Res increased these growth inhibitory effects. By flow cytometry experiment, after Res (5 µg/ml)+PA-H (10 µg/ml) treatment, the A549 cells showed the most apoptosic cells compared to other group treatments, and after additional treatment with Res, the apoptosic cells of both two PA concentrations were raised. Res+PA could reduce the mRNA and protein expressions of COX-2, and Res+PA could reduce the COX-2 related genes of VEGF, MMP-1, MMP-2, MMP-9, NF-κB, Bcl-2, Bcl-xL, procollagen I, collagen I, collagen III and CTGF, TNF-α, IL-1β, iNOS and raise the TIMP-1, TIMP-2, TIMP-3, IκB-α, p53, p21, caspase-3, caspase-8, caspase-9, Bax genes compared to the control cells and the PA treated cells. From these results, it can be suggested that Res could raise the anticancer effects of PA in A549 cells, thus Res might be used as a good sensitizing agent for PA.

  10. Construction and application of an inducible system for homogenous expression levels in bulk cell lines.

    Directory of Open Access Journals (Sweden)

    Jun Yu

    Full Text Available Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline-inducible elements, BAC (bacterial artificial chromosome and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein allows FACS (fluorescence activated cell sorting and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a beta-catenin-ERalpha fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-beta-catenin-ERalpha vector demonstrated sensitive inducibility by Tet or Dox (doxycycline in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.

  11. Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines.

    Directory of Open Access Journals (Sweden)

    Maricla Galetti

    Full Text Available BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism.The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes.Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake.Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

  12. Functional Expression of TRPV4 Cation Channels in Human Mast Cell Line (HMC-1).

    Science.gov (United States)

    Kim, Kyung Soo; Shin, Dong Hoon; Nam, Joo Hyun; Park, Kyung Sun; Zhang, Yin Hua; Kim, Woo Kyung; Kim, Sung Joon

    2010-12-01

    Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, 23~25℃ to a moderately high temperature (MHT, 37~39℃ to activate TRPV3/4, a high temperature (HT, 44~46℃ to activate TRPV1, or a very high temperature (VHT, 53~55℃ to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV4 activator 4α-phorbol 12,13-didecanoate (4αPDD, 1µM) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The [Ca(2+)](c) of HMC-1 cells was also increased by MHT or by 4αPDD. In summary, our present study indicates that HMC-1 cells express Ca(2+)-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.

  13. 3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

    International Nuclear Information System (INIS)

    Hatta, Mitsutoki; Naganuma, Kaori; Kato, Kenichi; Yamazaki, Jun

    2015-01-01

    In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.

  14. 3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Hatta, Mitsutoki, E-mail: hatta@college.fdcnet.ac.jp [Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka (Japan); Naganuma, Kaori [Department of Oral and Maxillofacial Surgery, Fukuoka Dental College, Fukuoka (Japan); Kato, Kenichi; Yamazaki, Jun [Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka (Japan)

    2015-12-04

    In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.

  15. Characterization of new cell line stably expressing CHI3L1 oncogene

    Directory of Open Access Journals (Sweden)

    Chekhonin V. P.

    2011-06-01

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

  16. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

    Science.gov (United States)

    Das, Krishna; Eisel, David; Lenkl, Clarissa; Goyal, Ashish; Diederichs, Sven; Dickes, Elke; Osen, Wolfram; Eichmüller, Stefan B

    2017-01-01

    In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

  17. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

    Directory of Open Access Journals (Sweden)

    Krishna Das

    Full Text Available In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY, were transfected with an expression plasmid encoding a β2m-specific single guide (sgRNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO clones did not give rise to tumors in syngeneic mice (C57BL/6N, unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

  18. Gene expression profiles in asbestos-exposed epithelial and mesothelial lung cell lines

    Directory of Open Access Journals (Sweden)

    Kaski Samuel

    2007-03-01

    Full Text Available Abstract Background Asbestos has been shown to cause chromosomal damage and DNA aberrations. Exposure to asbestos causes many lung diseases e.g. asbestosis, malignant mesothelioma, and lung cancer, but the disease-related processes are still largely unknown. We exposed the human cell lines A549, Beas-2B and Met5A to crocidolite asbestos and determined time-dependent gene expression profiles by using Affymetrix arrays. The hybridization data was analyzed by using an algorithm specifically designed for clustering of short time series expression data. A canonical correlation analysis was applied to identify correlations between the cell lines, and a Gene Ontology analysis method for the identification of enriched, differentially expressed biological processes. Results We recognized a large number of previously known as well as new potential asbestos-associated genes and biological processes, and identified chromosomal regions enriched with genes potentially contributing to common responses to asbestos in these cell lines. These include genes such as the thioredoxin domain containing gene (TXNDC and the potential tumor suppressor, BCL2/adenovirus E1B 19kD-interacting protein gene (BNIP3L, GO-terms such as "positive regulation of I-kappaB kinase/NF-kappaB cascade" and "positive regulation of transcription, DNA-dependent", and chromosomal regions such as 2p22, 9p13, and 14q21. We present the complete data sets as Additional files. Conclusion This study identifies several interesting targets for further investigation in relation to asbestos-associated diseases.

  19. The expression of oncogenes on the radiation-induced apoptosis in SCK mammary adenocarcinoma cell line

    International Nuclear Information System (INIS)

    Lee, Hyung Sik; Park, Hong Kyu; Moon, Chang Woo; Yoon, Seon Min; Hur, Won Joo; Jeong, Su Jin; Lee, Sang Hwa; Jeong, Min Ho; Park, Heon Joo

    1999-01-01

    The expression of p53, p21/WAF/CIP, Bcl-2, and Bax underlying the radiation-induced apoptosis in different pH environments using SCK mammary adenocarcinoma cell line was investigated. Mammary adenocarcinoma cells of A/J mice (SCK cells) in exponential growth phase were irradiated with a linear accelerator at room temperature. The cells were irradiated with 12 Gy and one hour later, the media was replaced with fresh media at a different pHs. After incubation at 37 .deg. for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. Western blot analysis was used to monitor p53, P21/WAF/CIP, BcI-2, and Bax protein levels. The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. The radiation-induced G2/M arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. Considerable amounts of p53 and p21 proteins already existed at pH 7.5 and increased the level of p53 and p21 significantly after 12 Gy X-irradiation. An incubation at pH6.6 after 12 Gy X-irradiation did not change the level of p53 and p21 protein levels significantly. BcI-2 proteins were not significantly affected by radiation and showed no correlation with cell susceptibility to radiation-induced apoptosis in different pHs. An exposure to 12 Gy of X-rays increased the level of Bax protein at pH 7.5 but at pH 6.6, it was slight. The molecular mechanism underlying radiation-induced apoptosis in different pH environments using SCK mammary adenocarcinoma cell line was dependent of the expression p53 and p21/WAF/CIP proteins. We may propose following hypothesis that an acidic stress augments the radiation-induced G2/M arrest, which inhibiting the irradiated cells undergo post-mitotic apoptosis. The effects of environmental acidity on anti-apoptotic and pro-apoptotic function of BcI-2

  20. Gene expression profile of colon cancer cell lines treated with SN-38

    DEFF Research Database (Denmark)

    Wallin, A; Francis, P; Nilbert, M

    2010-01-01

    the incidence in fact has increased. To improve chemotherapy and enable personalised treatment, the need of biomarkers is of great significance. In this study, we evaluated the gene expression profiles of the colon cancer cell lines treated with SN-38, the active metabolite of topoisomerase-1 inhibitor......Colorectal cancer is the third most common form of cancer in the industrial countries. Due to advances regarding the treatments, primarily development of improved surgical methods and the ability to make the earlier diagnosis, the mortality has remained constant during the past decades even though...

  1. Alteration of miRNA expression in a sulfur mustard resistant cell line.

    Science.gov (United States)

    Rothmiller, Simone; Wolf, Markus; Worek, Franz; Steinritz, Dirk; Thiermann, Horst; Schmidt, Annette

    2017-08-18

    MicroRNAs (miRNAs) are responsible for post-transcriptional control of protein expression. Numerous miRNAs have been identified to be responsible for the resistance of tumor cells to cytostatic drugs. Possibly, the same miRNAs also play a role in the sulfur mustard (SM)-resistance of the keratinocyte cell line HaCaT/SM as alkylating cytostatics exhibit similar cytotoxic effects as SM. Basal expression levels of 1920 miRNAs in total were analyzed in HaCaT/SM compared to the origin human keratinocyte cell line HaCaT. The effect for selected miRNAs on cell survival was analyzed using antagomirs for ectopic miRNA level decrease or miRNA mimics for increase. Cell survival was calculated as SM dose-dependent-curves. Out of 1920 miRNAs analyzed, 49 were significantly up- and 29 were significantly downregulated in HaCaT/SM when compared to HaCaT controls. Out of these, 36 could be grouped in miRNA families. Most of the 15 miRNA family members showed either a common increase or decrease. Only the members of miR-10, miR-154, miR-430 and miR-548 family showed an inconsistent picture. The ectopic increase of miR-181 in HaCaT/SM had a positive effect on cell survival in the presence of SM. In summary, the extensive differences in miRNA expression pattern between these cell lines indicate that specific miRNAs may play a role in the resistance mechanism against sulfur mustard. The miR-125b-2 and miR-181b alone are not responsible for the resistance development against SM, but an ectopic increase of miR-181 even enhances the SM resistance of HaCaT/SM. Improving the resistance in normal keratinocytes by treatment with either both miRNAs together or a different combination might be used as an initial step in development of an innovative new drug or prophylactic agent against SM. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Optimized signal peptides for the development of high expressing CHO cell lines.

    Science.gov (United States)

    Kober, Lars; Zehe, Christoph; Bode, Juergen

    2013-04-01

    Recombinant biotherapeutic proteins such as monoclonal antibodies are mostly produced in Chinese hamster ovary (CHO) cells and pharmaceutical companies are interested in an appropriate platform technology for the development of large-scale production processes. A major aim of our study was therefore to improve the secretion efficiency of a recombinant biotherapeutic antibody by optimizing signal peptides. Reporter molecules such as gaussia and vargula luciferase or secreted alkaline phosphatase are frequently used to this end. In striking contrast, we used a biotherapeutic antibody that was fused to 16 different signal peptides during our study. In this way, the secretion efficiency of the recombinant antibody has been analyzed by transient expression experiments in CHO cell lines. Compared to the control signal peptide, it was not possible to achieve higher efficiencies with signal peptides derived from a variety of species or even natural immunoglobulin G signal peptides. The best results were obtained with natural signal peptides derived from human albumin and human azurocidin. These results were confirmed by fed-batch experiments with stably transfected cell pools, in which cell-specific productivities up to 90 pg cell(-1) day(-1) and product concentrations up to 4 g L(-1) could be determined using the albumin signal peptide. Finally, the applicability of the identified signal peptides for both different antibodies and non-antibody products was demonstrated by transient expression experiments. In conclusion, it was found that signal peptides derived from human albumin and human azurocidin are most appropriate to generate cell lines with clearly improved production rates suitable for commercial purposes in a product-independent manner. Copyright © 2012 Wiley Periodicals, Inc.

  3. Hypoxia and Human Genome Stability: Downregulation of BRCA2 Expression in Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Daniele Fanale

    2013-01-01

    Full Text Available Previously, it has been reported that hypoxia causes increased mutagenesis and alteration in DNA repair mechanisms. In 2005, an interesting study showed that hypoxia-induced decreases in BRCA1 expression and the consequent suppression of homologous recombination may lead to genetic instability. However, nothing is yet known about the involvement of BRCA2 in hypoxic conditions in breast cancer. Initially, a cell proliferation assay allowed us to hypothesize that hypoxia could negatively regulate the breast cancer cell growth in short term in vitro studies. Subsequently, we analyzed gene expression in breast cancer cell lines exposed to hypoxic condition by microarray analysis. Interestingly, genes involved in DNA damage repair pathways such as mismatch repair, nucleotide excision repair, nonhomologous end-joining and homologous recombination repair were downregulated. In particular, we focused on the BRCA2 downregulation which was confirmed at mRNA and protein level. In addition, breast cancer cells were treated with dimethyloxalylglycine (DMOG, a cell-permeable inhibitor of both proline and asparaginyl hydroxylases able to induce HIF-1α stabilization in normoxia, providing results comparable to those previously described. These findings may provide new insights into the mechanisms underlying genetic instability mediated by hypoxia and BRCA involvement in sporadic breast cancers.

  4. Distinct lithium-induced gene expression effects in lymphoblastoid cell lines from patients with bipolar disorder.

    Science.gov (United States)

    Fries, Gabriel R; Colpo, Gabriela D; Monroy-Jaramillo, Nancy; Zhao, Junfei; Zhao, Zhongming; Arnold, Jodi G; Bowden, Charles L; Walss-Bass, Consuelo

    2017-11-01

    Lithium is the most commonly prescribed medication for the treatment of bipolar disorder (BD), yet the mechanisms underlying its beneficial effects are still unclear. We aimed to compare the effects of lithium treatment in lymphoblastoid cell lines (LCLs) from BD patients and controls. LCLs were generated from sixty-two BD patients (based on DSM-IV) and seventeen healthy controls matched for age, sex, and ethnicity. Patients were recruited from outpatient clinics from February 2012 to October 2014. LCLs were treated with 1mM lithium for 7 days followed by microarray gene expression assay and validation by real-time quantitative PCR. Baseline differences between groups, as well as differences between vehicle- and lithium-treated cells within each group were analyzed. The biological significance of differentially expressed genes was examined by pathway enrichment analysis. No significant differences in baseline gene expression (adjusted p-value < 0.05) were detected between groups. Lithium treatment of LCLs from controls did not lead to any significant differences. However, lithium altered the expression of 236 genes in LCLs from patients; those genes were enriched for signaling pathways related to apoptosis. Among those genes, the alterations in the expression of PIK3CG, SERP1 and UPP1 were validated by real-time PCR. A significant correlation was also found between circadian functioning and CEBPG and FGF2 expression levels. In summary, our results suggest that lithium treatment induces expression changes in genes associated with the apoptosis pathway in BD LCLs. The more pronounced effects of lithium in patients compared to controls suggest a disease-specific effect of this drug. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

  5. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    Directory of Open Access Journals (Sweden)

    Susanne C. Hammer

    2016-09-01

    Full Text Available Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

  6. Azidothymidine and cisplatin increase p14ARF expression in OVCAR-3 ovarian cancer cell line

    International Nuclear Information System (INIS)

    Vaskivuo, Liisa; Rysae, Jaana; Koivuperae, Johanna; Myllynen, Paeivi; Vaskivuo, Tommi; Chvalova, Katerina; Serpi, Raisa; Savolainen, Eeva-Riitta; Puistola, Ulla; Vaehaekangas, Kirsi

    2006-01-01

    p14 ARF tumor suppressor protein regulates p53 by interfering with mdm2-p53 interaction. p14 ARF is activated in response to oncogenic stimuli but little is known of the responses of endogenous p14 ARF to different types of cellular stress or DNA damage. Azidothymidine (AZT) is being tested in several clinical trials as an enhancer of anticancer chemotherapy. However, the knowledge of the relationship between AZT and cellular pathways, e.g. p53 pathway, is very limited. In this study, we show that AZT, cisplatin (CDDP) and docetaxel (DTX) all induce unique molecular responses in OVCAR-3 ovarian carcinoma cells carrying a mutated p53, while in A2780, ovarian carcinoma and MCF-7 breast carcinoma cells with wild type p53, all of these drugs cause similar p53 responses. We found that endogenous p14 ARF protein in OVCAR-3 cells is down-regulated by DTX but induced by AZT and a short CDDP pulse treatment. In HT-29 colon carcinoma cells with a mutated p53, all treatments down-regulated p14 ARF protein. Both CDDP and AZT increased the expression of p14ARF mRNA in OVCAR-3 cells. Differences in cell death induced by these drugs did not explain the differences in protein and mRNA expressions. No increase in the level of either c-Myc or H-ras oncoproteins was seen in OVCAR-3 cells after AZT or CDDP-treatment. These results suggest that p14 ARF can respond to DNA damage without oncogene activation in cell lines without functional p53

  7. Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin

    Science.gov (United States)

    2013-01-01

    Background Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified. Methods We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR. Results We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines. Conclusions This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific

  8. Tyrosinase-Expressing Neuronal Cell Line as in Vitro Model of Parkinson’s Disease

    Science.gov (United States)

    Hasegawa, Takafumi

    2010-01-01

    Oxidized metabolites of dopamine known as dopamine quinone derivatives are thought to play a pivotal role in the degeneration of nigrostriatal dopaminergic neurons in Parkinson’s disease. Although such quinone derivatives are usually produced via the autoxidation of catecholamines, tyrosinase, which is a key enzyme in melanin biosynthesis via the production of DOPA and subsequent molecules, can potentially accelerate the induction of catecholamine quinone derivatives by its oxidase activity. We have developed neuronal cell lines in which the expression of human tyrosinase was inducible. Overexpression of tyrosinase resulted in increased intracellular dopamine content in association with the formation of melanin pigments in neuronal somata, which eventually causes apoptotic cell death. This cellular model will provide a useful tool for detailed analyses of the neurotoxicity of oxidized catechol metabolites. PMID:20480001

  9. Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines

    Science.gov (United States)

    Galetti, Maricla; Petronini, Pier Giorgio; Fumarola, Claudia; Cretella, Daniele; La Monica, Silvia; Bonelli, Mara; Cavazzoni, Andrea; Saccani, Francesca; Caffarra, Cristina; Andreoli, Roberta; Mutti, Antonio; Tiseo, Marcello; Ardizzoni, Andrea; Alfieri, Roberta R.

    2015-01-01

    Background BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. Aim The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. Methods and Results Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. Conclusions Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells. PMID:26536031

  10. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    International Nuclear Information System (INIS)

    Tsujiuchi, Toshifumi; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

    2006-01-01

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells

  11. Gene expression in cell lines from propionic acidemia patients, carrier parents, and controls.

    Science.gov (United States)

    Chapman, Kimberly A; Bush, William S; Zhang, Zhe

    2015-08-01

    Propionic acidemia (PA) is an inborn of metabolism which usually presents with metabolic acidosis and accumulation of 3-hydroxypropionate among other toxins. Examining the gene expression in lymphoblastoid cell lines (LCLs) from PA patients, their carrier parents and age/sex-matched controls at normal glucose and low glucose growth conditions demonstrated differences among and between these groups. Using three-way ANOVA analysis, four DAVID clusters of response were identified of which three of the four clusters showed that LCLs from carrier parents had an intermediate response between healthy controls and PA patients. These differences included changes in expression of cell cycle regulatory genes, mitochondrial related genes, and transcriptional regulation. In addition, differences also were observed in expression of genes involved in transendothelial migration and focal adhesion at normal growth conditions when comparing the LCLs from PA patients and controls. These studies demonstrate transcriptional differences between LCLs from PA patients, their parents and biochemically normal controls. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Suppression of vascular endothelial growth factor expression by cannabinoids in a canine osteosarcoma cell line

    Directory of Open Access Journals (Sweden)

    Figueiredo AS

    2013-07-01

    Full Text Available Andreza S Figueiredo,1 Hiram J García-Crescioni,1 Sandra C Bulla,1 Matthew K Ross,2 Chelsea McIntosh,1 Kari Lunsford,3 Camilo Bulla11Department of Pathobiology and Population Medicine, 2Department of Basic Sciences, 3Department of Clinical Sciences and Animal Health Center, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, USAAbstract: Vascular endothelial growth factor (VEGF is a key regulator in both physiologic and pathologic angiogenesis, and cannabinoids decrease VEGF release in human and murine cancer cells. The aim of this study was to assess the in vitro effects of a synthetic cannabinoid, WIN-55,212-2, on the expression of the proangiogenic factor VEGF-A in the canine osteosarcoma cell line 8. After analysis of gene expression by quantitative real-time polymerase chain reaction, the compound decreased VEGF-A expression by 35% ± 10% (P < 0.0001 as compared with the control. This synthetic cannabinoid shows promise as a potential inhibitor of angiogenesis, and further studies are warranted to investigate its in vivo effects and to explore the potential of this and related compounds as adjuvant cancer therapy in the dog.Keywords: dog, cancer, angiogenesis, cannabinoids

  13. Lack of expression of preproorexin and orexin receptors genes in human normal and prostate cancer cell lines.

    Science.gov (United States)

    Szyszka, Marta; Paschke, Lukasz; Tyczewska, Marianna; Rucinski, Marcin; Grabowska, Paulina; Malendowicz, Ludwik K

    2015-01-01

    Studies on expression of orexins (OXs) and their receptors in human prostate gland and human prostatic cell lines are scanty and their results contradictory. Regarding this, we carefully reinvestigated this problem on human prostatic cell lines. Expression of preproorexin (ppOX) (6 primer pairs), and orexin receptors 1 and 2 (OXR1, OXR2) (4 and 2 primer pairs, respectively) was assessed by conventional PCR and QPCR in human normal (PrEC, PrSc, PrSmC) and prostate carcinoma (Du145, LNCaP, and PC3) cell lines. We designed intron spanning primers and also we applied primers from earlier publications and commercially available ones. With the designed primer pairs, in all studied cell lines we failed to demonstrate expression of ppOX, OXR1 and OXR2 genes at the mRNA level, while reaction products were observed in control tissues (human placenta and adrenals). Primers applied in earlier studies did not form amplification products specific for preproorexin or orexin 1 receptor. Some commercially available primers for orexin receptor 1 produced false positive results. We found no evidence for the presence of preproorexin-orexin receptors system genes' mRNAs in human prostate cell lines. The reported premises for these genes’ expression in prostate and prostatic cell lines may have arisen either from the presence of non-prostate cells included in the samples or from faulty PCR settings.

  14. Ficus carica latex prevents invasion through induction of let-7d expression in GBM cell lines.

    Science.gov (United States)

    Tezcan, Gulcin; Tunca, Berrin; Bekar, Ahmet; Yalcin, Murat; Sahin, Saliha; Budak, Ferah; Cecener, Gulsah; Egeli, Unal; Demir, Cevdet; Guvenc, Gokcen; Yilmaz, Gozde; Erkan, Leman Gizem; Malyer, Hulusi; Taskapilioglu, Mevlut Ozgur; Evrensel, Turkkan; Bilir, Ayhan

    2015-03-01

    Glioblastoma multiforme (GBM) is one of the deadliest human malignancies. A cure for GBM remains elusive, and the overall survival time is less than 1 year. Thus, the development of more efficient therapeutic approaches for the treatment of these patients is required. Induction of tumor cell death by certain phytochemicals derived from medicinal herbs and dietary plants has become a new frontier for cancer therapy research. Although the cancer suppressive effect of Ficus carica (fig) latex (FCL) has been determined in a few cancer types, the effect of this latex on GBM tumors has not been investigated. Therefore, in the current study, the anti-proliferative activity of FCL and the effect of the FCL-temozolomide (TMZ) combination were tested in the T98G, U-138 MG, and U-87 MG GBM cell lines using the WST-1 assay. The mechanism of cell death was analyzed using Annexin-V/FITC and TUNEL assays, and the effect of FCL on invasion was tested using the chick chorioallantoic membrane assay. To determine the effect of FCL on GBM progression, the expression levels of 40 GBM associated miRNAs were analyzed in T98G cells using RT-qPCR. According to the obtained data, FCL causes cell death in GBM cells with different responses to TMZ, and this effect is synergistically increased in combination with TMZ. In addition, the current study is the first to demonstrate the effect of FCL on modulation of let-7d expression, which may be an important underlying mechanism of the anti-invasive effect of this extract.

  15. Zingiber officinale, Piper retrofractum and Combination Induced Apoptosis and p53 Expression in Myeloma and WiDr Cell Lines

    Directory of Open Access Journals (Sweden)

    HENY EKOWATI

    2012-09-01

    Full Text Available In previous studies, Zingiber officinale, Piper retrofractum, and the combination showed cytotoxic activity, induced apoptosis, and p53 expression of HeLa, T47D, and MCF-7 cell lines. This study was conducted to investigate the cytotoxic and apoptotic activity of Zingiber officinale (ZO, Piper retrofractum (PR, and the combination as well as their effect to p53 expression on Myeloma and WiDr cells. The powder of ZO, PR, and ZO + PR combination (1:1 were macerated with 96% ethanol for 3 x 24 hours. MTT cytotoxic assay was performed on Myeloma and WiDr cell lines. Apoptotic cells were stained with ethidium bromide and acridine orange. Imunohistochemical expression of p53 was examined on Myeloma and WiDr cell lines. Doxorubicin was used as positive control in all assays. Results showed that ZO, PR, and ZO + PR combination had cytotoxic activity on Myeloma cells with IC50 of 28, 36, and 55 mg/ml respectively and WiDr cell lines with IC50 of 74, 158, and 64 mg/ml respectively, induced apoptotic activity, and increased p53 expression on Myeloma and WiDr cells. These results suggest that ZO, PR, and their combination induced Myeloma and WiDr cells in apoptosis through p53 expression.

  16. Frequent hemizygous deletion at 1p36 and hypermethylation downregulate RUNX3 expression in human lung cancer cell lines.

    Science.gov (United States)

    Yanada, Masashi; Yaoi, Takeshi; Shimada, Junichi; Sakakura, Chouhei; Nishimura, Motohiro; Ito, Kazuhiro; Terauchi, Kunihiko; Nishiyama, Katsuhiko; Itoh, Kyoko; Fushiki, Shinji

    2005-10-01

    Runt-related transcription factor 3 (RUNX3) has been recognized as a tumor suppressor gene in gastric cancer because its expression level was reduced or disappeared due to epigenetic changes. To evaluate the usefulness of the RUNX3 gene as a biomarker of lung cancer, we have analyzed the expression of the RUNX3 gene in 15 lung cancer cell lines by real-time reverse transcription-polymerase chain reaction (RT-PCR), and demonstrated that RUNX3 gene expression was reduced or disappeared in all cell lines examined (100%). In addition, we have attempted to classify all the cell lines into three groups according to the expression level; less than 10% (group I), 10-30% (group II) and approximately 50% (group III). We further investigated methylation status of the CpG sites in the exon 1 region of RUNX3 by methylation specific PCR (MSP), and studied the correlation between the expression level and hemizygous deletion as revealed by bicolor fluorescence in situ hybridization (FISH). The CpG sites were hypermethylated in 8 cell lines (53%) and the RUNX3 loci were hemizygously deleted in another 8 cell lines (53%). Furthermore group I, II, and III corresponded well to methylation-positive cell lines, cell lines showing hemizygous deletion, and the rest of cell lines without methylation or hemizygous deletion, respectively. These results suggest that a comprehensive study on RUNX3 using real-time RT-PCR, MSP, and FISH could be beneficial in understanding the pathogenetic mechanisms of human lung cancer at the molecular level.

  17. Oncogene Expression Modulation in Cancer Cell Lines by DNA G-Quadruplex-Interactive Small Molecules.

    Science.gov (United States)

    Francisco, Ana Paula; Paulo, Alexandra

    2017-01-01

    Nucleic acids are prone to structural polymorphism and a number of structures may be formed in addition to the well-known DNA double helix. Among these is a family of nucleic acid four-stranded structures known as G-quadruplexes (G4). These quadruplex structures can be formed by sequences containing repetitive guanine-rich tracks and the analysis of Non-B-DNA database indicated an enrichment of these sequences in genomic regions controlling cellular proliferation, such as for example in the promoter regions of c- MYC, k-RAS, c-KIT, HSP90 and VEGF among others. The broad concept of G4 targeting with small molecules is now generally accepted as a promising novel approach to anticancer therapy and several small molecules with antiproliferative activity in cancer cell lines have also been shown to stabilize these DNA structures, thus suggesting a potential application of G4-interactive small molecules as new anticancer drugs. Herein we review, by targeted oncogene and main chemical scaffold, those G4-interactive small molecules with reported gene expression modulatory activity in cancer cell lines. The data obtained so far are encouraging but further efforts are needed to validate G4 as drug targets and optimize the structure of G4- interactive small molecules into new anticancer drugs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Thymosin beta-10 expression in melanoma cell lines and melanocytic lesions: a new progression marker for human cutaneous melanoma

    NARCIS (Netherlands)

    Weterman, M. A.; van Muijen, G. N.; Ruiter, D. J.; Bloemers, H. P.

    1993-01-01

    When screening a subtraction library for sequences that were specifically expressed in highly metastatic human melanoma cell lines, a cDNA clone was isolated encoding thymosin beta-10. We found that expression of thymosin beta-10 mRNA was associated with metastatic behavior of various human melanoma

  19. 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

  20. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    International Nuclear Information System (INIS)

    Sustarsic, Elahu G.; Junnila, Riia K.; Kopchick, John J.

    2013-01-01

    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

  1. Modulation of integrin-linked kinase (ILK expression in human oesophageal squamous cell carcinoma cell lines by the EGF and TGFβ1 growth factors

    Directory of Open Access Journals (Sweden)

    Veale Robin B

    2006-04-01

    Full Text Available Abstract Background Integrin-linked kinase (ILK is a ubiquitously expressed protein kinase that has emerged as one of the points of convergence between integrin- and growth factor-signalling pathways. Results In this study we identify the ILK isoform expressed in five human oesophageal squamous cell carcinoma cell lines of South African origin as ILK1, and demonstrate its cellular distribution. ILK expression, although similar in the majority of the cell lines, did show variation. Furthermore, the ILK expressed was shown to be catalytically functional. The effect of growth factors on ILK expression was examined. An increase in ILK expression, following EGF and TGFβ1 exposure, was a trend across all the five oesophageal carcinoma cell lines tested. Conclusion These results suggest that growth factor modulation of ILK expression relies on the internalisation/recycling of growth factor receptors and stimulation of the PI3K pathway, which may have implications with regards to cell adhesion and tumourigenesis.

  2. Bortezomib and Arsenic Trioxide Activity on a Myelodysplastic Cell Line (P39: A Gene Expression Study

    Directory of Open Access Journals (Sweden)

    Hakan Savlı

    2015-09-01

    Full Text Available Objective: We aimed to understand the molecular pathways affected by bortezomib and arsenic trioxide treatment on myelomonocytoid cell line P39. Materials and Methods: Oligonucleotide microarray platforms were used for gene expression and pathway analysis. Confirmation studies were performed using quantitative real time PCR. Results: Bortezomib treatment has shown upregulated DIABLO and NF-κBIB (a NF-κB inhibitor and downregulated NF-κB1, NF-κB2, and BIRC1 gene expressions. Combination treatment of the two compounds showed gene expression deregulations in concordance by the results of single bortezomib treatment. Especially, P53 was a pathway more significantly modified and a gene network centralized around the beta estradiol gene. Beta estradiol, BRCA2, and FOXA1 genes were remarkable deregulations in our findings. Conclusion: Results support the suggestions about possible use of proteasome inhibitors in the treatment of high-risk myelodysplastic syndrome (MDS. NF-κB was observed as an important modulator in leukemic transformation of MDS.

  3. Differential Expression of HERV-K (HML-2 Proviruses in Cells and Virions of the Teratocarcinoma Cell Line Tera-1

    Directory of Open Access Journals (Sweden)

    Neeru Bhardwaj

    2015-03-01

    Full Text Available Human endogenous retrovirus (HERV-K (HML-2 proviruses are among the few endogenous retroviral elements in the human genome that retain coding sequence. HML-2 expression has been widely associated with human disease states, including different types of cancers as well as with HIV-1 infection. Understanding of the potential impact of this expression requires that it be annotated at the proviral level. Here, we utilized the high throughput capabilities of next-generation sequencing to profile HML-2 expression at the level of individual proviruses and secreted virions in the teratocarcinoma cell line Tera-1. We identified well-defined expression patterns, with transcripts emanating primarily from two proviruses located on chromosome 22, only one of which was efficiently packaged. Interestingly, there was a preference for transcripts of recently integrated proviruses, over those from other highly expressed but older elements, to be packaged into virions. We also assessed the promoter competence of the 5’ long terminal repeats (LTRs of expressed proviruses via a luciferase assay following transfection of Tera-1 cells. Consistent with the RNASeq results, we found that the activity of most LTRs corresponded to their transcript levels.

  4. Expression of human organic cation transporter 3 in kidney carcinoma cell lines increases chemosensitivity to melphalan, irinotecan, and vincristine.

    Science.gov (United States)

    Shnitsar, Volodymyr; Eckardt, Ronny; Gupta, Shivangi; Grottker, Julia; Müller, Gerhard A; Koepsell, Hermann; Burckhardt, Gerhard; Hagos, Yohannes

    2009-02-15

    Renal cell carcinoma (RCC) is usually chemoresistant. This chemoresistance could be overcome if specific cytostatics are applied for which the RCC expresses an uptake transporter. In the present study, we investigated the expression of solute carrier (SLC) transporters in different RCC lines and their ability to interact with chemotherapeutics. We tested five RCC lines for the expression of different SLCs by reverse transcription-PCR and TaqMan real-time PCR. In two of five RCC lines, A498 and 7860, we observed a highly significant expression of SLC22A3 (hOCT3). Uptake of the organic cation [(3)H]MPP (4-methyl-pyridinium iodide) into these cells and also into hOCT3 stably transfected Chinese hamster ovary (CHO) cells was inhibited by irinotecan, vincristine, and melphalan. The K(i) values [determined from Dixon plots] for irinotecan, vincristine, and melphalan were 1.72 +/- 0.45 micromol/L, 17 +/- 4.81 micromol/L, and 366 +/- 51 micromol/L, respectively. Cytotoxic activities of the selected drugs were tested by [(3)H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays on CHO-hOCT3, A498 (high expression of hOCT3), and ACHN cell lines (low expression of hOCT3). The growth of CHO-hOCT3 was inhibited by 20% more with irinotecan and by 50% more with vincristine compared with nontransfected CHO cells. Melphalan produced 20% to 30% more inhibition in hOCT3-expressing cells compared with nonexpressing control cells. Similar results were obtained for A498 and ACHN cells. Thus, our data support the hypothesis that the sensitivity of tumor cells to chemotherapeutic treatment depends on the expression of transporter proteins mediating specific drug accumulation into target cells.

  5. Assessment of Cytokeratin-19 Gene Expression in Peripheral Blood of Breast Cancer Patients and Breast Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Saeideh Keyvani

    2016-01-01

    Full Text Available Detection of cytokeratin-19 (CK19 expression as an epithelial-specific marker in circulating tumor cells (CTCs of breast cancer patients can be important for diagnostic purposes. Comparison of CK19 expression in breast cancer cell lines can indicate that expression of this marker is different in various breast cancer cell lines based on their category. Thirty-five breast cancer patients were evaluated for detection of CK19 mRNA in their peripheral blood using CK19-specific primers and a nested reverse transcriptase polymerase chain reaction (RT-PCR technique. CK19 expression levels were detected in MCF7, T47D, SK-BR-3, and MDA-MB-231 cell lines by semiquantitative RT-PCR and Western blot analyses. Statistical analysis of our data indicates that there is no significant difference between CK19 expression and histopathological parameters and some molecular markers, including Ki-67, HER-2, and P53, but there are statistically significant correlations between estrogen receptor (P = 0.040 and progesterone receptor ( P = 0.046 with CK19 expression. CK19 expression was detected in MCF7, T47D, and SK-BR-3 cell lines but not in MDA-MB-231 cell line. More studies are needed to determine the relationship between this marker and other markers in the diagnosis and treatment of breast cancer. On the other hand, the study of different markers using breast cancer cell lines as experimental models of breast cancer could have an impact on improving the health outcomes of patients with breast cancer.

  6. A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines

    OpenAIRE

    Boegel, Sebastian; L?wer, Martin; Bukur, Thomas; Sahin, Ugur; Castle, John C

    2014-01-01

    Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel?opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antige...

  7. CD133 expression is not selective for tumor initiating or radioresistant cell populations in the CRC line HCT-116

    International Nuclear Information System (INIS)

    Seidel, Claudia; Dietrich, Antje; Wondrak, Marit; Kunz-Schughart, Leoni A.; Grade, Marian; Ried, Thomas

    2009-01-01

    The hypothesis of certain subpopulations of cancer cells with stem-cell like characteristics that might be responsible for treatment resistance and recurrence of disease is still challenging and under quite controversial discussion. In most studies, surrogate cell surface antigens such as the 92-110 kDa transmembrane glycoprotein CD133 (human Prominin-1) were labeled to isolate particular small cancer cell populations for studying their tumorigenic potential. In colorectal carcinomas (CRC) for example, a small CD133 positive (CD133 + ) cell population has recently been described to be enriched for tumor-initiating/cancer stem cells (TIC/CSC) as compared to the CD133 negative (CD133) population. Furthermore, it was documented that the CD133 + subpopulation could exclusively be maintained in culture as spheres under serum-free conditions. Addition of serum resulted in cell differentiation, growth in 2-D and downregulation of CD133 expression. This would imply that established colorectal cancer (CRC) cell lines that have been grown under adherent, serum-supplemented conditions for years should be devoid of CD133 + cells and TIC/CSC, respectively, which seems contradictory to the finding that many CRC lines produce tumors in nude mice models. In order to gain insight into this paradox, we studied the expression of CD133 in numerous established CRC lines under standard culture conditions and chose one particular cell line based on its expression pattern to study the behavior of CD133 + / CD133 - subpopulations

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

    Directory of Open Access Journals (Sweden)

    Quadros Edward V

    2009-05-01

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

  9. Comparison of Oct4, Sox2 and Nanog Expression in Pancreatic Cancer Cell Lines and Human Pancreatic Tumor

    Directory of Open Access Journals (Sweden)

    Vahideh Assadollahi

    2015-12-01

    Full Text Available Background: Genes are involved in the control of stem cell self-renewal as a new class of molecular markers of cancer. Objectives: In this study, the expression of Oct4, Nanog and Sox2 in cell lines MIA Paca-2, PA-TU-8902 and AsPC-1 and pancreatic cancer tissue were examined. Materials and Methods: In this experimental study, cell lines, MIA Paca-2, PA-TU-8902 and AsPC-1, were cultured in DMEM (Dulbecco’s Modified Eagles Medium and RPMI-1640 (Roswell Park Memorial Institute containing FBS 10% (fetal bovine serum in a 37°C incubator containing Co2 5% and humidity 90%. Samples of tumor and non-cancer pancreatic tumor were purchased Iran tumor bank. Extraction of RNA and synthesis of cDNA was performed. Expression levels of Oct4, Nanog and Sox2 were determined using Real-time PCR. The protein expression levels of target genes in the cell lines were studied by flow cytometry and immunocytochemistry. Results: The expression rate of Oct4, Nanog and Sox2 is more in the cancer cell lines than those in the control (normal tissue samples. The protein expression levels of target genes in the cell lines were confirmed by flow cytometry and immunocytochemistry. Conclusions: The genes are involved in stem cell self-renewal as a new class of molecular markers of cancer that detected in the pancreatic cell lines. Maybe, these genes play important role in the uncontrolled proliferation of cancer cells.

  10. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment.

    Science.gov (United States)

    Sustarsic, Elahu G; Junnila, Riia K; Kopchick, John J

    2013-11-08

    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 this data, GH could be a new therapeutic target in melanoma. Published by Elsevier Inc.

  11. Protection against UVA-induced photooxidative damage in mammalian cell lines expressing increased levels of metallothionein

    International Nuclear Information System (INIS)

    Dudek, E.J.; Roth, R.M.

    1990-01-01

    Metallothionein (MT) is an endogenous low molecular weight protein that is inducible in a variety of eukaryotic cells and has the ability to selectivity bind heavy metal ions such as zinc and the cadmium. Although the exact physiological role of MT is still not understood, there is strong evidence that MT is involved in providing cellular resistance against the damaging effects of heavy metals and in the regulation of intracellular zinc and copper. Recently, it has been demonstrated that MT can scavenge radiation-induced reactive oxygen intermediates in vitro, specifically hydroxyl and superoxide radicals, and because of these observations it has been suggested that MT may provide protection against radiation-induced oxidative stress in vivo. Cell lines expressing increased levels of MT have demonstrated resistance to ionizing radiation, to ultraviolet radiation, and also to various DNA damaging agents including melphalan and cis-diaminedichloroplatinum. It is therefore important to gain some insight into the relationship between cellular MT content and cellular resistance to radiation and other DNA damaging agents. In this study we investigated the role of MT in providing protection against monochromatic 365-nm UVA radiation, which is known to generate intracellular reactive oxygen species that are involved in both DNA damage and cell killing. For this purpose, we used zinc acetate, a potent inducer of MT, to elevate MT levels in V79 Chinese hamster fibroblasts prior to UVA exposure and determined cell survival for uninduced and induced cultures. In order to eliminate any zinc effects other than MT induction, we also isolated and characterized cadmium chloride-resistant clones of V79 cells that have increased steady-state levels of both MT mRNA and protein, and we examined their survival characteristics against 365-nm radiation in the absence of zinc acetate. 14 refs., 3 figs

  12. SATB1 regulates SPARC expression in K562 cell line through binding to a specific sequence in the third intron

    International Nuclear Information System (INIS)

    Li, K.; Cai, R.; Dai, B.B.; Zhang, X.Q.; Wang, H.J.; Ge, S.F.; Xu, W.R.; Lu, J.

    2007-01-01

    Special AT-rich binding protein 1 (SATB1), a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, tethers to a specific DNA sequence and regulates gene expression through chromatin remodeling and HDAC (histone deacetylase complex) recruitment. In this study, a SATB1 eukaryotic expression plasmid was transfected into the human erythroleukemia K562 cell line and individual clones that stably over-expressed the SATB1 protein were isolated. Microarray analysis revealed that hundreds of genes were either up- or down-regulated in the SATB1 over-expressing K562 cell lines. One of these was the extra-cellular matrix glycoprotein, SPARC (human secreted protein acidic and rich in cysteine). siRNA knock-down of SATB1 also reduced SPARC expression, which was consistent with elevated SPARC levels in the SATB1 over-expressing cell line. Bioinformatics software Mat-inspector showed that a 17 bp DNA sequence in the third intron of SPARC possessed a high potential for SATB1 binding; a finding confirmed by Chromatin immunoprecipitation (ChIP) with anti-SATB1 antibody. Our results show for the first time that forced-expression of SATB1 in K562 cells triggers SPARC up-regulation by binding to a 17 bp DNA sequence in the third intron

  13. Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT, MMR, P-Glycoprotein and CD133 Expression

    Science.gov (United States)

    Prados, Jose; Caba, Octavio; Cabeza, Laura; Berdasco, Maria; Gónzalez, Beatriz; Melguizo, Consolación

    2015-01-01

    Background The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated. Methods Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed. Results Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC50 showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC50 did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC50, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines. Conclusions These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma

  14. Effect Of Interferon-γ and TNF-α on MUCl MUCIN Expression in Ovarian Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Sean Clark

    1994-01-01

    Full Text Available In view of the potential uses of cell surface tumour associated antigens in novel anticancer treatment. a study was designed to investigate whether the biological response modifiers interferon-gamma (IFN-γ and tumour necrosis factor-alpha (TNF-α could effect the expression of an epitope on the tumour associated MUC I epithelial mucin. Four ovarian carcinoma cell lines showing high (OAW42 and GG and low (JAM and PEO1 basal expression of MUC1 were treated with 10-1000 U/mL of IFN-γor TNF-α for one or five days. Changes in MUC1 expression in cells exposed to IFN-γ or TNF-α were monitored using an ELISA technique with the monoclonal antibody BC2 which reacts with a core protein epitope on the MUC1 mucin, and then corrected for the number of viable cells present. TNF-α had little effect on MUC1 expression, but one or five days exposure to IFN-γ significantly increased MUC1 expression (p < 0.01 in all cell lines including the two cell lines that initially showed little or no expression.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

  16. Transporter mRNA expression in a conditionally immortalized rat small intestine epithelial cell line (TR-SIE).

    Science.gov (United States)

    Hosoya, Ken-ichi; Tomi, Masatoshi; Takayama, Megumi; Komokata, Yuko; Nakai, Daisuke; Tokui, Taro; Nishimura, Kenji; Ueda, Masatsugu; Obinata, Masuo; Hori, Satoko; Ohtsuki, Sumio; Amidon, Gordon L; Terasaki, Tetsuya

    2004-08-01

    Small intestine epithelial cell lines (TR-SIE), which are established from the small intestine of transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), were used to characterize the mRNA expression of small intestine transporters. TR-SIE cells had a polygonal morphology and expressed cytokeratin protein and villin mRNA. Although the large T-antigen was strongly expressed at 33 degrees C, this was reduced at 37 and 39 degrees C. Concomitantly, the cell growth was arrested at 37 and 39 degrees C compared with that at 33 degrees C, suggesting that TR-SIE cells are conditionally immortalized cell lines. RT-PCR analysis revealed that TR-SIE cells expressed ABCB1 (mdr1a and mdr1b), ABCB4 (mdr2), ABCC2 (mrp2), ABCC6 (mrp6), ABCG1, ABCG2 (bcrp/mxr), Slc21a7 (Oatp3), Slc15a1 (PepT1), and Slc16a1 (Mct1). Conditionally immortalized rat small intestine epithelial cell lines were established from tsA58 Tg rats and expressed the mRNA of intestinal transporters.

  17. Acquired TGF beta 1 sensitivity and TGF beta 1 expression in cell lines established from a single small cell lung cancer patient during clinical progression

    DEFF Research Database (Denmark)

    Nørgaard, P; Damstrup, L; Rygaard, K

    1996-01-01

    Three small cell lung cancer cell lines established from a single patient during longitudinal follow-up were examined for in vitro expression of TGF beta and TGF beta receptors, i.e. the components of an autocrine loop. GLC 14 was established prior to treatment, GLC 16 on relapse after chemotherapy...... was found in GLC 16 and GLC 19. These cell lines were also growth inhibited by exogenously administrated TGF beta 1. TGF beta 1 mRNA and protein in its latent form was only expressed in the radiotherapy-resistant cell line, GLC 19. The results indicate that disease progression in this patient was paralleled...... II receptor gene, as examined by Southern blotting. Also, the type I receptor could not be detected by ligand binding assay in this cell line, despite expression of mRNA for this receptor. This agrees with previous findings that type I receptor cannot bind TGF beta 1 without co-expression of the type...

  18. Expression of E-cadherin in human mast cell line HMC-1.

    Science.gov (United States)

    Nishida, Minoru; Kawai, Kenzo; Tanaka, Makoto; Tegoshi, Tatsuya; Arizono, Naoki

    2003-11-01

    E-cadherin is one of the cell adhesion molecules normally expressed on epithelial cells. We previously reported that murine bone marrow-derived mast cells express E-cadherin that could be involved in homophilic binding with epithelial cell E-cadherin. In the present study we examined whether E-cadherin is also expressed in human mast cell HMC-1. Gene expression of E-cadherin and beta-catenin was observed in HMC-1 by reverse transcription-polymerase chain reaction (RT-PCR), while N-cadherin expression was undetectable. cDNA sequencing of HMC-1 E-cadherin revealed no deletions or mutations. E-cadherin expression in HMC-1 was confirmed by immunoblotting as well as by flow cytometric analyses. In the presence of E-cadherin blocking antibody or a synthetic E-cadherin decapeptide with HAV sequence in culture medium, adhesion of HMC-1 cells to the A431 epithelial cell monolayer was slightly but significantly suppressed. In contrast, N- or P-cadherin decapeptides did not suppress the binding. These results indicated that human mast cell HMC-1 expresses E-cadherin, and is possibly involved in cellular interactions with epithelial cells, while other functions still remain to be elucidated.

  19. Establishment and characterization of a spontaneously immortalized trophoblast cell line (HPT-8) and its hepatitis B virus-expressing clone.

    Science.gov (United States)

    Zhang, Lei; Zhang, Weilu; Shao, Chen; Zhang, Jingxia; Men, Ke; Shao, Zhongjun; Yan, Yongping; Xu, Dezhong

    2011-08-01

    Most trophoblast cell lines currently available to study vertical transmission of hepatitis B virus (HBV) are immortalized by viral transformation. Our goal was to establish and characterize a spontaneously immortalized human first-trimester trophoblast cell line and its HBV-expressing clone. Chorionic villi of Asian human first-trimester placentae were digested with trypsin and collagenase I to obtain the primary trophoblast cell culture. A spontaneously immortalized trophoblast cell line (HPT-8) was analyzed by scanning and transmission electron microscopy, cell cycle analysis, immunohistochemistry and immunofluorescence. HPT-8 cells were stably transfected with the adr subtype of HBV (HPT-8-HBV) and characterized by PCR and enzyme-linked immunosorbent assay. We obtained a clonal derivative of a spontaneously immortalized primary cell clone (HPT-8). HPT-8 cells were epithelioid and polygonal, and formed multinucleate, giant cells. They exhibited microvilli, distinct desmosomes between adjacent cells, abundant endoplasm, lipid inclusions and glycogen granules, which are all characteristic of cytotrophoblasts. HPT-8 cells expressed cytokeratin 7, cytokeratin 18, vimentin, cluster of differentiation antigen 9, epidermal growth factor receptor, stromal cell-derived factor 1 and placental alkaline phosphatase. They secreted prolactin, estradiol, progesterone and hCG, and were positive for HLA-G, a marker of extravillous trophoblasts. HPT-8-HBV cells were positive for HBV relaxed-circular, covalently closed circular DNA and pre-S sequence. HPT-8-HBV cells also produced and secreted HBV surface antigen and HBV e antigen. We established a trophoblast cell line, HPT-8 and its HBV-expressing clone which could be valuable in exploring the mechanism of HBV viral integration in human trophoblasts during intrauterine infection.

  20. Investigation of hTERT gene expression levels in two cell lines infected by high-risk human papilloma virus

    Directory of Open Access Journals (Sweden)

    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.

  1. Different Expression and Localization of Phosphoinositide Specific Phospholipases C in Human Osteoblasts, Osteosarcoma Cell Lines, Ewing Sarcoma and Synovial Sarcoma

    Directory of Open Access Journals (Sweden)

    V.Vasco

    2017-06-01

    Full Text Available Background: Bone hardness and strength depends on mineralization, which involves a complex process in which calcium phosphate, produced by bone-forming cells, was shed around the fibrous matrix. This process is strictly regulated, and a number of signal transduction systems were interested in calcium metabolism, such as the phosphoinositide (PI pathway and related phospholipase C (PLC enzymes. Objectives: Our aim was to search for common patterns of expression in osteoblasts, as well as in ES and SS. Methods: We analysed the PLC enzymes in human osteoblasts and osteosarcoma cell lines MG-63 and SaOS-2. We compared the obtained results to the expression of PLCs in samples of patients affected with Ewing sarcoma (ES and synovial sarcoma (SS. Results: In osteoblasts, MG-63 cells and SaOS-2 significant differences were identified in the expression of PLC δ4 and PLC η subfamily isoforms. Differences were also identified regarding the expression of PLCs in ES and SS. Most ES and SS did not express PLCB1, which was expressed in most osteoblasts, MG-63 and SaOS-2 cells. Conversely, PLCB2, unexpressed in the cell lines, was expressed in some ES and SS. However, PLCH1 was expressed in SaOS-2 and inconstantly expressed in osteoblasts, while it was expressed in ES and unexpressed in SS. The most relevant difference observed in ES compared to SS regarded PLC ε and PLC η isoforms. Conclusion: MG-63 and SaOS-2 osteosarcoma cell lines might represent an inappropriate experimental model for studies about the analysis of signal transduction in osteoblasts

  2. Cancer antigens are expressed in a carcinogen-transformed Bloom syndrome B-lymphoblastoid cell line

    International Nuclear Information System (INIS)

    Shiraishi, Yukimasa; Soma, Hiroaki

    1988-01-01

    The authors have cloned malignant cells carrying specific antigens associated with ovarian cancer (OVC) and malignant lymphoma (ML) from BS-SHI-4M cells, a line derived from a 1-methyl-3-nitro-1-nitrosoguanidine-treated B-lymphoblastoid cell line isolated from a patient with Bloom syndrome. Since BS-SHI-4M cells react with sera from various individual cancer patients at relatively low frequencies (2-9%), as detected by an indirect immunofluorescence technique, cell clones that specifically react with sera from patients with OVC and ML were separated by the panning method in which polystyrene dishes were coated with sera from OVC and ML patients and cells with the corresponding antigens bound to the dishes. Subsequent cloning by limiting dilution provided cell clones highly enriched for OVC- and ML-associated antigens. Karyotype analyses revealed that cell clones with OVC and ML antigens had common marker chromosomes. Interestingly, in cell clones with a strong OVC antigen response, the distal part of the Y chromosome (Yq11) was missing in 100% of the cells. Therefore the cell line BS-SHI-4M appears to be a reservoir of cell clones each of which carries a specific tumor antigen and thus provides a potential tool for rapid serological diagnosis of cancer

  3. 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.

  4. Vav-1 expression correlates with NFkappaB activation and CD40-mediated cell death in diffuse large B-cell lymphoma cell lines

    DEFF Research Database (Denmark)

    Hollmann, Annette; Aloyz, Raquel; Baker, Kristi

    2010-01-01

    Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy with a variable response to therapy. We have previously shown that DLBCL cell lines differ in their susceptibility to CD40-mediated cell death, and that resistance to CD40-targeted antibodies correlated with increased expression...... as a potential marker to identify tumours likely to respond to CD40-targeted therapies. Copyright (c) 2010 John Wiley & Sons, Ltd....

  5. A novel cytochrome P450 gene from Catharanthus roseus cell line C20hi: cloning and characterization of expression

    Directory of Open Access Journals (Sweden)

    Lihong He

    2012-06-01

    Full Text Available An expressed sequence tag (EST obtained from a subtractive-suppression hybridization cDNA library constructed using Catharanthus roseus cell line C20hi and its parental cell line C20D was used to clone a full-length cytochrome P450 cDNA of cyp71d1. The encoded polypeptide contained 507 amino acids with 39–56% identity to other CYP71D subfamily members at the amino acid level. Expression characteristics of cyp71d1 were determined using semi-quantitative RT-PCR. The cyp71d1 transcript was expressed in all three cell lines with the highest level in the cell line C20hi. In the mature C. roseus plant, the cyp71d1 cDNA was highly expressed in petals, roots and stems, but very weakly expressed in young leaves. Its transcription level increased with the development of flowers. 2,4-D could down-regulate the transcription of cyp71d1, as did KT, but only to a minor degree. Neither light nor yeast elicitor could induce the transcription of cyp71d1.

  6. [Correlation between histone H3-K9 methylation, DNA methylation and expression of gene MGMT in Hep-2 cell line].

    Science.gov (United States)

    Yang, Jing; He, Liria; Ji, Wenyue; Jin, Mingzhu; Zhao, Xudong

    2012-11-01

    To explore the correlation between histone H3-K9 methylation, DNA methylation and expression of carcinoma suppressor gene MGMT in laryngeal carcinoma Hep-2 cell line. 5-Aza-dC was used to deal with Hep-2 cell cultured in vitro. ChIP, MSP and Realtime-PCR were used to detect H3-K9 methylation, DNA methylation, of MGMT gene promoter region and MGMT gene expression before and after treatment with drugs. (1) In Hep-2 cell line, gene MGMT was characterized by DNA methylation and histone H3-K9 hypermethylation. (2) 5-Aza-dC was able to reduce H3-K9 methylation of MGMT gene histone in Hep-2 cell line, 5-Aza-dC was able to reverse DNA methylation of MGMT gene histone in Hep-2 cell line, 5-Aza-dC was able to upregulate the down-regulated gene expression of tumor suppressor genes MGMT. Promoter methylation of cancer suppressor gene MGMT may induce the gene inactivity. DNA methylation may increase H3-K9 methylation. 5-Aza-dC can reduce H3-K9 methylation of tumor suppressor gene MGMT histone by reversing DNA methylation of tumor suppressor gene MGMT, and then the expression of tumor suppressor genes is increased and tumor development is inhibited.

  7. Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Qingbei Zhang

    Full Text Available Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3 and basal (MDA-MB-231 types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL and glioblastoma cell lines (U87 and D54. These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.

  8. Preparation of oligonucleotide microarray for radiation-associated gene expression detection and its application in lung cancer cell lines

    International Nuclear Information System (INIS)

    Guo Wanfeng; Lin Ruxian; Huang Jian; Guo Guozhen; Wang Shengqi

    2005-01-01

    Objective: The response of tumor cell to radiation is accompanied by complex change in patterns of gene expression. It is highly probable that a better understanding of molecular and genetic changes can help to sensitize the radioresistant tumor cells. Methods: Oligonucleotide microarray provides a powerful tool for high-throughput identifying a wider range of genes involved in the radioresistance. Therefore, the authors designed one oligonucleotide microarray according to the biological effect of IR. By using different radiosensitive lung cancer cell lines, the authors identified genes showing altered expression in lung cancer cell lines. To provide independent confirmation of microarray data, semi-quantitative RT-PCR was performed on a selection of genes. Results: In radioresistant A549 cell lines, a total of 18 genes were selected as having significant fold-changes compared to NCI-H446, 8 genes were up-regulated and 10 genes were down-regulated. Subsequently, A549 and NCI-H446 cells were delivered by ionizing radiation. In A549 cell line, we found 22 (19 up-regulated and 3 down-regulated) and 26 (8 up-regulated and 18 down-regulated) differentially expressed genes at 6h and 24h after ionizing radiation. In NCI-H446 cell line, we identified 17 (9 up-regulated and 8 down-regulated) and 18 (6 up-regulated and 12 down-regulated) differentially expressed genes at 6 h and 24 h after ionizing radiation. The authors tested seven genes (MDM2, p53, XRCC5, Bcl-2, PIM2, NFKBIA and Cyclin B1) for RT-PCR, and found that the results were in good agreement with those from the microarray data except for NFKBIA gene, even though the value for each mRNA level might be different between the two measurements. In present study, the authors identified some genes with cell proliferation and anti-apoptosis, such as MdM2, BCL-2, PKCz and PIM2 expression levels increased in A549 cells and decreased in NCI-H446 cells after radiation, and other genes with DNA repair, such as XRCC5, ERCC5

  9. Progesterone-induced inhibition of growth and differential regulation of gene expression in PRA- and/or PRB-expressing endometrial cancer cell lines.

    Science.gov (United States)

    Smid-Koopman, Ellen; Kuhne, Liesbeth C M; Hanekamp, Eline E; Gielen, Susanne C J P; De Ruiter, Petra E; Grootegoed, J Anton; Helmerhorst, Theo J M; Burger, Curt W; Brinkmann, Albert O; Huikeshoven, Frans J; Blok, Leen J

    2005-05-01

    Progesterone plays an important role in controlling proliferation and differentiation of the human endometrium. Because there are two progesterone receptor isoforms (PRA and PRB), it was important to generate tools to be able to study the role of these two progesterone receptors separately. Using stable transfection techniques, both human progesterone receptor isoforms (hPRA and hPRB) were reintroduced into a hPR-negative subclone of the well-differentiated endometrial cancer cell line Ishikawa. Several Ishikawa subcell lines were constructed, each expressing different levels of hPRA, hPRB, or hPRA and hPRB, respectively. These Ishikawa subcell lines showed a marked progesterone-induced growth inhibition with induction of apoptosis after long-term culture in the presence of hormone. Upon measuring gene regulation, a clear difference in regulation of expression of the selected genes by progesterone treatment was observed between the PRA-, PRB-, or PRA/B-expressing cell lines. Integrin beta4 (ITGB4) was only regulated in PRA-expressing cells; amphiregulin was highly regulated in PRB-expressing cells; insulin-like growth factor binding protein 3 (IGFBP3) was only regulated in PRB- and PRA/B-expressing cells; and metallothionein 1L (MT1L) was highly regulated in PRA/B-expressing cells. Interestingly, based on literature data, these genes can be implicated in induction of apoptosis, but are modulated here in such a way that suggests induction of resistance against apoptosis. Reintroduction of PRs into Ishikawa cells rescued progesterone responsiveness in these cells. Furthermore, using these human endometrial cancer subcell lines, clear and distinct functional differences between the PR isoforms were observed.

  10. Gene expression changes associated with Barrett's esophagus and Barrett's-associated adenocarcinoma cell lines after acid or bile salt exposure

    Directory of Open Access Journals (Sweden)

    Sahbaie Peyman

    2007-06-01

    Full Text Available Abstract Background Esophageal reflux and Barrett's esophagus represent two major risk factors for the development of esophageal adenocarcinoma. Previous studies have shown that brief exposure of the Barrett's-associated adenocarcinoma cell line, SEG-1, or primary cultures of Barrett's esophageal tissues to acid or bile results in changes consistent with cell proliferation. In this study, we determined whether similar exposure to acid or bile salts results in gene expression changes that provide insights into malignant transformation. Methods Using previously published methods, Barrett's-associated esophageal adenocarcinoma cell lines and primary cultures of Barrett's esophageal tissue were exposed to short pulses of acid or bile salts followed by incubation in culture media at pH 7.4. A genome-wide assessment of gene expression was then determined for the samples using cDNA microarrays. Subsequent analysis evaluated for statistical differences in gene expression with and without treatment. Results The SEG-1 cell line showed changes in gene expression that was dependent on the length of exposure to pH 3.5. Further analysis using the Gene Ontology, however, showed that representation by genes associated with cell proliferation is not enhanced by acid exposure. The changes in gene expression also did not involve genes known to be differentially expressed in esophageal adenocarcinoma. Similar experiments using short-term primary cultures of Barrett's esophagus also did not result in detectable changes in gene expression with either acid or bile salt exposure. Conclusion Short-term exposure of esophageal adenocarcinoma SEG-1 cells or primary cultures of Barrett's esophagus does not result in gene expression changes that are consistent with enhanced cell proliferation. Thus other model systems are needed that may reflect the impact of acid and bile salt exposure on the esophagus in vivo.

  11. Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Kawaguchi Makoto

    2010-01-01

    Full Text Available Abstract Background Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD, squamous cell carcinoma (SQ, large cell carcinoma (LC, and small cell carcinoma (SC. Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR. Results We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA and a normal control lung cell line (MRC-9. From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L. Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2. The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression. Conclusions These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results

  12. A comparison of cell survival and heat shock protein expression after radiation in normal dermal fibroblasts, microvascular endothelial cells, and different head and neck squamous carcinoma cell lines.

    Science.gov (United States)

    Muschter, Dominique; Geyer, Fabian; Bauer, Richard; Ettl, Tobias; Schreml, Stephan; Haubner, Frank

    2018-01-06

    Head and neck squamous cell carcinoma (HNSCC) shows increased radioresistance due to the manipulation of homeostatic mechanisms like the heat shock response. This study intended to comparatively analyze effects of ionizing radiation on different HNSCC cell lines (PCI) and normal human dermal fibroblasts (NHFs) and human dermal microvascular endothelial cells (HDMECs) to uncover differences in radiation coping strategies. Proliferation (BrdU assay), apoptosis (caspase 3/7) and intracellular protein expression of heat shock protein (HSP)-70, and phosphorylated and total HSP27, determined by enzyme-linked immunosorbent assay (ELISA), were analyzed after exposure to increasing doses of ionizing radiation (2, 6, and 12 Gray, Gy). Cell count decreased dose-dependently, but PCI cell lines consistently showed higher numbers compared to NHF and HDMEC. Likewise, high doses reduced cell proliferation, but low-dose radiation (2 Gy) instead increased proliferation in PCI 9 and 52. Apoptosis was not detectable in PCI cell lines. Basic HSP70 expression was high in PCI cells with little additional increase by irradiation. PCI cells yielded high basic total HSP27 concentrations but irradiation dose-dependently increased HSP27 in HDMEC, NHF, and PCI cells. Phosphorylated HSP27 concentrations were highest in NHF. PCI cell lines showed higher resistance to dose-dependent reduction in cell number, proliferation, and protection from apoptosis compared to NHF and HDMEC. In parallel, we observed a high basic and radiation-induced expression of intracellular HSP70 leading to the assumption that the radioresistance of PCI cells is conferred by HSP70. HNSCC use HSP to escape radiation-induced apoptosis and certain subtypes might increase proliferation after low-dose irradiation.

  13. A rank-based algorithm of differential expression analysis for small cell line data with statistical control.

    Science.gov (United States)

    Li, Xiangyu; Cai, Hao; Wang, Xianlong; Ao, Lu; Guo, You; He, Jun; Gu, Yunyan; Qi, Lishuang; Guan, Qingzhou; Lin, Xu; Guo, Zheng

    2017-10-13

    To detect differentially expressed genes (DEGs) in small-scale cell line experiments, usually with only two or three technical replicates for each state, the commonly used statistical methods such as significance analysis of microarrays (SAM), limma and RankProd (RP) lack statistical power, while the fold change method lacks any statistical control. In this study, we demonstrated that the within-sample relative expression orderings (REOs) of gene pairs were highly stable among technical replicates of a cell line but often widely disrupted after certain treatments such like gene knockdown, gene transfection and drug treatment. Based on this finding, we customized the RankComp algorithm, previously designed for individualized differential expression analysis through REO comparison, to identify DEGs with certain statistical control for small-scale cell line data. In both simulated and real data, the new algorithm, named CellComp, exhibited high precision with much higher sensitivity than the original RankComp, SAM, limma and RP methods. Therefore, CellComp provides an efficient tool for analyzing small-scale cell line data. © The Author 2017. Published by Oxford University Press.

  14. Expression of tumor-specific antigen MAGE, GAGE and BAGE in ovarian cancer tissues and cell lines

    International Nuclear Information System (INIS)

    Zhang, Shiqian; Zhou, Xiaoliang; Yu, Hao; Yu, Yunhai

    2010-01-01

    To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer. mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR) in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1. MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20). In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41) and 36.6% (15/41), while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41) and 14.6% (6/41). In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7) and 14.3% (1/7). The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (P < 0.05). Gene expression rate was not correlated with menopause or lymph node metastasis. Positive expression of MAGE-1 and MAGE-3 was positively correlated with tumor differentiation and the clinical stage of the ovarian cancer. In addition, the positive expression rate of BAGE was significantly higher in ovarian cancer patients with ascites (P < 0.05). The mRNA expression profiles of MAGE, GAGE and BAGE in ovarian carcinoma cell lines SKOV3, A2780 and COC1 varied, but there was at least one gene expressed in each cell line. Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  16. Lung Adenocarcinomas and Lung Cancer Cell Lines Show Association of MMP-1 Expression With STAT3 Activation

    Directory of Open Access Journals (Sweden)

    Alexander Schütz

    2015-04-01

    Full Text Available Signal transducer and activator of transcription 3 (STAT3 is constitutively activated in the majority of lung cancer. This study aims at defining connections between STAT3 function and the malignant properties of non–small cell lung carcinoma (NSCLC cells. To address possible mechanisms by which STAT3 influences invasiveness, the expression of matrix metalloproteinase-1 (MMP-1 was analyzed and correlated with the STAT3 activity status. Studies on both surgical biopsies and on lung cancer cell lines revealed a coincidence of STAT3 activation and strong expression of MMP-1. MMP-1 and tyrosine-phosphorylated activated STAT3 were found co-localized in cancer tissues, most pronounced in tumor fronts, and in particular in adenocarcinomas. STAT3 activity was constitutive, although to different degrees, in the lung cancer cell lines investigated. Three cell lines (BEN, KNS62, and A549 were identified in which STAT3 activitation was inducible by Interleukin-6 (IL-6. In A549 cells, STAT3 activity enhanced the level of MMP-1 mRNA and stimulated transcription from the MMP-1 promoter in IL-6–stimulated A549 cells. STAT3 specificity of this effect was confirmed by STAT3 knockdown through RNA interference. Our results link aberrant activity of STAT3 in lung cancer cells to malignant tumor progression through up-regulation of expression of invasiveness-associated MMPs.

  17. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    International Nuclear Information System (INIS)

    Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique; Villa, Luisa Lina; Rahal, Paula

    2016-01-01

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

  18. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Calmon, Marilia Freitas [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil); Sichero, Laura [Molecular Biology Laboratory, Centre for Translational Research in Oncology, Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo (Brazil); Boccardo, Enrique [Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo., São Paulo (Brazil); Villa, Luisa Lina [Department of Radiology and Oncology, Faculdade de Medicina, Universidade de São Paulo, São Paulo (Brazil); Rahal, Paula, E-mail: rahalp@yahoo.com.br [Department of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São Jose do Rio Preto (Brazil)

    2016-09-15

    Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16 E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.

  19. Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling.

    Science.gov (United States)

    Mwangi, William N; Vasoya, Deepali; Kgosana, Lydia B; Watson, Mick; Nair, Venugopal

    2017-04-01

    Marek's disease virus (MDV), an alphaherpesvirus of poultry, causes Marek's disease and is characterized by visceral CD4+TCRαβ+ T-cell lymphomas in susceptible hosts. Immortal cell lines harbouring the viral genome have been generated from ex vivo cultures of MD tumours. As readily available sources of large numbers of cells, MDV-transformed lymphoblastoid cell lines (LCLs) are extremely valuable for studies of virus-host interaction. While the viral genome in most cells is held in a latent state, minor populations of cells display spontaneous reactivation identifiable by the expression of lytic viral genes. Spontaneous reactivation in these cells presents an opportunity to investigate the biological processes involved in the virus reactivation. For detailed characterization of the molecular events associated with reactivation, we used two lymphoblastoid cell lines derived from lymphomas induced by pRB1B-UL47eGFP, a recombinant MDV engineered to express enhanced green fluorescent protein (EGFP) fused with the UL47. We used fluorescence-activated cell sorting to purify the low-frequency EGFP-positive cells with a spontaneously activating viral genome from the majority EGFP-negative cells and analysed their gene expression profiles by RNA-seq using Illumina HiSeq2500. Ingenuity pathway analysis on more than 2000 differentially expressed genes between the lytically infected (EGFP-positive) and latently infected (EGFP-negative) cell populations identified the biological pathways involved in the reactivation. Virus-reactivating cells exhibited differential expression of a significant number of viral genes, with hierarchical differences in expression levels. Downregulation of a number of host genes including those directly involved in T-cell activation, such as CD3, CD28, ICOS and phospholipase C, was also noticed in the LCL undergoing lytic switch.

  20. Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

    DEFF Research Database (Denmark)

    Jacobsen, Kari Stougaard; Nielsen, Kirstine Overgaard; Nordmann Winther, Thilde

    2016-01-01

    expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection...

  1. Gene expression profiling in chemoresistant variants of three cell lines of different origin

    DEFF Research Database (Denmark)

    Johnsson, Anders; Vallon-Christensson, Johan; Strand, Carina

    2005-01-01

    lines (K562 leukemia, MCF-7 breast cancer and S1 colon cancer) with acquired resistance against five cytostatic drugs; daunorubicin (DNR), doxorubicin (DOX), vincristine (VCR), etoposide (VP) and mitoxantrone (MX). RESULTS: The resistant cell lines clustered together based on their type of origin...... was also seen in, e.g., GSTs, topoisomerases, caveolins, annexins and CD44. CONCLUSION: These results will constitute a platform for further studies on specific pathways and biological processes involved in chemotherapy resistance....

  2. Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line

    International Nuclear Information System (INIS)

    Dallas, Peter B; Egli, Simone; Terry, Philippa A; Kees, Ursula R

    2007-01-01

    The mammalian FOXO (forkhead box, O subclass) proteins are a family of pleiotropic transcription factors involved in the regulation of a broad range of cellular processes critical for survival. Despite the essential and diverse roles of the FOXO family members in human cells and their involvement in tumor pathogenesis, the regulation of FOXO expression remains poorly understood. We have addressed the mechanisms underlying the high level of expression of the FOXO1A gene in a cell line, PER-453, derived from a primitive neuroectodermal tumor of the central nervous system (CNS-PNET). The status of the FOXO1A locus in the PER-453 CNS-PNET cell line was investigated by Southern blotting and DNA sequence analysis of the proximal promoter, 5'-UTR, open reading frame and 3'-UTR. FOXO1A expression was assessed by conventional and quantitative RT-PCR, Northern and Western blotting. Quantitative real-time RT-PCR (qRT-PCR) data indicated that after normalization to ACTB mRNA levels, canonical FOXO1A mRNA expression in the PER-453 cell line was 124-fold higher than the average level of five other CNS-PNET cell lines tested, 24-fold higher than the level in whole fetal brain, and 3.5-fold higher than the level in fetal brain germinal matrix cells. No mutations within the FOXO1A open reading frame or gross rearrangements of the FOXO1A locus were detected. However, a single nucleotide change within the proximal promoter and several nucleotide changes within the 3'-UTR were identified. In addition, two novel FOXO1A transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR. The CNS-PNET cell line, PER-453, expresses FOXO1A at very high levels relative to most normal and cancer cells from a broad range of tissues. The FOXO1A open reading frame is wild type in the PER-453 cell line and the abnormally high FOXO1A mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter. Over expression

  3. Mouse thymic epithelial cell lines expressing "Aire" and peripheral tissue-specific antigens reproduce in vitro negative selection of T cells.

    Science.gov (United States)

    Yamaguchi, Yoshitaka; Takayanagi, Atsushi; Chen, Jiabing; Sakai, Kosuke; Kudoh, Jun; Shimizu, Nobuyoshi

    2011-08-15

    In the human thymus, AIRE (autoimmune regulator) gene is expressed in a very limited type of medullary thymic epithelial cells (mTECs) and no cognate cell lines are available, hence the molecular analysis of AIRE gene function has been difficult. To improve this situation, we attempted to isolate Aire-expressing cells and established three cell lines (Aire⁺TEC1, Aire⁺TEC2, Aire⁺DC) from the abnormally enlarged thymus, which was developed in the transgenic mice expressing SV40 T-antigen driven by the mouse Aire gene promoter. When these Aire⁺ cell lines were co-cultured with fresh thymocytes, they adhered to the majority of thymocytes and induced apoptosis as if negative selection of T-cells in the thymus is occurring in vitro. Further analysis revealed that these Aire⁺ cell lines are derived from mTECs and exhibit characteristic natures of "antigen presenting cells" including several distinct abilities: to express a variety of peripheral tissue-specific antigens, to produce immunoproteasome and immunological synapse, and to express some of TNFSFs (tumor necrosis factor super families). Thus, the newly established Aire⁺ cell lines will be invaluable for the further detailed analysis of AIRE gene function in the central tolerance of immunity and autoimmune disease. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. In vitro and in situ intercellular adhesion molecule-1 (ICAM-1) expression by endothelial cells lining a polyester fabric.

    Science.gov (United States)

    Rémy, M; Valli, N; Brethes, D; Labrugère, C; Porté-Durrieu, M C; Dobrova, N B; Novikova, S P; Gorodkov, A J; Bordenave, L

    1999-02-01

    In order to improve long-term patency of vascular grafts, the promising concept of endothelial cell seeding is actually under investigation. Our laboratory tested a polyester coated with albumin and chitosan which permits a rapid colonization by human umbilical vein endothelial cells (HUVEC) and it seems relevant to test in vitro the expression of adhesive molecules expressed by cells with regard to the inflammatory process. We studied intercellular adhesion molecule-1 (ICAM-1) expression and focused our work on the determination of ICAM-1 sites expressed per adherent cell lining the biomaterial, thus in situ, in comparison to control HUVEC on plastic wells: the results obtained by binding experiments were correlated to flow cytometry analyses and showed that the polyester does not induce a proinflammatory state and that HUVEC covering the structure are able to respond to a stimulus.

  5. Urotensin-II receptor is over-expressed in colon cancer cell lines and in colon carcinoma in humans.

    Science.gov (United States)

    Federico, Alessandro; Zappavigna, Silvia; Romano, Marco; Grieco, Paolo; Luce, Amalia; Marra, Monica; Gravina, Antonietta Gerarda; Stiuso, Paola; D'Armiento, Francesco Paolo; Vitale, Giovanni; Tuccillo, Concetta; Novellino, Ettore; Loguercio, Carmela; Caraglia, Michele

    2014-01-01

    Urotensin (U)-II receptor (UTR) has been previously reported to be over-expressed in a number of tumours. Whether UTR-related pathway plays a role in colon carcinogenesis is unknown. We evaluated UTR protein and mRNA expression in human epithelial colon cancer cell lines and in normal colon tissue, adenomatous polyps and colon cancer. U-II protein expression was assessed in cancer cell lines. Moreover, we evaluated the effects of U-II(4-11) (an UTR agonist), antagonists and knockdown of UTR protein expression through a specific shRNA, on proliferation, invasion and motility of human colon cancer cells. Cancer cell lines expressed U-II protein and UTR protein and mRNA. By immunohistochemistry, UTR was expressed in 5-30% of epithelial cells in 45 normal controls, in 30-48% in 21 adenomatous polyps and in 65-90% in 48 colon adenocarcinomas. UTR mRNA expression was increased by threefold in adenomatous polyps and eightfold in colon cancer, compared with normal colon. U-II(4-11) induced a 20-40% increase in cell growth while the blockade of the receptor with specific antagonists caused growth inhibition of 20-40%. Moreover, the knock down of UTR with a shRNA or the inhibition of UTR with the antagonist urantide induced an approximately 50% inhibition of both motility and invasion. UTR appears to be involved in the regulation of colon cancer cell invasion and motility. These data suggest that UTR-related pathway may play a role in colon carcinogenesis and that UTR may function as a target for therapeutic intervention in colon cancer. © 2013 Stichting European Society for Clinical Investigation Journal Foundation.

  6. c-Myc over-expression in Ramos Burkitt's lymphoma cell line predisposes to iron homeostasis disruption in vitro

    International Nuclear Information System (INIS)

    Habel, Marie-Eve; Jung, Daniel

    2006-01-01

    Burkitt's lymphoma is an aggressive B-cell neoplasm resulting from deregulated c-myc expression. We have previously shown that proliferation of Burkitt's lymphoma cell lines such as Ramos is markedly reduced by iron treatment. It has been shown that iron induces expression of c-myc which, owing to its transcriptional regulatory functions, regulates genes involved in iron metabolism. Transient enhancement of c-myc expression by iron could increase the expression of genes involved in iron incorporation, which could lead to an accumulation of intracellular free iron. Here, we have investigated whether cells with a high basal level of c-Myc were more likely to accumulate free iron. Our results suggest that the basal level of c-Myc in Ramos cells is twofold higher than what is seen in HL-60 cells. Moreover, in Ramos cells, where c-Myc is expressed at a high level, H-ferritin expression is down-regulated, transferrin receptor (CD71) expression is increased, and ferritin translation is inhibited. These modifications in iron metabolism, resulting from the strong basal expression of c-Myc, and amplified by iron addition, could lead to a disruption in homeostasis and consequently to growth arrest

  7. Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

    Science.gov (United States)

    Große-Kreul, Jan; Busch, Maike; Winter, Claudia; Pikos, Stefanie; Stephan, Harald; Dünker, Nicole

    2016-01-01

    Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.

  8. Flatworm stem cells and the germ line: developmental and evolutionary implications of macvasa expression in Macrostomum lignano.

    Science.gov (United States)

    Pfister, Daniela; De Mulder, Katrien; Hartenstein, Volker; Kuales, Georg; Borgonie, Gaetan; Marx, Florentine; Morris, Joshua; Ladurner, Peter

    2008-07-01

    We have isolated and identified the vasa homologue macvasa, expressed in testes, ovaries, eggs and somatic stem cells of the flatworm Macrostomum lignano. Molecular tools such as in situ hybridization and RNA interference were developed for M. lignano to study gene expression and function. Macvasa expression was followed during postembryonic development, regeneration and in starvation experiments. We were able to follow gonad formation in juveniles and the reformation of gonads from stem cells after amputation by in situ hybridization and a specific Macvasa antibody. Expression of macvasa in the germ cells was highly affected by feeding conditions and correlated with the decrease and regrowth of the gonads. RNA interference showed specific down-regulation of macvasa mRNA and protein. The absence of Macvasa did not influence gonad formation and stem cell proliferation. Our results corroborate the exclusive nature of the flatworm stem cell system but challenge the concept of a solely postembryonic specification of the germ line in Platyhelminthes. We address the transition of somatic stem cells to germ cells and speculate on Macrostomum as a system to unravel the mechanisms of preformation or epigenesis in the evolution of germ line specification from somatic stem cells.

  9. Low or undetectable TPO receptor expression in malignant tissue and cell lines derived from breast, lung, and ovarian tumors

    Directory of Open Access Journals (Sweden)

    Erickson-Miller Connie L

    2012-09-01

    Full Text Available Abstract Background Numerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues. Methods Breast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR, and for TPO-R protein expression by immunohistochemistry (IHC. Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot. Results MPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43% and malignant (3-17% breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines. Conclusions Multiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and

  10. Low or undetectable TPO receptor expression in malignant tissue and cell lines derived from breast, lung, and ovarian tumors.

    Science.gov (United States)

    Erickson-Miller, Connie L; Pillarisetti, Kodandaram; Kirchner, Jennifer; Figueroa, David J; Ottesen, Lone; Martin, Anne-Marie; Liu, Yuan; Kamel, Yasser Mostafa; Messam, Conrad

    2012-09-11

    Numerous efficacious chemotherapy regimens may cause thrombocytopenia. Thrombopoietin receptor (TPO-R) agonists, such as eltrombopag, represent a novel approach for the treatment of chemotherapy-induced thrombocytopenia. The TPO-R MPL is expressed on megakaryocytes and megakaryocyte precursors, although little is known about its expression on other tissues. Breast, lung, and ovarian tumor samples were analyzed for MPL expression by microarray and/or quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and for TPO-R protein expression by immunohistochemistry (IHC). Cell line proliferation assays were used to analyze the in vitro effect of eltrombopag on breast, lung, and ovarian tumor cell proliferation. The lung carcinoma cell lines were also analyzed for TPO-R protein expression by Western blot. MPL mRNA was not detectable in 118 breast tumors and was detectable at only very low levels in 48% of 29 lung tumors studied by microarray analysis. By qRT-PCR, low but detectable levels of MPL mRNA were detectable in some normal (14-43%) and malignant (3-17%) breast, lung, and ovarian tissues. A comparison of MPL to EPOR, ERBB2, and IGF1R mRNA demonstrates that MPL mRNA levels were far lower than those of EPOR and ERBB2 mRNA in the same tissues. IHC analysis showed negligible TPO-R protein expression in tumor tissues, confirming mRNA analysis. Culture of breast, lung, and ovarian carcinoma cell lines showed no increase, and in fact, showed a decrease in proliferation following incubation with eltrombopag. Western blot analyses revealed no detectable TPO-R protein expression in the lung carcinoma cell lines. Multiple analyses of breast, lung, and ovarian tumor samples and/or cell lines show no evidence of MPL mRNA or TPO-R protein expression. Eltrombopag does not stimulate growth of breast, lung, or ovarian tumor cell lines at doses likely to exert their actions on megakaryocytes and megakaryocyte precursors.

  11. Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

    Directory of Open Access Journals (Sweden)

    JIANG Hua-wei

    2014-09-01

    Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed >90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

  12. Reversal of resistance by GF120918 in cell lines expressing the ABC half-transporter, MXR

    DEFF Research Database (Denmark)

    de Bruin, M; Miyake, K; Litman, Thomas

    1999-01-01

    -80, a subline expressing a newly identified mitoxantrone transporter, MXR. GF120918 was ineffective in sensitizing MRP-overexpressing MCF-7 VP-16 cells to etoposide as determined by cytotoxicity studies. In flow cytometry experiments, rhodamine 123 efflux in S1-B1-20 cells was decreased at GF120918...

  13. EGFR over-expression and activation in high HER2, ER negative breast cancer cell line induces trastuzumab resistance.

    Science.gov (United States)

    Dua, Rajiv; Zhang, Jianhuan; Nhonthachit, Phets; Penuel, Elicia; Petropoulos, Chris; Parry, Gordon

    2010-08-01

    HER2 is gene amplified or over-expressed in 20-25% of breast cancers resulting in elevated HER2 activation. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, despite prolonged survival, treated breast cancer patients develop resistance. Resistance to trastuzumab occurs upon inactivation of HER2 regulatory proteins or upon up-regulation of alternative receptors. In particular, elevated levels of EGFR, present in estrogen receptor (ER) positive, trastuzumab-resistant BT-474 xenografts caused, a trastuzumab-resistant phenotype (Ritter et al. Clin Cancer Res 13:4909-4919, 2007). However, the role of EGFR in acquired trastuzumab resistance in ER negative cell models is not well defined. In this study, SKBR3 cell line clones expressing EGFR were generated to examine the role of EGFR over-expression on trastuzumab sensitivity in an, ER-negative breast carcinoma cell line. A stable clone, SKBR3/EGFR (clone 4) expressing moderate levels of EGFR remained sensitive to trastuzumab, whereas a stable clone, SKBR3/EGFR (clone 5) expressing high levels of EGFR, became resistant to trastuzumab. Depletion of EGFR by EGFR small-interfering RNAs in the SKBR3/EGFR (clone 5) reversed trastuzumab resistance. However, the SKBR3/EGFR (clone 5) cell line remained sensitive to lapatinib, an EGFR/HER2 inhibitor. Biochemical analysis using co-immunoprecipitation and proximity-based quantitative VeraTag assays demonstrated that high levels of EGFR phosphorylation, EGFR/EGFR homo-dimerization, and EGFR/HER2 hetero-dimerization were present in the trastuzumab-resistant cells. We conclude that EGFR over-expression can mediate trastuzumab resistance in both ER positive and ER negative cells and hypothesize that a threshold level of EGFR, in the absence of autocrine ligand production, is required to induce the resistant phenotype.

  14. A HLA-A2 restricted human CTL line recognizes a novel tumor cell expressed p53 epitope

    DEFF Research Database (Denmark)

    Würtzen, Peter A; Claesson, Mogens H

    2002-01-01

    , the CTL line, which expressed relatively low affinity for the HLA-A2/peptide complex, was able to kill 3 different HLA-A2(+) p53 mutated tumor cell lines. The present and our previous observations expand the number of p53-derived peptides suitable for vaccination protocols for cancer patients with p53......A p53 peptide-specific CTL line was generated through stimulation with autologous monocyte-derived dendritic cells (DC) pulsed with wild-type HLA-A2 binding p53 derived peptides. A p53 peptide-specific CD8(+) CTL line was established from a healthy HLA-A2 positive donor. The CTL line...... was characterized with respect to specificity, affinity and killing of cell lines derived from p53 mutated spontaneous tumors. The CTL line demonstrated lysis of p53(139-147) pulsed target cells and cold target inhibition experiments as well as antibody blocking confirmed that the killing was epitope-specific, HLA...

  15. Effects of Smac gene over-expression on radiotherapeutic sensitivity of cervical cancer cell line HeLa

    International Nuclear Information System (INIS)

    Zheng Liduan; Wang Liang; Tong Qiangsong; Fei Shihong; Xiong Yufang; Wu Gang

    2005-01-01

    Objective: To study the effects of extrinsic Smac gene transfection and its over-expression on radiotherapeutic sensitivity of cervical cancer cells, in order to explore a novel strategy for ameliorating radiotherapy of cervical cancer. Methods: After Smac gene was transferred into cells of cervical cancer cell line HeLa, the subclone cells were obtained by persistent G 418 selection. Cellular Smac gene expression was determined by RT-PCR and Western blot. After treatment with X-ray irradiation, cellular growth activity in vitro was investigated by MTT colorimetry. Cellular apoptosis and its rate were determined by electron microscopy, Annexin V-FITC and propidium iodide staining flow cytometry. Cellular Caspase-3 protein expression and its activity were assayed by Western blot and colorimetry. Results: RT-PCR and Western blot demonstrated that Smac mRNA and protein levels of HeLa/Smac cells, the selected subclone cells of cervical cancer cell line, were significantly higher than those of HeLa cells (P<0.01). After treated with 8 Gy X-ray irradiation, growth activity of HeLa/Smac cells reduced by 10.19%(P<0.01), as compared with that of HeLa cells. Partial HeLa/Smac cancer cells presented characteristic morphological changes of apoptosis under electron microscope, with an apoptosis rate of 16.4%, which was significantly higher than that of HeLa cells(6.2%, P<0.01). Compared with HeLa cells, Caspase-3 expression level in HeLa/Smac was improved significantly (P<0.01), while its activity was 3.42 times as much as that of HeLa cells (P<0.01). Conclusion: Stable transfection of extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance cellular caspase-3 expression and activity, ameliorate apoptosis-inducing effects of radiation on cancer cells, which would be a novel strategy to improve radiotherapeutic effects for cervical cancer. (authors)

  16. Molecular portrait of cisplatin induced response in human testis cancer cell lines based on gene expression profiles

    Directory of Open Access Journals (Sweden)

    Soderlund Erik J

    2007-08-01

    Full Text Available Abstract Background Testicular germ cell tumors (TGCTs respond well to cisplatin-based chemotherapy and show a low incidence of acquired resistance compared to most somatic tumors. The reasons for these specific characteristics are not known in detail but seem to be multifactorial. We have studied gene expression profiles of testicular and colon cancer derived cell lines treated with cisplatin. The main goal of this study was to identify novel gene expression profiles with their functional categories and the biochemical pathways that are associated with TGCT cells' response to cisplatin. Results Genes that were differentially expressed between the TGCT cell lines vs the (somatic HCT116 cell line, after cisplatin treatment, were identified using the significance analysis of microarrays (SAM method. The response of TGCT cells was strikingly different from that of HCT116, and we identified 1794 genes that were differentially expressed. Functional classification of these genes showed that they participate in a variety of different and widely distributed functional categories and biochemical pathways. Database mining showed significant association of genes (n = 41 induced by cisplatin in our study, and genes previously reported to by expressed in differentiated TGCT cells. We identified 37 p53-responsive genes that were altered after cisplatin exposure. We also identified 40 target genes for two microRNAs, hsa-mir-372 and 373 that may interfere with p53 signaling in TGCTs. The tumor suppressor genes NEO1 and LATS2, and the estrogen receptor gene ESR1, all have binding sites for p53 and hsa-mir-372/373. NEO1 and LATS2 were down-regulated in TGCT cells following cisplatin exposure, while ESR1 was up-regulated in TGCT cells. Cisplatin-induced genes associated with terminal growth arrest through senescence were identified, indicating associations which were not previously described for TGCT cells. Conclusion By linking our gene expression data to

  17. Phenotype-dependent effects of EpCAM expression on growth and invasion of human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Martowicz Agnieszka

    2012-10-01

    Full Text Available Abstract Background The epithelial cell adhesion molecule (EpCAM has been shown to be overexpressed in breast cancer and stem cells and has emerged as an attractive target for immunotherapy of breast cancer patients. This study analyzes the effects of EpCAM on breast cancer cell lines with epithelial or mesenchymal phenotype. Methods For this purpose, shRNA-mediated knockdown of EpCAM gene expression was performed in EpCAMhigh breast cancer cell lines with epithelial phenotype (MCF-7, T47D and SkBR3. Moreover, EpCAMlow breast carcinoma cell lines with mesenchymal phenotype (MDA-MB-231, Hs578t and inducible overexpression of EpCAM were used to study effects on proliferation, migration and in vivo growth. Results In comparison to non-specific silencing controls (n/s-crtl knockdown of EpCAM (E#2 in EpCAMhigh cell lines resulted in reduced cell proliferation under serum-reduced culture conditions. Moreover, DNA synthesis under 3D culture conditions in collagen was significantly reduced. Xenografts of MCF-7 and T47D cells with knockdown of EpCAM formed smaller tumors that were less invasive. EpCAMlow cell lines with tetracycline-inducible overexpression of EpCAM showed no increased cell proliferation or migration under serum-reduced growth conditions. MDA-MB-231 xenografts with EpCAM overexpression showed reduced invasion into host tissue and more infiltrates of chicken granulocytes. Conclusions The role of EpCAM in breast cancer strongly depends on the epithelial or mesenchymal phenotype of tumor cells. Cancer cells with epithelial phenotype need EpCAM as a growth- and invasion-promoting factor, whereas tumor cells with a mesenchymal phenotype are independent of EpCAM in invasion processes and tumor progression. These findings might have clinical implications for EpCAM-based targeting strategies in patients with invasive breast cancer.

  18. Phenotype-dependent effects of EpCAM expression on growth and invasion of human breast cancer cell lines

    International Nuclear Information System (INIS)

    Martowicz, Agnieszka; Spizzo, Gilbert; Gastl, Guenther; Untergasser, Gerold

    2012-01-01

    The epithelial cell adhesion molecule (EpCAM) has been shown to be overexpressed in breast cancer and stem cells and has emerged as an attractive target for immunotherapy of breast cancer patients. This study analyzes the effects of EpCAM on breast cancer cell lines with epithelial or mesenchymal phenotype. For this purpose, shRNA-mediated knockdown of EpCAM gene expression was performed in EpCAM high breast cancer cell lines with epithelial phenotype (MCF-7, T47D and SkBR3). Moreover, EpCAM low breast carcinoma cell lines with mesenchymal phenotype (MDA-MB-231, Hs578t) and inducible overexpression of EpCAM were used to study effects on proliferation, migration and in vivo growth. In comparison to non-specific silencing controls (n/s-crtl) knockdown of EpCAM (E#2) in EpCAM high cell lines resulted in reduced cell proliferation under serum-reduced culture conditions. Moreover, DNA synthesis under 3D culture conditions in collagen was significantly reduced. Xenografts of MCF-7 and T47D cells with knockdown of EpCAM formed smaller tumors that were less invasive. EpCAM low cell lines with tetracycline-inducible overexpression of EpCAM showed no increased cell proliferation or migration under serum-reduced growth conditions. MDA-MB-231 xenografts with EpCAM overexpression showed reduced invasion into host tissue and more infiltrates of chicken granulocytes. The role of EpCAM in breast cancer strongly depends on the epithelial or mesenchymal phenotype of tumor cells. Cancer cells with epithelial phenotype need EpCAM as a growth- and invasion-promoting factor, whereas tumor cells with a mesenchymal phenotype are independent of EpCAM in invasion processes and tumor progression. These findings might have clinical implications for EpCAM-based targeting strategies in patients with invasive breast cancer

  19. Effect of Silibinin on Maspin and ERα Gene Expression in MCF-7 Human Breast Cancer Cell Line.

    Science.gov (United States)

    Karimi, Maryam; Babaahmadi-Rezaei, Hossein; Mohammadzadeh, Ghorban; Ghaffari, Mohammad-Ali

    2017-01-01

    According to reports, a serine protease inhibitor (Maspin) suppresses metastasis, invasion and angiogenesis in breast and prostate cancers. Silibinin is a natural polyphenolic flavonoid with anti-cancer activity. We assessed the effects of silibinin on cell viability, maspin and ERα gene expression in MCF-7 cell line. The human MCF-7 breast cancer cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with different concentrations of silibinin (100-600 μg/mL) for 24, 48 and 72 hours. The cytotoxic effect of silibinin on MCF-7 viability was determined using Methyl-Thiazolyl-Tetrazolium (MTT) assay by IC50 determination. The fold changes of Maspin and ERα expression were determined by reverse-transcription real-time Polymerase Chain Reaction (PCR). All experiments on the cells were performed in triplicates. The maximum inhibitory effect of silibinin on cell viability was observed at 600 μg/mL after 72-hour incubation (p = 0.001). Incubation of the cells with silibinin for 48 and 72 hours significantly decreased IC50 values to 250 and 207 μg/mL (p = 0.005 and p= 0.006), respectively. The expression of maspin and ERα in the treated cells compared to controls was significantly decreased following treatment with different concentrations of silibinin during a 24-hour period. Silibinin reduces both maspin and ERα gene expression in MCF-7 cell line. The therapeutic effect of silibinin on the treatment of breast cancer may be mediated by the reduction of ERα expression. For verifying this hypothesis and the possible therapeutic implication of silibinin on breast cancer, further studies in this direction are necessary.

  20. Regulation of laminin beta2 chain gene expression in human cancer cell lines

    DEFF Research Database (Denmark)

    Durkin, M E; Nielsen, F C; Loechel, F

    2001-01-01

    by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides -164 to -667 and between...

  1. Doxycyclin induces p53 expression in SaOs (osteosarcoma) cell line ...

    African Journals Online (AJOL)

    The p53 tumour suppressor gene plays an important role in preventing cancer development. This study determined if p53 can be induced in osteosarcoma cell line upon treatment ... represent an important component of the p53 tumor suppressor pathway. Keywords: Tumor suppressor, oncogene, mdm2, cyclinE, apoptosis ...

  2. Translational control of TWIST1 expression in MCF-10A cell lines recapitulating breast cancer progression

    DEFF Research Database (Denmark)

    Nairismägi, Maarja-Liisa; Vislovukh, Andrii; Meng, Q

    2012-01-01

    TWIST1 is a highly conserved basic helix-loop-helix transcription factor that promotes epithelial–mesenchymal transition (EMT). Its misregulation has been observed in various types of tumors. Using the MCF-10A-series of cell lines that recapitulate the early stages of breast cancer formation...

  3. Comparison of the gene expression profile of undifferentiated human embryonic stem cell lines and differentiating embryoid bodies

    Directory of Open Access Journals (Sweden)

    Rao Mahendra S

    2005-10-01

    Full Text Available Abstract Background The identification of molecular pathways of differentiation of embryonic stem cells (hESC is critical for the development of stem cell based medical therapies. In order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing 16,659 seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hESC lines and differentiating embryoid bodies. Results Previously identified "stemness" genes in undifferentiated hESC lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. In addition, a subset of 194 genes showed overexpression of greater than ≥ 3 folds in human embryoid bodies (hEB. These included 37 novel and 157 known genes. Gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cDNA microarrays, massively parallel signature sequencing (MPSS analysis and immunocytochemisty. Several novel hEB specific expressed sequence tags (ESTs were mapped to the human genome database and their expression profile characterized. A hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hESC and confirmed that microarray analysis could readily distinguish them. Conclusion These results present a detailed characterization of a unique set of genes, which can be used to assess the hESC differentiation.

  4. Comparison of the gene expression profile of undifferentiated human embryonic stem cell lines and differentiating embryoid bodies.

    Science.gov (United States)

    Bhattacharya, Bhaskar; Cai, Jingli; Luo, Youngquan; Miura, Takumi; Mejido, Josef; Brimble, Sandii N; Zeng, Xianmin; Schulz, Thomas C; Rao, Mahendra S; Puri, Raj K

    2005-10-05

    The identification of molecular pathways of differentiation of embryonic stem cells (hESC) is critical for the development of stem cell based medical therapies. In order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing 16,659 seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hESC lines and differentiating embryoid bodies. Previously identified "stemness" genes in undifferentiated hESC lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. In addition, a subset of 194 genes showed overexpression of greater than > or = 3 folds in human embryoid bodies (hEB). These included 37 novel and 157 known genes. Gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cDNA microarrays, massively parallel signature sequencing (MPSS) analysis and immunocytochemisty. Several novel hEB specific expressed sequence tags (ESTs) were mapped to the human genome database and their expression profile characterized. A hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hESC and confirmed that microarray analysis could readily distinguish them. These results present a detailed characterization of a unique set of genes, which can be used to assess the hESC differentiation.

  5. Expression of tumor-specific antigen MAGE, GAGE and BAGE in ovarian cancer tissues and cell lines.

    Science.gov (United States)

    Zhang, Shiqian; Zhou, Xiaoliang; Yu, Hao; Yu, Yunhai

    2010-04-27

    To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer. mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR) in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1. MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20). In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41) and 36.6% (15/41), while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41) and 14.6% (6/41). In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7) and 14.3% (1/7). The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (P BAGE was significantly higher in ovarian cancer patients with ascites (P BAGE in ovarian carcinoma cell lines SKOV3, A2780 and COC1 varied, but there was at least one gene expressed in each cell line. Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer. These genes can be used as one of the important indicators for early diagnosis, efficacy evaluation and prognostic determination of ovarian cancer.

  6. Upregulation of Lhx8 increase VAChT expression and ACh release in neuronal cell line SHSY5Y.

    Science.gov (United States)

    Li, Haoming; Jin, Guohua; Zhu, Peipei; Zou, Linqing; Shi, Jinhong; Yi, Xin; Zhang, Xinhua; Tian, Meiling; Qin, Jianbing

    2014-01-24

    Lhx8 is a transcription factor for cholinergic differentiation. Our previous experiments found upregulation of Lhx8 promoted cholinergic neuronal differentiation of hippocampal neural stem/progenitor cells or hippocampal newborn neurons in vitro. However, the role of Lhx8 in VAChT expression and ACh release is still less understood. In this report, we transfected Lhx8 cDNA into neuronal cell line SHSY5Y by lentiviral vectors to acquire the cells which stably expressed high level of Lhx8. Using this cell model, we provided experimental evidence that increasing Lhx8 upregulated the expression of ChAT and VAChT, and also increased the ACh release in culture medium. We suggested that Lhx8 overexpression is a useful strategy to increase the release of ACh and maybe of therapeutic value to neurodegenerative diseases. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. 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

  8. Gene expression changes as markers of early lapatinib response in a panel of breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    O’Neill Fiona

    2012-06-01

    Full Text Available Abstract Background Lapatinib, a tyrosine kinase inhibitor of HER2 and EGFR and is approved, in combination with capecitabine, for the treatment of trastuzumab-refractory metastatic breast cancer. In order to establish a possible gene expression response to lapatinib, a panel of breast cancer cell lines with varying sensitivity to lapatinib were analysed using a combination of microarray and qPCR profiling. Methods Co-inertia analysis (CIA, a data integration technique, was used to identify transcription factors associated with the lapatinib response on a previously published dataset of 96 microarrays. RNA was extracted from BT474, SKBR3, EFM192A, HCC1954, MDAMB453 and MDAMB231 breast cancer cell lines displaying a range of lapatinib sensitivities and HER2 expression treated with 1 μM of lapatinib for 12 hours and quantified using Taqman RT-PCR. A fold change ≥ ± 2 was considered significant. Results A list of 421 differentially-expressed genes and 8 transcription factors (TFs whose potential regulatory impact was inferred in silico, were identified as associated with lapatinib response. From this group, a panel of 27 genes (including the 8 TFs were selected for qPCR validation. 5 genes were determined to be significantly differentially expressed following the 12 hr treatment of 1 μM lapatinib across all six cell lines. Furthermore, the expression of 4 of these genes (RB1CC1, FOXO3A, NR3C1 and ERBB3 was directly correlated with the degree of sensitivity of the cell line to lapatinib and their expression was observed to “switch” from up-regulated to down-regulated when the cell lines were arranged in a lapatinib-sensitive to insensitive order. These included the novel lapatinib response-associated genes RB1CC1 and NR3C1. Additionally, Cyclin D1 (CCND1, a common regulator of the other four proteins, was also demonstrated to observe a proportional response to lapatinib exposure. Conclusions A panel of 5 genes were determined to

  9. Gene expression changes as markers of early lapatinib response in a panel of breast cancer cell lines

    LENUS (Irish Health Repository)

    O’Neill, Fiona

    2012-06-18

    AbstractBackgroundLapatinib, a tyrosine kinase inhibitor of HER2 and EGFR and is approved, in combination with capecitabine, for the treatment of trastuzumab-refractory metastatic breast cancer. In order to establish a possible gene expression response to lapatinib, a panel of breast cancer cell lines with varying sensitivity to lapatinib were analysed using a combination of microarray and qPCR profiling.MethodsCo-inertia analysis (CIA), a data integration technique, was used to identify transcription factors associated with the lapatinib response on a previously published dataset of 96 microarrays. RNA was extracted from BT474, SKBR3, EFM192A, HCC1954, MDAMB453 and MDAMB231 breast cancer cell lines displaying a range of lapatinib sensitivities and HER2 expression treated with 1 μM of lapatinib for 12 hours and quantified using Taqman RT-PCR. A fold change ≥ ± 2 was considered significant.ResultsA list of 421 differentially-expressed genes and 8 transcription factors (TFs) whose potential regulatory impact was inferred in silico, were identified as associated with lapatinib response. From this group, a panel of 27 genes (including the 8 TFs) were selected for qPCR validation. 5 genes were determined to be significantly differentially expressed following the 12 hr treatment of 1 μM lapatinib across all six cell lines. Furthermore, the expression of 4 of these genes (RB1CC1, FOXO3A, NR3C1 and ERBB3) was directly correlated with the degree of sensitivity of the cell line to lapatinib and their expression was observed to “switch” from up-regulated to down-regulated when the cell lines were arranged in a lapatinib-sensitive to insensitive order. These included the novel lapatinib response-associated genes RB1CC1 and NR3C1. Additionally, Cyclin D1 (CCND1), a common regulator of the other four proteins, was also demonstrated to observe a proportional response to lapatinib exposure.ConclusionsA panel of 5 genes were determined to be differentially

  10. Tributyltin (TBT) increases TNFα mRNA expression and induces apoptosis in the murine macrophage cell line in vitro.

    Science.gov (United States)

    Nakano, Ken; Tsunoda, Masashi; Konno, Nobuhiro

    2004-11-01

    Tributyltin (TBT) compounds have been widely used as antifouling agents for shipbottom paint. The immune system is a target of TBT intoxication. We evaluated the effects of TBT chloride in macrophages, which have critical roles in the immune system, using a murine macrophage lineage cell line, J774.1,in vitro. We examined tumor necrosis factor α (TNFα), interleukin-1β (IL-1β) andc-jun mRNA expression in J774.1 cells. The effects of TBT on the apoptosis of J774.1 cells were examined by determining the percentage of TUNEL-positive cells and caspase-3 activity. The mean values of the viabilities of J774.1 cells exposed to TBT decreased dose-dependently. The relative mRNA expression of TNFα increased dose-dependently, however, that of IL-1β was not significantly different among the groups. The mean percentage of TUNEL-positive cells increased dose-dependently. Increases in the caspase-3 activities of J774.1 cells were observed in the groups exposed to higher concentrations of TBT. The mean value of relative mRNA expression of c-Jun transcription factor increased dose-dependently. The increases in the percentage of TUNEL-positive cells and in caspase-3 activity suggested that exposure to TBT induces apoptosis of J774.1 cells. The increases in the mRNA expression of TNFα andc-jun by TBT may be related to apoptosis in macrophages.

  11. Circadian Gene CLOCK Affects Drug-Resistant Gene Expression and Cell Proliferation in Ovarian Cancer SKOV3/DDP Cell Lines Through Autophagy.

    Science.gov (United States)

    Sun, Yang; Jin, Long; Sui, Yu-Xia; Han, Li-Li; Liu, Jia-Hua

    2017-05-01

    Abnormal autophagy regulation affects the chemoresistance of ovarian cancer, during which the circadian gene clock may play a major role. In this study, RNA interference plasmid pSUPER-Clock and overexpression plasmid pcDNA3.1-Clock of CLOCK were used to stably transfect the SKOV3/DDP cells by lipofection. Upon screening, the in vitro transfected cell lines with pSUPER-Clock, the autophagy level, and G 0 /G 1 phase cells were significantly reduced, and the expression levels of Clock, LC3, P-gp, and MRP2 were inhibited. In contrast, the autophagy level and G 0 /G 1 phase cells in cell lines transfected with pcDNA3.1-Clock were significantly increased, and the expressions of Clock, LC3, P-gp, and MRP2 were enhanced. In comparison with the untransfected control group showed the percentage of apoptotic cells in SKOV3/DDP cell lines of Clock interfering expression group after cisplatin treatment was significantly increased while the survival was substantially reduced. These results indicated that inhibiting the circadian gene Clock expression can reverse the cisplatin resistance of ovarian cancer SKOV3/DDP cell lines by affecting the protein expression of drug resistance genes during which autophagy plays an important role. The CLOCK gene may be designated as a novel candidate for targeted gene therapy in drug-resistant ovarian cancer.

  12. Validation of housekeeping genes for the normalization of RT-qPCR expression studies in oral squamous cell carcinoma cell line treated by 5 kinds of chemotherapy drugs.

    Science.gov (United States)

    Song, W; Zhang, W H; Zhang, H; Li, Y; Zhang, Y; Yin, W; Yang, Q

    2016-11-30

    Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become a frequently used strategy in gene expression studies. The relative quantification method is an important and commonly used method for the evaluation of RT-qPCR data. The key of this method is to identify an applicable internal control gene because the usage of different internal control genes may lead to distinct conclusions. Herein, we report the validation of 12 common housekeeping genes for RT-qPCR for gene expression analysis in the Oral squamous cell carcinoma (OSCC) cell line (KB and Tca-8113) treated by 5 kinds of Chemotherapy Drugs. The gene expression stability and applicability of the 12 housekeeping gene candidates were determined using the geNorm, NormFinder, and BestKeeper software programs. Comprehensive analyzing the results of the three software, ALAS1/GAPDH, ALAS1 and GUSB were suggested to be the most stable candidate genes for the study of both KB and Tca-8113 cell line together, KB cell line, and Tca-8113 cell line, respectively. This study provides useful information to normalize gene expression accurately for the investigation of target gene profiling in cell lines of OSCC. Further clarification of tumor molecular expression markers with our recommended housekeeping genes may improve the accuracy of diagnosis and estimation of prognostic factors as well as provide novel personalized treatments for OSCC patients.

  13. Cervical cancer cell lines expressing NKG2D-ligands are able to down-modulate the NKG2D receptor on NKL cells with functional implications

    Directory of Open Access Journals (Sweden)

    Jimenez-Perez Miriam I

    2012-02-01

    Full Text Available Abstract Background Cervical cancer represents the third most commonly diagnosed cancer and the fourth leading cause of cancer-related deaths in women worldwide. Natural killer (NK cells play an important role in the defense against viruses, intracellular bacteria and tumors. NKG2D, an activating receptor on NK cells, recognizes MHC class I chain-related molecules, such as MICA/B and members of the ULBP/RAET1 family. Tumor-derived soluble NKG2D-ligands have been shown to down-modulate the expression of NKG2D on NK cells. In addition to the down-modulation induced by soluble NKG2D-ligands, it has recently been described that persistent cell-cell contact can also down-modulate NKG2D expression. The goal of this study was to determine whether the NKG2D receptor is down-modulated by cell-cell contact with cervical cancer cells and whether this down-modulation might be associated with changes in NK cell activity. Results We demonstrate that NKG2D expressed on NKL cells is down-modulated by direct cell contact with cervical cancer cell lines HeLa, SiHa, and C33A, but not with non-tumorigenic keratinocytes (HaCaT. Moreover, this down-modulation had functional implications. We found expression of NKG2D-ligands in all cervical cancer cell lines, but the patterns of ligand distribution were different in each cell line. Cervical cancer cell lines co-cultured with NKL cells or fresh NK cells induced a marked diminution of NKG2D expression on NKL cells. Additionally, the cytotoxic activity of NKL cells against K562 targets was compromised after co-culture with HeLa and SiHa cells, while co-culture with C33A increased the cytotoxic activity of the NKL cells. Conclusions Our results suggest that differential expression of NKG2D-ligands in cervical cancer cell lines might be associated with the down-modulation of NKG2D, as well as with changes in the cytotoxic activity of NKL cells after cell-cell contact with the tumor cells.

  14. Alterations in gene expression profiles correlated with cisplatin cytotoxicity in the glioma U343 cell line

    OpenAIRE

    Patricia Oliveira Carminati; Stephano Spano Mello; Ana Lucia Fachin; Cristina Moraes Junta; Paula Sandrin-Garcia; Carlos Gilberto Carlotti; Eduardo Antonio Donadi; Geraldo Aleixo Silva Passos; Elza Tiemi Sakamoto-Hojo

    2010-01-01

    Gliomas are the most common tumors in the central nervous system, the average survival time of patients with glioblastoma multiforme being about 1 year from diagnosis, in spite of harsh therapy. Aiming to study the transcriptional profiles displayed by glioma cells undergoing cisplatin treatment, gene expression analysis was performed by the cDNA microarray method. Cell survival and apoptosis induction following treatment were also evaluated. Drug concentrations of 12.5 to 300 μM caused ...

  15. Estrogen stimulates adenosine receptor expression subtypes in human breast cancer MCF-7 cell line.

    Science.gov (United States)

    Mohamadi, Azam; Aghaei, Mahmoud; Panjehpour, Mojtaba

    2018-02-01

    Estrogen is a steroid hormone that plays a key role in the development and regulation of reproductive system. It has been shown that estrogen is related to breast cancer development through binding to its receptors. In order to uncover the estrogen effects on adenosine receptor expression, estrogen-positive MCF-7 cells were used to treat with agonist and antagonist of estrogen and then the mRNA expression of adenosine receptor subtypes were evaluated. Estrogen-positive MCF-7 cells were treated with various concentrations of 17β estradiol (E2) as an estrogen agonist, and ICI 182,780 as an estrogen antagonist. The gene expression of adenosine receptor subtypes were detected by real time RT-PCR. The results of MTT assay showed that E2 increased cell viability in a dose dependent manner. The expression pattern of all adenosine receptor subtypes are as follow; A2b > A1 > A2a > A3 in untreated MCF-7 cells. Obtained results showed that E2 incubation at 0.001-0.01 μM led to up-regulation of A1ARs, A2aARs and A3ARs dose dependently. E2 at 0.001 μM also had no significant effect on A2bARs expression but, at higher doses induced a considerable decrease in mRNA A2bARs expression. Treatment with antagonist confirmed that up-regulation of these receptors is mediated by estrogen receptor. Taken together, our results indicate that treatment of MCF-7 cells with E2 led to up-regulation of adenosine receptors. However, these effects were partially restored by treatment with antagonist suggesting that such effects are mediated by estrogen receptors.

  16. Expression of tumor-specific antigen MAGE, GAGE and BAGE in ovarian cancer tissues and cell lines

    Directory of Open Access Journals (Sweden)

    Yu Hao

    2010-04-01

    Full Text Available Abstract Background To observe mRNA expression of tumor-specific antigen MAGE, BAGE and GAGE in epithelial ovarian cancer tissues and cell lines, to explore the relationship between gene expression and diagnosis, treatment and prognosis of ovarian cancer, and to evaluate the feasibility of their gene products as markers, and an immunotherapy target for ovarian cancer. Methods mRNA expression of MAGE-1, MAGE-3, GAGE-1/2 and BAGE were determined by reverse transcription polymerase chain reaction (RT-PCR in 14 cases of normal ovarian tissue, 20 cases of ovarian benign tumor specimens, 41 cases of ovarian cancer specimens, and ovarian cancer cell lines SKOV3, A2780, and COC1. Results MAGE, GAGE and BAGE genes were not expressed in normal ovarian tissue. In benign tumors, only the MAGE gene was expressed; the expression rate of this gene in benign tumors was 15% (3/20. In ovarian cancer tissues, MAGE-1 and MAGE-3 was highly expressed, with expression rates of 53.7% (22/41 and 36.6% (15/41, while GAGE-1/2 and BAGE had relatively low expression, with rates of 26.8% (11/41 and 14.6% (6/41. In metastatic lesions of ovarian cancer, only MAGE-1 and BAGE were expressed, with expression rates of 28.6% (2/7 and 14.3% (1/7. The positive expression rates of MAGE-1 and MAGE-3 in serous cystadenocarcinoma were significantly higher than that in other types of ovarian cancer (P P Conclusion Tumor-specific antigen MAGE, BAGE and GAGE may play a role in the occurrence and development of ovarian cancer. These genes can be used as one of the important indicators for early diagnosis, efficacy evaluation and prognostic determination of ovarian cancer.

  17. Poliovirus modulates Bcl-xl expression in the human U937 promonocytic cell line.

    Science.gov (United States)

    Calandria, C; López-Guerrero, J A

    2002-12-01

    Poliovirus induces bcl-2-independent apoptosis in the human U937 promonocytic cell line [28]. Here we describe that this cell death, induced after viral infection, correlates with the modulation of the protooncoprotein Bcl-xl. Furthermore, poliovirus infection decreases the detected Bcl-xl in a U937 clone that overexpresses this protein (U937bcl-xl). Although in U937bcl-xl cells, Bcl-xl was not as highly regulated as in parental U937 cells, correlation between Bcl-xl modulation and the cleavage of poly(ADP-ribose)polymerase could be observed. Nevertheless, the induction of shutoff after infection of transfected control U937neo or U937bcl-xl clones was not significantly altered. Finally, production of new viral particles was slightly restricted in Bcl-xl-overexpressing U937 cells. Taken together, these results suggest that Bcl-xl is modulated during the induction of apoptosis in the promonocytic cell line U937 after poliovirus infection, although modulation of this protooncogene was not sufficient to modify the course of infection.

  18. Abnormalities in structure and expression of the retinoblastoma gene in small cell lung cancer cell lines and xenografts in nude mice

    DEFF Research Database (Denmark)

    Rygaard, K; Sorenson, G D; Pettengill, O S

    1990-01-01

    the Rb protein, we investigated the expression of the Mr 110,000-116,000 Rb protein in SCLC tumors grown as xenografts in nude mice and/or as cell lines. Rb messenger RNA expression was determined by Northern blotting, and gross structural gene alterations were investigated by Southern blotting. Tumors...

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

    Directory of Open Access Journals (Sweden)

    Erica L Cain

    2011-04-01

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

  20. The human megakaryocytic cell line UT-7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor.

    Science.gov (United States)

    Kawaguchi, Tatsuya; Hashimoto, Ryuji; Yokota, Hiroshi

    2010-09-01

    We have demonstrated that a unique megakaryocytic cell line UT-7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT-7/TPO was found to express GPVI and its associate signal molecule of FcRgamma (Fc receptor gamma chain). When UT-7/TPO was stimulated with the GPVI agonist convulxin, the [Ca2+]i (intracellular Ca2+) was elevated in a convulxin concentration-dependent manner, and [Ca2+]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT-7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)-1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca2+]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin-stimulated platelets failed to produce TX, it is suggested that UT-7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT-7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.

  1. Cloning and Stable Expression of cDNA Coding For Platelet Endothelial Cell Adhesion Molecule -1 (PECAM-1, CD31 in NIH-3T3 Cell Line

    Directory of Open Access Journals (Sweden)

    Hamed Salehi-Lalemarzi

    2015-06-01

    Full Text Available Purpose: PECAM-1 (CD31 is a glycoprotein expressed on endothelial and bone marrow precursor cells. It plays important roles in angiogenesis, maintenance and integration of the cytoskeleton and direction of leukocytes to the site of inflammation. We aimed to clone the cDNA coding for human CD31 from KG1a for further subcloning and expression in NIH- 3T3 mouse cell line. Methods: CD31 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 2235 bp specific band was aligned completely to human CD31 reference sequence in NCBI database. Transient and stable expression of human CD31 on transfected NIH-3T3 mouse fibroblast cells was achieved (23% and 96%, respectively as shown by flow cytometry. Conclusion: Due to murine origin of NIH-3T3 cell line, CD31-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD31, with no need for purification of recombinant proteins.

  2. TGF-β1 and IL-10 expression in epithelial ovarian cancer cell line ...

    African Journals Online (AJOL)

    TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol 1991; 115: 1091-. 1105. 21. Gordinier ME, Zhang HZ, Patenia R, Levy LB, Atkinson. EN, Nash MA, Katz RL, Platsoucas CD, Freedman. RS. Quantitative analysis of transforming growth.

  3. Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines

    Directory of Open Access Journals (Sweden)

    Nidal Ghosheh

    2016-01-01

    Full Text Available Human pluripotent stem cells- (hPSCs- derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4 which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.

  4. Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines

    Science.gov (United States)

    Ghosheh, Nidal; Olsson, Björn; Edsbagge, Josefina; Küppers-Munther, Barbara; Van Giezen, Mariska; Asplund, Annika; Andersson, Tommy B.; Björquist, Petter; Carén, Helena; Simonsson, Stina; Sartipy, Peter; Synnergren, Jane

    2016-01-01

    Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models. PMID:26949401

  5. Temporal regulation of HTLV-2 expression in infected cell lines and patients: evidence for distinct expression kinetics with nuclear accumulation of APH-2 mRNA

    Directory of Open Access Journals (Sweden)

    Bender Cecilia

    2012-09-01

    Full Text Available Abstract Background Human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2 are delta retroviruses with similar genetic organization. Although both viruses immortalize T-cells in vitro, they exhibit distinct pathogenic potential in vivo. To search for possible differences in its expression strategy with respect to HTLV-1, we investigated the pattern of HTLV-2 expression in infected cell lines and peripheral blood mononuclear cells (PBMCs from infected patients using splice site-specific quantitative RT-PCR. Findings A novel alternative splice acceptor site for exon 2 was identified; its usage in env transcripts was found to be subtype-specific. Time-course analysis revealed a two-phase expression kinetics in an infected cell line and in PBMCs of two of the three patients examined; this pattern was reminiscent of HTLV-1. In addition, the minus-strand APH2 transcript was mainly detected in the nucleus, a feature that was similar to its HTLV-1 orthologue HBZ. In contrast to HTLV-1, expression of the mRNA encoding the main regulatory proteins Tax and Rex and that of the mRNAs encoding the p28 and truncated Rex inhibitors is skewed towards p28/truncated Rex inhibitors in HTLV-2. Conclusion Our data suggest a general converging pattern of expression of HTLV-2 and HTLV-1 and highlight peculiar differences in the expression of regulatory proteins that might influence the pathobiology of these viruses.

  6. Plasmodium falciparum parasites expressing pregnancy-specific variant surface antigens adhere strongly to the choriocarcinoma cell line BeWo

    DEFF Research Database (Denmark)

    Haase, Rikke N; Megnekou, Rosette; Lundquist, Maja

    2006-01-01

    Placenta-sequestering Plasmodium falciparum parasites causing pregnancy-associated malaria express pregnancy-specific variant surface antigens (VSA(PAM)). We report here that VSA(PAM)-expressing patient isolates adhere strongly to the choriocarcinoma cell line BeWo and that the BeWo line can...... be used to efficiently select for VSA(PAM) expression in vitro....

  7. Transepithelial resistance and claudin expression in trout RTgill-W1 cell line

    DEFF Research Database (Denmark)

    T. Trubitt, Rebecca; Rabeneck, D. Brett; Bujak, Joanna

    2015-01-01

    In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1–2 days (20–80 Ω⋅cm2), which was then mai......In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1–2 days (20–80 Ω⋅cm2), which...... was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6 h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to b15% of pre-treatment level. Cortisol...

  8. Glioblastoma cancer stem cell lines express functional acid sensing ion channels ASIC1a and ASIC3

    DEFF Research Database (Denmark)

    Tian, Yuemin; Bresenitz, Pia; Reska, Anna

    2017-01-01

    Acidic microenvironment is commonly observed in tumour tissues, including glioblastoma (GBM), the most aggressive and lethal brain tumour in adults. Acid sensing ion channels (ASICs) are neuronal voltage-insensitive sodium channels, which are sensors of extracellular protons. Here we studied...... and functionally characterized ASICs in two primary glioblastoma stem cell lines as cell culture models. We detected transcripts of the ACCN2 and ACCN3 genes, coding for ASIC1 and ASIC3, respectively, but not transcripts of ACCN1 (coding for ASIC2). Available microarray data confirmed that ACCN1 is downregulated...... in glioma. Western blotting confirmed expression of ASIC1 and ASIC3, the most proton-sensitive ASICs, in both GBM cell lines. We characterized ASICs functionally using whole-cell patch clamp and detected different types of acid-sensitive currents. Some of these currents had kinetics typical for ASICs...

  9. The endosymbiont Wolbachia pipientis induces the expression of host antioxidant proteins in an Aedes albopictus cell line.

    Directory of Open Access Journals (Sweden)

    Lesley J Brennan

    Full Text Available Wolbachia are obligate intracellular bacteria which commonly infect arthropods. They are maternally inherited and capable of altering host development, sex determination, and reproduction. Reproductive manipulations include feminization, male-killing, parthenogenesis, and cytoplasmic incompatibility. The mechanism by which Wolbachia avoid destruction by the host immune response is unknown. Generation of antimicrobial peptides (AMPs and reactive oxygen species (ROS by the host are among the first lines of traditional antimicrobial defense. Previous work shows no link between a Wolbachia infection and the induction of AMPs. Here we compare the expression of protein in a cell line naturally infected with Wolbachia and an identical cell line cured of the infection through the use of antibiotics. Protein extracts of each cell line were analyzed by two dimensional gel electrophoresis and LC/MS/MS. Our results show the upregulation of host antioxidant proteins, which are active against ROS generated by aerobic cell metabolism and during an immune response. Furthermore, flow cytometric and microscopic analysis demonstrates that ROS production is significantly greater in Wolbachia-infected mosquito cells and is associated with endosymbiont-containing vacuoles located in the host cell cytoplasm. This is the first empirical data supporting an association between Wolbachia and the insect antioxidant system.

  10. Dendrosomal curcumin nanoformulation modulate apoptosis-related genes and protein expression in hepatocarcinoma cell lines.

    Science.gov (United States)

    Montazeri, Maryam; Sadeghizadeh, Majid; Pilehvar-Soltanahmadi, Yones; Zarghami, Faraz; Khodi, Samaneh; Mohaghegh, Mina; Sadeghzadeh, Hadi; Zarghami, Nosratollah

    2016-07-25

    The side-effects observed in conventional therapies have made them unpromising in curing Hepatocellular carcinoma; therefore, developing novel treatments can be an overwhelming significance. One of such novel agents is curcumin which can induce apoptosis in various cancerous cells, however, its poor solubility is restricted its application. To overcome this issue, this paper employed dendrosomal curcumin (DNC) was employed to in prevent hepatocarcinoma in both RNA and protein levels. Hepatocarcinoma cells, p53 wild-type HepG2 and p53 mutant Huh7, were treated with DNC and investigated for toxicity study using MTT assay. Cell cycle distribution and apoptosis were analyzed using Flow-cytometry and Annexin-V-FLUOS/PI staining. Real-time PCR and Western blot were employed to analyze p53, BAX, Bcl-2, p21 and Noxa in DNC-treated cells. DNC inhibited the growth in the form of time-dependent manner, while the carrier alone was not toxic to the cell. Flow-cytometry data showed the constant concentration of 20μM DNC during the time significantly increases cell population in SubG1 phase. Annexin-V-PI test showed curcumin-induced apoptosis was enhanced in Huh7 as well as HepG2, compared to untreated cells. Followed by treatment, mRNA expression of p21, BAX, and Noxa increased, while the expression of Bcl-2 decreased, and unlike HepG2, Huh7 showed down-regulation of p53. In summary, DNC-treated hepatocellular carcinoma cells undergo apoptosis by changing the expression of genes involved in the apoptosis and proliferation processes. These findings suggest that DNC, as a plant-originated therapeutic agent, could be applied in cancer treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. A Comparitive Assessement of Cytokine Expression in Human-Derived Cell Lines Exposed to Alpha Particles and X-Rays

    Directory of Open Access Journals (Sweden)

    Vinita Chauhan

    2012-01-01

    Full Text Available Alpha- (α- particle radiation exposure has been linked to the development of lung cancer and has been identified as a radiation type likely to be employed in radiological dispersal devices. Currently, there exists a knowledge gap concerning cytokine modulations associated with exposure to α-particles. Bio-plex technology was employed to investigate changes in proinflammatory cytokines in two human-derived cell lines. Cells were irradiated at a dose of 1.5 Gy to either α-particles or X-rays at equivalent dose rates. The two cell lines exhibited a unique pattern of cytokine expression and the response varied with radiation type. Of the 27 cytokines assessed, only vascular endothelin growth factor (VEGF was observed to be modulated in both cell lines solely after α-particle exposure, and the expression of VEGF was shown to be dose responsive. These results suggest that certain proinflammatory cytokines may be involved in the biological effects related to α- particle exposure and the responses are cell type and radiation type specific.

  12. TERRA Expression Levels Do Not Correlate With Telomere Length and Radiation Sensitivity in Human Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Alexandra eSmirnova

    2013-05-01

    Full Text Available Mammalian telomeres are transcribed into long non-coding telomeric RNA molecules (TERRA that seem to play a role in the maintenance of telomere stability. In human cells, CpG island promoters drive TERRA transcription and are regulated by methylation. It was suggested that the amount of TERRA may be related to telomere length. To test this hypothesis we measured telomere length and TERRA levels in single clones isolated from five human cell lines: HeLa (cervical carcinoma, BRC-230 (breast cancer, AKG and GK2 (gastric cancers and GM847 (SV40 immortalized skin fibroblasts. We observed great clonal heterogeneity both in TRF (Terminal Restriction Fragment length and in TERRA levels. However, these two parameters did not correlate with each other. Moreover, cell survival to γ-rays did not show a significant variation among the clones, suggesting that, in this cellular system, the intra-population variability in telomere length and TERRA levels does not influence sensitivity to ionizing radiation. This conclusion was supported by the observation that in a cell line in which telomeres were greatly elongated by the ectopic expression of telomerase, TERRA expression levels and radiation sensitivity were similar to the parental HeLa cell line.

  13. TERRA Expression Levels Do Not Correlate with Telomere Length and Radiation Sensitivity in Human Cancer Cell Lines.

    Science.gov (United States)

    Smirnova, Alexandra; Gamba, Riccardo; Khoriauli, Lela; Vitelli, Valerio; Nergadze, Solomon G; Giulotto, Elena

    2013-01-01

    Mammalian telomeres are transcribed into long non-coding telomeric repeat-containing RNA (TERRA) molecules that seem to play a role in the maintenance of telomere stability. In human cells, CpG-island promoters drive TERRA transcription and are regulated by methylation. It was suggested that the amount of TERRA may be related to telomere length. To test this hypothesis we measured telomere length and TERRA levels in single clones isolated from five human cell lines: HeLa (cervical carcinoma), BRC-230 (breast cancer), AKG and GK2 (gastric cancers), and GM847 (SV40 immortalized skin fibroblasts). However, these two parameters did not correlate with each other. Moreover, cell survival to γ-rays did not show a significant variation among the clones, suggesting that, in this cellular system, the intra-population variability in telomere length and TERRA levels does not influence sensitivity to ionizing radiation. This conclusion was supported by the observation that in a cell line in which telomeres were greatly elongated by the ectopic expression of telomerase, TERRA expression levels and radiation sensitivity were similar to the parental HeLa cell line.

  14. Enhanced expression of unique gangliosides with GM2-determinant in human uterine cervical carcinoma-derived cell lines.

    Science.gov (United States)

    Tanaka, Kyoko; Miyazawa, Masaki; Mikami, Mikio; Aoki, Daisuke; Kiguchi, Kazushige; Iwamori, Masao

    2016-10-01

    Monoclonal antibody YHD-06 generated by immunization with GM2 reacted with gangliosides with GM2-determinant, i.e., GM2, GalNAc-GM1b and GalNAc-GD1a, among which GalNAc-GD1a was characterized as an antigen of autoimmune peripheral neuropathies including Guillain-Barré syndrome. When glycolipids were examined by TLC-immunostaining with YHD-06 in seven human cervical carcinoma-derived cell lines, GM2 was found in all cell lines, amounting to 15.5 % to 57.5 % of total gangliosides. Whereas GalNAc-GD1a was present in three cell lines, amounting to 5.4-17.5 % of total gangliosides, and GalNAc-GM1b in four cell lines in amounts of less than 2 %. The elevated amounts of gangliosides with GM2 determinant were closely correlated with the relative intensities of gene expression of GalNAc transferase, this being characteristic of cervical carcinoma-derived cells. However, in tissues from patients with several histological types of cervical carcinomas, GM3 was ubiquitously expressed in amounts of more than 66 % of total gangliosides, GM2 was expressed in only five of 15 tissues, and both GalNAc-GM1b and GalNAc-GD1a were not even detected in trace amounts. Since GM1 was detected in all tissues in amounts of less than 0.06 μg/mg dried tissue, all cervical carcinoma tissues were revealed to exhibit GM2 synthesis, indicating that enhanced synthesis of gangliosides with GM2 determinant is a characteristic of cultivated cells in vitro. Similarly, although I(3)SO3-GalCer was not present in the squamous cell carcinoma (SCC) tissues, SCC-derived cells selectively expressed II(3)SO3-LacCer. Since enhanced synthesis of GM2 has been reported in SV-40 virus-transfected fibroblasts, papilloma virus might be involved in the expression of GM2 in cervical carcinoma-derived cells.

  15. Expression of transcription factor Klf8 in lung cancer tissue and the biological effect of downregulation of Klf8 expression in lung cancer cell lines

    Directory of Open Access Journals (Sweden)

    Jing Wang

    2016-01-01

    Full Text Available Objective: To study the expression of transcription factor Klf8 in lung cancer tissue and the biological effect of downregulation of Klf8 expression in lung cancer cell lines. Methods: Cancer tissue and adjacent normal lung tissue were collected and mRNA contents of Klf8 were detected; lung cancer A549 cell lines were cultured, and after transfection of Klf8 siRNA, cell cycle, cell invasion and epithelial-mesenchymal transition were detected. Results: mRNA contents of Klf8 in lung cancer tissue were higher than those in adjacent normal lung tissue; after transfection of Klf8 siRNA, Klf8 mRNA inhibition rate was 74.31%; G0/G1 phase ratio of Klf8 siRNA group was higher than that of negative control siRNA group; ratios of S-phase and G2/M phase cells, mRNA contents of Cyclin D1 and number of cells invading to the outer side of the transwell microporous membrane were lower than those of negative control siRNA group; mRNA contents of CDH1 and CK18 as well as Snail and Slug of Klf8 siRNA group were higher than those of negative control siRNA group; mRNA contents of VIM and N-cadherin were lower than those of negative control siRNA group. Conclusion: The expression of Klf8 in lung cancer tissue abnormally elevates; downregulation of Klf8 expression in lung cancer cell lines can inhibit malignant biological effect of cells, manifested as cell cycle arrest as well as the inhibition of cell invasion and epithelial-mesenchymal transition processes.

  16. Analysis of miRNA expression profiles in melatonin-exposed GC-1 spg cell line.

    Science.gov (United States)

    Zhu, Xiaoling; Chen, Shuxiong; Jiang, Yanwen; Xu, Ying; Zhao, Yun; Chen, Lu; Li, Chunjin; Zhou, Xu

    2018-02-05

    Melatonin is an endocrine neurohormone secreted by pinealocytes in the pineal gland. It exerts diverse physiological effects, such as circadian rhythm regulator and antioxidant. However, the functional importance of melatonin in spermatogenesis regulation remains unclear. The objectives of this study are to: (1) detect melatonin affection on miRNA expression profiles in GC-1 spg cells by miRNA deep sequencing (DeepSeq) and (2) define melatonin affected miRNA-mRNA interactions and associated biological processes using bioinformatics analysis. GC-1 spg cells were cultured with melatonin (10 -7 M) for 24h. DeepSeq data were validated using quantitative real-time reverse transcription polymerase chain reaction analysis (qRT-PCR). A total of 176 miRNA expressions were found to be significantly different between two groups (fold change of >2 or melatonin could regulate the expression of miRNA to perform its physiological effects in GC-1 spg cells. These results should be useful to investigate the biological function of miRNAs regulated by melatonin in spermatogenesis and testicular germ cell tumor. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. ERVK Polyprotein Processing and Reverse Transcriptase Expression in Human Cell Line Models of Neurological Disease

    Directory of Open Access Journals (Sweden)

    Mamneet Manghera

    2015-01-01

    Full Text Available Enhanced expression of the reverse transcriptase (RT protein encoded by human endogenous retrovirus-K (ERVK is a promising biomarker for several inflammatory and neurological diseases. However, unlike RT enzymes encoded by exogenous retroviruses, little work has been done to identify ERVK RT isoforms, their expression patterns, and cellular localization. Using Western blot, we showcase the ERVK gag-pro-pol polyprotein processing leading to the production of several ERVK RT isoforms in human neuronal (ReNcell CX and astrocytic (SVGA models of neuroinflammatory disease. Since the pro-inflammatory cytokine IFNγ plays a key role in the pathology of several ERVK-associated neurological diseases, we sought to determine if IFNγ can drive ERVK RT expression. IFNγ signalling markedly enhanced ERVK polyprotein and RT expression in both human astrocytes and neurons. RT isoforms were expressed in a cell-type specific pattern and the RT-RNase H form was significantly increased with IFNγ treatment. Fluorescent imaging revealed distinct cytoplasmic, perinuclear and nuclear ERVK RT staining patterns upon IFNγ stimulation of astrocytes and neurons. These findings indicate that ERVK expression is inducible under inflammatory conditions such as IFNγ exposure—and thus, these newly established in vitro models may be useful in exploring ERVK biology in the context of neuroinflammatory disease.

  18. Expression of CD44 on two lines of transplantable melanoma cells--relationship with cytokine secretion and tumor progression.

    Directory of Open Access Journals (Sweden)

    Sławomir Wójcik

    2004-03-01

    Full Text Available In the present work it was investigated if a spontaneous alteration of the native melanotic transplantable melanoma form into amelanotic form, connected with the tumor progression, is accompanied by changes of CD44 surface glycoprotein expression. We also tried to find out if there exists any correlation between changes in CD44 expression and IL-6, TNF-alpha, and IL-10 secretion. Cells of two hamster transplantable melanoma lines: melanotic and amelanotic were used. The levels of TNF-alpha, IL-6, IL-10 in supernatants were determined by the ELISA test. For the detection of CD44 expression by flow cytometry, isolated melanoma cells were stained with the rat anti-mouse CD44 monoclonal antibody. The stained cells were also examined using a fluorescence microscope and a confocal microscopy system. The obtained results indicate that a spontaneous alteration of the native melanotic form into amelanotic form and the associated tumor progression was accompanied by a decrease in CD44 glycoprotein expression on the cell surface and a decrease in IL-6, TNF-alpha and especially IL-10 secretion by amelanotic melanoma cells. Our observations suggest a relationship between CD44 expression and locally secreted cytokines in the course of transplantable melanoma progression.

  19. Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines

    DEFF Research Database (Denmark)

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

    1993-01-01

    A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis......(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell...... lines express TGF beta-receptors and also produce TGF beta mRNAs....

  20. Clofarabine Has Apoptotic Effect on T47D Breast Cancer Cell Line via P53R2 Gene Expression.

    Science.gov (United States)

    Rahmati-Yamchi, Mohammad; Zarghami, Nosratollah; Nozad Charoudeh, Hojjatollah; Ahmadi, Yasin; Baradaran, Behzad; Khalaj-Kondori, Mohammad; Milani, Morteza; Akbarzadeh, Abolfazl; Shaker, Maghsud; Pourhassan-Moghaddam, Mohammad

    2015-11-01

    Clofarabine, a purine nucleoside analogue and inhibitor of Ribonucleotide Reductase (RR), is used for treatment of leukemia. Clofarabine-induced defect in DNA replication, induces p53 and subsequently P53R2 genes as subunit of RR. clofarabine deregulated P53R2 gene expression leading to the elevated levels of P53R2 which impose resistance to DNA damaging drugs. In this study the apoptotic and cytotoxic effects of clofarabine has been investigated on breast cancer cell line. Cofarabine cytotoxicity on T47D cells has been studied by MTT assay. T47D cells were exposed to the different concentrations of clofarabine for 24, 48 and 72 hours intervals. Relative expression of P53R2 gene has been studied using real-time PCR. Moreover, after treating with clofarabine the apoptotic and necrotic cells were detected using Annexin V and propodium iodide (PI) reagents by flowcytometry technique. MTT assay results showed that the clofarabine IC50 on T47D cell line were 3 and 2.5µM after 48 and 72 h exposure, respectively. Clofarabine did not show any significant cytotoxic effect after 24 h exposure. The analysis of qRT-PCR showed a significant increase in P53R2 gene expression in treated cells with both 2.5 and 3 μM doses and also, the results of flowcytometry revealed 26.91 and 74.46 percent apoptosis induction in 48 and 72h treatments respectively in comparison to the control groups. Our results showed that apoptotic and cytotoxic effects of clofarabine on T47D cell line were in time and dose dependent manner; therefore it could be considered a new candidate in breast cancer therapy.

  1. Effect of saffron extract on VEGF-A expression in MCF7 cell line

    Directory of Open Access Journals (Sweden)

    Marzieh Mousavi

    2014-03-01

    Full Text Available Background: Various studies have focused on the anticancer effects of saffron. Angiogenesis or new blood vessel formation, which is required for embryonic development and many physiological events, plays a crucial role in many pathological conditions such as tumor growth. One of the main genes which is involved in the process of angiogenesis is VEGF-A. In this in vitro study, the effects of saffron extract on VEGF-A expression were examined. Methods: In this experimental study, the saffron extract was obtained by Soxhlet extractor and then the powder was frozen and dried in vacuum (lyophilisation using a freeze dryer. MCF7 cells were grown in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS and incubated at 37˚C with 5% CO2. After 24 h of cell culture, their adhesion to the bottom flasks was investigated, then, they were treated by the aqueous extract of saffron at concentrations of 100, 200, 400 and 800 µg/ml. 48 hours after treatment, total RNA was extracted and cDNA was synthesized using the sequence of target gene. Finally, the synthesized products were analysed by Real Time PCR to determine the expression level of VEGF-A. Results: The results of data analysis showed the inhibitory effect of saffron extract in concentrations of 100, 200, 400 and 800 µg/ml on VEGF-A expression in MCF7 cells in comparison with control group, indicating the highest reduction of gen expression for the highest concentration of saffron extract (800 µg/ml. Conclusion: Results indicated a decrease in the expression of VEGF-A, specific biomarker of angiogenesis, in the treated samples compared to the control group.

  2. Down-regulated βIII-tubulin Expression Can Reverse Paclitaxel Resistance in A549/Taxol Cells Lines

    Directory of Open Access Journals (Sweden)

    Yinling ZHUO

    2014-08-01

    Full Text Available Background and objective Chemotherapy drug resistance is the primary causes of death in patients with pulmonary carcinoma which make tumor recurrence or metastasis. β-tubulin is the main cell targets of anti-microtubule drug. Increased expression of βIII-tubulin has been implicated in non-small cell lung cancer (NSCLC cell lines. To explore the relationship among the expression level of βIII-tubulin and the sensitivity of A549/Taxolcell lines to Taxol and cell cycles and cell apoptosis by RNA interference-mediated inhibition of βIII-tubulin in A549/Taxol cells. Methods Three pairs of siRNA targetd βIII-tubulin were designed and prepared, which were transfected into A549/Taxol cells using LipofectamineTM 2000. We detected the expression of βIII-tubulin mRNA using Real-time fluorescence qRT-PCR. Tedhen we selected the most efficient siRNA by the expression of βIII-tubulin mRNA in transfected group. βIII-tubulin protein level were mesured by Western blot. The taxol sensitivity in transfected group were evaluated by MTT assay. And the cell apoptosis and cell cycles were determined by flow cytometry. Results βIII-tubulin mRNA levels in A549/Taxol cells were significantly decreased in transfected grop by Real-time qRT-PCR than control groups. And βIII-tubulin siRNA-1 sequence showed the highest transfection efficiency, which was (87.73±4.87% (P<0.01; Western blot results showed that the expressional level of BIII tublin protein was significantly down-reulated in the transfectant cells than thant in the control cells. By MTT assay, we showed that the inhibition ratio of Taxol to A549/Taxol cells transfeced was higher than that of control group (51.77±4.60% (P<0.01. The early apoptosis rate of A549/Taxol cells in transfected group were significantly higher than that of control group (P<0.01; G2-M content in taxol group obviously increased than untreated samples by the cell cycle (P<0.05. Conclusion βIII-tubulin down-regulated significantly

  3. TNF-α-induced IL-6 and MMP-9 expression in immortalized ameloblastoma cell line established by hTERT.

    Science.gov (United States)

    Ohta, K; Naruse, T; Ishida, Y; Shigeishi, H; Nakagawa, T; Fukui, A; Nishi, H; Sasaki, K; Ogawa, I; Takechi, M

    2017-03-01

    Ameloblastoma (AM) shows locally invasive behaviour. However, biological investigations regarding regulation of gene expression associated with AM pathological features are difficult to perform, because AM cells can be passaged for a few generations due to senescence. We report a newly established immortalized AM cell line, AMB cells, by transfection with human telomerase reverse transcriptase (hTERT). Furthermore, we examined whether TNF-α modulates bone resorption-related genes, IL-6 and MMP-9 in cooperation with TGF-β or IFN-γ. Following transfection of an hTERT expression vector into AM cells using a non-viral method, the effects of cytokines on the expressions of IL-6 and MMP-9 mRNA were examined using real-time PCR. TNF-α-induced NF-κB activity was examined by western blotting and transcription factor assays. AMB cells continued to grow for more than 100 population doublings. Stimulation with TNF-α increased IL-6 and MMP-9 mRNA expressions, as well as NF-κB activation. Furthermore, TGF-β and IFN-γ dramatically increased TNF-α-mediated expressions of MMP-9 and IL-6 mRNA, respectively, while those responses were suppressed by NF-κB inhibitor. We established an immortalized AM cell line by hTERT transfection. TNF-α-mediated regulation of MMP-9 and IL-6 via NF-κB may play an important role in the pathological behaviour of AMs, such as bone resorption. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Amplification of rabbit hepatocyte growth factor and detection of its expression in COS-7 cell line.

    Science.gov (United States)

    Yao, H; Han, J; Wang, J; Wang, L; Gong, C; Li, L; Liang, Z; Tian, Y

    2015-11-25

    We used RT-PCR, nested PCR to acquire the partial 5'- race fragment of rabbit HGF cDNA and the partial 3'- race fragment of rabbit HGF cDNA. Then, we used recombination PCR to acquire rabbit HGF successfully. Homology analysis was conducted among the sequence of RABHGF and known human and rat HGF by DNAStar. It was proved that high level of homology existed among the sequences of those three HGF genes. We used the acquired gene of RABHGF to construct its recombinant eukaryotic expression vector pcDNA3.1(+)-RABHGF (pRABHGF). The identification of the eukaryotic expression vector pRABHGF by PCR, restriction enzyme and sequencing analysis showed that rabbit HGF gene was correctly inserted into the vector. pRABHGF and pcDNA3.1(+) as controls were transfected into COS-7 cells by lipofectamine. It takes 24h-36h after transfection to detect the expression of RABHGF protein by indirect immunofluorescence assay (IFA). The proliferation of cos-7 cells were evaluated by MTT assay. The result displayed positive effect of RABHGF protein on the proliferation of COS-7 cells. This study lays the foundation for a new gene therapy method for ischemic heart disease.

  5. Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

    Science.gov (United States)

    Nakamura, Tsuyoshi; Omasa, Takeshi

    2015-09-01

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Sulforaphane causes epigenetic repression of hTERT expression in human breast cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Syed M Meeran

    Full Text Available BACKGROUND: Sulforaphane (SFN, an isothiocyanate found in cruciferous vegetables, is a common dietary component that has histone deacetylase inhibition activity and exciting potential in cancer prevention. The mechanisms by which SFN imparts its chemopreventive properties are of considerable interest and little is known of its preventive potential for breast cancer. PRINCIPAL FINDINGS: We found that SFN significantly inhibits the viability and proliferation of breast cancer cells in vitro while it has negligible effects on normal breast cells. Inhibition of telomerase has received considerable attention because of its high expression in cancer cells and extremely low level of expression in normal cells. SFN treatment dose- and time-dependently inhibited human telomerase reverse transcriptase (hTERT, the catalytic regulatory subunit of telomerase, in both MCF-7 and MDA-MB-231 human breast cancer cells. DNA methyltransferases (DNMTs, especially DNMT1 and DNMT3a, were also decreased in SFN-treated breast cancer cells suggesting that SFN may repress hTERT by impacting epigenetic pathways. Down-regulation of DNMTs in response to SFN induced site-specific CpG demethylation occurring primarily in the first exon of the hTERT gene thereby facilitating CTCF binding associated with hTERT repression. Chromatin immunoprecipitation (ChIP analysis of the hTERT promoter revealed that SFN increased the level of active chromatin markers acetyl-H3, acetyl-H3K9 and acetyl-H4, whereas the trimethyl-H3K9 and trimethyl-H3K27 inactive chromatin markers were decreased in a dose-dependent manner. SFN-induced hyperacetylation facilitated the binding of many hTERT repressor proteins such as MAD1 and CTCF to the hTERT regulatory region. Depletion of CTCF using siRNA reduced the SFN-induced down-regulation of hTERT mRNA transcription in these breast cancer cells. In addition, down-regulation of hTERT expression facilitated the induction of cellular apoptosis in human breast

  7. Regulation of laminin beta2 chain gene expression in human cancer cell lines

    DEFF Research Database (Denmark)

    Durkin, M E; Nielsen, F C; Loechel, F

    2001-01-01

    and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested...... nucleotides -667 to -1724. Genomic DNA at the 3' end of the gene also appeared to have enhancer activity, as a 1.1-kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides -667/+297 promoter segment approximately threefold. Alternative splicing of the first intron...... of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain m...

  8. The consequences of chromosomal aneuploidy on gene expression profiles in a cell line model for prostate carcinogenesis.

    Science.gov (United States)

    Phillips, J L; Hayward, S W; Wang, Y; Vasselli, J; Pavlovich, C; Padilla-Nash, H; Pezullo, J R; Ghadimi, B M; Grossfeld, G D; Rivera, A; Linehan, W M; Cunha, G R; Ried, T

    2001-11-15

    Here we report the genetic characterization of immortalized prostate epithelial cells before and after conversion to tumorigenicity using molecular cytogenetics and microarray technology. We were particularly interested to analyze the consequences of acquired chromosomal aneuploidies with respect to modifications of gene expression profiles. Compared with nontumorigenic but immortalized prostate epithelium, prostate tumor cell lines showed high levels of chromosomal rearrangements that led to gains of 1p, 5, 11q, 12p, 16q, and 20q and losses of 1pter, 11p, 17, 20p, 21, 22, and Y. Of 5700 unique targets on a 6.5K cDNA microarray, approximately 3% were subject to modification in expression levels; these included GRO-1, -2, IAP-1,- 2, MMP-9, and cyclin D1, which showed increased expression, and TRAIL, BRCA1, and CTNNA, which showed decreased expression. Thirty % of expression changes occurred in regions the genomic copy number of which remained balanced. Of the remainder, 42% of down-regulated and 51% of up-regulated genes mapped to regions present in decreased or increased genomic copy numbers, respectively. A relative gain or loss of a chromosome or chromosomal arm usually resulted in a statistically significant increase or decrease, respectively, in the average expression level of all of the genes on the chromosome. However, of these genes, very few (e.g., 5 of 101 genes on chromosome 11q), and in some instances only two genes (MMP-9 and PROCR on chromosome 20q), were overexpressed by > or =1.7-fold when scored individually. Cluster analysis by gene function suggests that prostate tumorigenesis in these cell line models involves alterations in gene expression that may favor invasion, prevent apoptosis, and promote growth.

  9. RAGE Expression and NF-κB Activation Attenuated by Extracellular Domain of RAGE in Human Salivary Gland Cell Line

    OpenAIRE

    Chuong, Christopher; Katz, Joseph; Pauley, Kaleb M.; Bulosan, Marievic; Cha, Seunghee

    2009-01-01

    The receptor for advanced-glycation-end-products (RAGE) has been implicated as a pro-inflammatory factor in chronic inflammatory conditions such as diabetes mellitus and rheumatoid arthritis. The aim of this study was to investigate the inhibitory effect of the soluble-RAGE (sRAGE), the extracellular domain of RAGE, on RAGE expression and NF-κB translocation in human-salivary gland-cell-lines (HSG). Cells were stimulated with agonist S100A4, fusion protein of RAGE encompassing the extracellul...

  10. 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.

  11. DNA Methyltransferase Expression and Proliferation Status of Metastatic Breast Cancer Cell Line After Prolonged and Repeated Rapamycin and Melatonin Application

    Directory of Open Access Journals (Sweden)

    Esra Gökmen

    2015-08-01

    Full Text Available Objective: The aim of this study was to investigate the effects of Rapamycin and Melatonin and their combination on deoxyribonucleic acid (DNA methylation and cell proliferation in a estrogen receptor (ER-negative breast cancer cell line (4T1 cell line. Materials and Methods: Four groups were designed with 4T1 cell line depending on drug combination (control, Rapamycin, Melatonin, Rapamycin + Melatonin and their administration on different time periods (24, 48 and 72 hours. The drugs were administrated for 1, 2 and 3 times, respectively for these time periods. All samples were counted; immunostained (Ki67, DNA methyltransferase-1 (DNMT-1, DNA methyltransferase-3a (DNMT-3a and p53 and real-time polymerase chain reaction (PCR (DNMT-1 and DNMT-3a was performed. Results: The live/dead cell ratios were decreased in the Rapamycin and Rapamycin + Melatonin applied groups. Ki67 immunostaining showed that there was a decreased proliferation in the drug applied groups at 48th hours compared to the 24th hours. Also DNMT-1 expressions were decreased at 72th hour compared to that at 24th hour in all groups, especially in the Rapamycin administrated group. Adversely, DNMT-3a expression was increased at 72th hour compared to that at 24th hour in the groups, especially in the Rapamycin administrated group. Furthermore, an increased expression of p53 was seen in the drug given groups (highest in the Rapamycin applied group when the time prolonged. Real-time RT-PCR analysis of DNMT-1 gene expression showed a decreased expression level in the Melatonin given group compared to the control group and an increased expression level was seen in the Rapamycin and Rapamycin + Melatonin administrated groups compared to the control group. Conclusion: As a result, it was found that Rapamycin is more effective in metastatic breast cancer cells than Melatonin, both in the manner of cell viability and expressional changes of Ki67, DNMT-1, DNMT-3a and p53.

  12. Effect of the standardized Cimicifuga foetida extract on Hsp 27 expression in the MCF-7 cell line

    Directory of Open Access Journals (Sweden)

    Maritza C Soler

    2011-01-01

    Full Text Available Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose of this research was to study the expression of this protein in MCF-7 breast cancer cells. To accomplish this aim, MCF-7 cells were exposed to different concentrations of Cimicifuga foetida extract showing a reduction in cell number measured by the sulforhodamine assay. In addition, the expression of Hsp-27 mRNA detected by RT-PCR and Hsp-27 protein detected by immnofluorescence was present in all conditions, except when using the highest concentration of Cimicifuga foetida extract (2,000 jig /L. We conclude that Hsp 27 expression at 2,000 jig /L Cimicifuga foetida extract is diminished. This is the first report showing the Hsp-27 expression after exposure to Cimicifuga foetida extract in MCF-7 cells.

  13. Effect of the standardized Cimicifuga foetida extract on Hsp 27 expression in the MCF-7 cell line.

    Science.gov (United States)

    Soler, Maritza C; Molina, Jessica L; Díaz, Hugo A; Pinto, Vivian C; Barrios, Yasenka L; He, Kan; Roller, Marc; Weinstein-Oppenheimer, Caroline R

    2011-01-01

    Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose of this research was to study the expression of this protein in MCF-7 breast cancer cells. To accomplish this aim, MCF-7 cells were exposed to different concentrations of Cimicifuga foetida extract showing a reduction in cell number measured by the sulforhodamine assay. In addition, the expression of Hsp-27 mRNA detected by RT-PCR and Hsp-27 protein detected by immnofluorescence was present in all conditions, except when using the highest concentration of Cimicifuga foetida extract (2,000 jig /L). We conclude that Hsp 27 expression at 2,000 jig /L Cimicifuga foetida extract is diminished. This is the first report showing the Hsp-27 expression after exposure to Cimicifuga foetida extract in MCF-7 cells.

  14. Effects of X-ray irradiation on the expression of Pokemon gene in human lung adenocarcinoma cell line

    International Nuclear Information System (INIS)

    Liang Xiaofang; Zou Yue; Wang Lu; Jiang Qisheng; Li Fengsheng

    2012-01-01

    Objective: To study the dose and time effects of X-ray radiation on the expression of Pokemon gene and protein in human lung adenocarcinoma cell line A 549 . Methods: A 549 cells was exposed to different doses of X-ray (2, 4, 6 and 8 Gy), and the expression of Pokemon mRNA and protein of the cells was detected by using Quantitative real-time PCR and western-blotting at 2, 4, 8, 12, 24, and 48 h after irradiation. 3-( 4, 5-Dimethylthiazole-2-yl )-2, 5-diphenyltetrazolium bromide was used to detect the proliferation of A 549 cells at 1, 2, 3, 4, and 5 d after 2 Gy X-ray irradiation. The mock treated A 549 cells were used as the control. Results: The expression of Pokemon mRNA trended to decrease after irradiated with 4, 6 and 8 Gy in the earlier period and increased in the later period with statistical difference at the most time points (t =3.40 -154.76, P =0.000 -0.041). The expression of Pokemon protein trended to increase and reached the peak at 8 h after irradiated of 2, 4, 6 and 8 Gy with statistical difference at the most time points (t =4.18 - 89.64, P =0.000 - 0.039). Compared with the control, the proliferation of A 549 cells was significantly inhibited during 3 to 5 d after irradiation of 2 Gy (t =2.34 - 18.19, P =0.000 -0.040). Conclusions: X-ray irradiation may increase the expression of Pokemon mRNA and protein in A 549 cells, which might be correlated with radiation-resistance of A 549 cells. (authors)

  15. miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

    Science.gov (United States)

    Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

    2017-07-01

    In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. Expression of blood group I and i active carbohydrate sequences on cultured human and animal cell lines assessed by radioimmunoassays with monoclonal cold agglutinins

    International Nuclear Information System (INIS)

    Childs, R.A.; Kapadia, A.; Feizi, T.

    1980-01-01

    Human monoclonal anti-I and anti-i, reactive with known carbohydrate sequences, have been used as reagents to quantitate (by radioimmunoassay) and visualize (by immunofluorescence) the expression of the various blood group I and i antigenic determinants in a variety of cultured cell lines commonly used in laboratory investigations. It has been shown that the antigens they recognize are widely distributed on the surface of human and animal cell lines, expressed in varying amounts in different cell lines and on individual cells within a given cell line. In two cell lines, a transformation-associated increase in the expression of I antigen was observed. Because of their precise specificity for defined carbohydrate chain domains, these autoantibodies have become valuable reagents in biological chemistry. (orig.) [de

  17. Effect of bixin and norbixin on the expression of cytochrome P450 in HepG2 cell line.

    Science.gov (United States)

    Matuo, Míriam Cristina Sakuragui; de Oliveira Takamoto, Rafael Teruiti; Kikuchi, Irene Satiko; de Jesus Andreoli Pinto, Terezinha

    2013-08-01

    Bixin and norbixin are the main components of annatto, which is extracted from Bixa orellana and largely used as natural colorant in the food and pharmaceutical industries. Annatto can enhance CYP1A and CYP2B activity in rats; however, the inducer effect has not been investigated in human cell lines. In this study, the ability of bixin and norbixin to induce the cytochrome P450 (CYP) enzymes was assessed in HepG2 human hepatoma cell line. HepG2 cells were treated with bixin and norbixin, and the expression of the CYP genes quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). Expression of CYP1A1 and CYP1A2 was significantly increased by bixin treatment, while CYP2B6, 2C9, 2E1 and 3A4 were unaffected. Cells were treated with norbixin showed no inducer effect. The results suggest that the inducer potential of annatto is attributed to bixin, but not to norbixin, despite their similarities in molecular structure. © 2013 International Federation for Cell Biology.

  18. Thyroid cell lines in research on goitrogenesis.

    Science.gov (United States)

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

    1991-12-01

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

  19. Gene expression time-series analysis of camptothecin effects in U87-MG and DBTRG-05 glioblastoma cell lines.

    Science.gov (United States)

    Morandi, Elena; Severini, Cinzia; Quercioli, Daniele; D'Ario, Giovanni; Perdichizzi, Stefania; Capri, Miriam; Farruggia, Giovanna; Mascolo, Maria Grazia; Horn, Wolfango; Vaccari, Monica; Serra, Roberto; Colacci, Annamaria; Silingardi, Paola

    2008-08-11

    The clinical efficacy of camptothecin (CPT), a drug specifically targeting topoisomerase I (TopoI), is under evaluation for the treatment of malignant gliomas. Due to the high unresponsiveness of these tumours to chemotherapy, it would be very important to study the signalling network that drives camptothecin outcome in this type of cancer cells. To address this issue, we had previously compared the expression profile of human U87-MG glioblastoma cells with that of a CPT-resistant counterpart, giving evidence that the development of a robust inflammatory response was the main transcriptional effect associated with CPT resistance. Here we report time-related changes and cell line specific patterns of gene expression after CPT treatment by using two p53 wild-type glioblastoma cell lines, U87-MG and DBTRG-05, with different sensitivities to TopoI inhibition. First, we demonstrated that CPT treatment brings the two cell lines to completely different outcomes: accelerated senescence in U87-MG and apoptosis in DBTRG-05 cells. Then, to understand the different susceptibility to CPT, we used oligo-microarray to identify the genes whose expression was regulated during a time-course treatment, ranging from 2 h to 72 h. The statistical analysis of microarray data by MAANOVA (MicroArray ANalysis Of VAriance) showed much less modulated genes in apoptotic DBTRG-05 cells (155) with respect to the senescent U87-MG cells (3168), where the number of down-regulated genes largely exceeded that of the up-regulated ones (80% vs. 20%). Despite this great difference, the two data-sets showed a large overlapping (60% circa) mainly due to the expression of early stress responsive genes. The use of High-Throughput GoMINER and EASE tools, for functional analysis of significantly enriched GO terms, highlighted common cellular processes and showed that U87-MG and DBTRG-05 cells shared many GO terms, which are related to the down-regulation of cell cycle and mitosis and to the up-regulation of

  20. LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    International Nuclear Information System (INIS)

    Ghislin, Stephanie; Obino, Dorian; Middendorp, Sandrine; Boggetto, Nicole; Alcaide-Loridan, Catherine; Deshayes, Frederique

    2012-01-01

    Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration

  1. LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro

    Directory of Open Access Journals (Sweden)

    Ghislin Stephanie

    2012-10-01

    Full Text Available Abstract Background Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18 expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. Methods A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. Results We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. Conclusion Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.

  2. Quantitative analysis of human leucocyte antigen expression during culture of Epstein-Barr virus-transformed cell lines using the dako QIFIKIT.

    Science.gov (United States)

    Battle, R; Clark, B

    2007-01-01

    Pre-existing donor-specific human leucocyte antigen (HLA) antibodies in renal allograft recipients result in hyperacute and accelerated graft failure. These antibodies can be detected in flow cytometric assay systems using HLA-characterised Epstein-Barr virus (EBV)-transformed B-lymphocyte cell lines. Confident assay performance is predicated by the expression of HLAs on the EBV-transformed B-cell line surface. Surface HLA expression of three EBV-transformed B-cell lines that had previously been used as part of a potential organ recipient serum screening panel at St James' University Hospital, Leeds, are assessed for changes in the level of HLA expression over the nominal culture duration of eight days using the QIFIKIT (Dako, Denmark), a quantitative flow cytometry kit for assessing cell surface antigens. A comparison of the mean fluorescence intensity (MFI) of the known antigen levels of the beads via a calibration graph permits determination of the antibody binding capacity of the cell lines. Results showed that HLA expression is not consistent throughout the cell culture, with optimal expression occurring during day 2 of culture. Inconsistent HLA expression demonstrated during the cell culture means that no assumption of the level of HLA expression can be made, and that cell lines used as part of a screening panel should have their HLA expression in cell culture determined.

  3. FLI1 Expression in Breast Cancer Cell Lines and Primary Breast Carcinomas is Correlated with ER, PR and HER2

    Directory of Open Access Journals (Sweden)

    Inam Jasim Lafta

    2017-12-01

    Full Text Available FLI1 is a member of ETS family of transcription factors that regulate a variety of normal biologic activities including cell proliferation, differentiation, and apoptosis. The expression of FLI1 and its correlation with well-known breast cancer prognostic markers (ER, PR and HER2 was determined in primary breast tumors as well as four breast cancer lines including: MCF-7, T47D, MDA-MB-231 and MDA-MB-468 using RT-qPCR with either 18S rRNA or ACTB (β-actin for normalization of data. FLI1 mRNA level was decreased in the breast cancer cell lines under study compared to the normal breast tissue; however, Jurkat cells, which were used as a positive control, showed overexpression compared to the normal breast. Regarding primary breast carcinomas, FLI1 is significantly under expressed in all of the stages of breast cancer upon using 18S as an internal control. This FLI1 expression was correlated with ER, PR and HER2 status. In conclusion FLI1 can be exploited as a preliminary marker that can predict the status of ER, PR and HER2 in primary breast tumors.

  4. 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)

  5. Cytotoxicity and analysis of apoptosis gene expression in colon cancer cell line treated with cell extract of Lactobacillus casei as indigenous probiotic bacterium

    Directory of Open Access Journals (Sweden)

    Amir Mirzaie

    2017-03-01

    Full Text Available Background and aim: Nowadays, the probiotic bacteria such as lactobacilli are known as prevention factor for various disease especially cancer. The aim of this study was to investigate the cytotoxic effect of Lactobacillus casei PTCC 1608 cell extract as probiotic bacteria on colon cancer cell line (HT29 and analysis of Bax and Bcl2 apoptosis gene expression. Methods: In this experimental study, the cell extract of heat killed L. casei was prepared in 0.01, 0.1, 1, 10, 100 and 1000 µg/ml concentration and subsequently, the cytotoxicity of various cell extracts on HT29 and HEC293 cell lines were evaluated in 24 hours using MTT assay. Moreover, the Bax and Bcl2 apoptosis gene expression level in HT29 cell line was analyzed using Real Time PCR. The apoptotic effects of cell extract was determined using Flow-cytometry technique. Finally, the collected data were statistically analyzed using one-way anal­ysis of variance with the SPSS/18 software. Results: The results of MTT test show that cell extracts of L. casei is able to reduce the survival rate of HT29 cell line to 0.95±0.44, 73.45±0.21, 51.49±0.87, 39.5±0.45 and 19.7±0.55. In addition to, the Real Time PCR results indicated the expression level of Bax and Bcl2 was increased and decreased respectively, in HT29 cell line (2.76 ± 0.54 (P<0.05, 0.21 ± 0.43 (P< 0.05 in 24 h. Moreover, the flow cytometry results indicated the 35.62 % apoptosis in HT29 cell line treated with IC50 value. Conclusion: The results show that the cell extract of L. casei PTCC 1608 could induced the apoptosis in HT29 cell line and it had low toxicity on HEC293 cell line. Therefore, it seems that L. casei has potential uses as probiotic for pharmaceutical applications including prevention and treatment of colon cancer.

  6. Effect of culture at low oxygen tension on the expression of heat shock proteins in a panel of melanoma cell lines.

    Directory of Open Access Journals (Sweden)

    Christopher Shipp

    Full Text Available Tumours are commonly hypoxic and this can be associated with aggressive tumour type, metastasis and resistance to therapy. Heat shock proteins (hsps are induced in response to hypoxia, provide cancer cells with protection against tumour-associated stressors and chaperone oncoproteins that drive tumour proliferation. This study examined the effect of different oxygen concentrations on the expression of hsps in melanoma cell lines.Melanoma cell lines were cultured in 2% and 20% O(2. Expression of Hsp90, Hsp70, Hsp60, Hsp40 and Hsp32 proteins were determined by flow cytometry.Growth rates and viability were reduced in the majority of cell lines by culture in 2% O(2. Hsp expression was different in 2% compared to 20% O(2 and changes in Hsp90 expression correlated with cell line generation time (P<0.005 and viability (P<0.01. Greater total hsp expression correlated with improved viability in 2% but not 20% O(2 (P<0.05. Relative expression of the different hsps was consistent across cell lines and each correlated with the others (P = 0.0001 but not with Hsp32. Hsp expression was inversely correlated with cell line adhesion to laminin as well as collagen type IV and Breslow depth of the original primary tumour tissue (P<0.05, but not with Clark level or patient survival. All five hsps were identified on the cell surface.Culture in 2% O(2 variably altered hsp expression in a panel of melanoma cell lines. Hsp expression was associated with certain cell line characteristics and clinical parameters of the originating tumour.

  7. Effect of culture at low oxygen tension on the expression of heat shock proteins in a panel of melanoma cell lines.

    Science.gov (United States)

    Shipp, Christopher; Derhovanessian, Evelyna; Pawelec, Graham

    2012-01-01

    Tumours are commonly hypoxic and this can be associated with aggressive tumour type, metastasis and resistance to therapy. Heat shock proteins (hsps) are induced in response to hypoxia, provide cancer cells with protection against tumour-associated stressors and chaperone oncoproteins that drive tumour proliferation. This study examined the effect of different oxygen concentrations on the expression of hsps in melanoma cell lines. Melanoma cell lines were cultured in 2% and 20% O(2). Expression of Hsp90, Hsp70, Hsp60, Hsp40 and Hsp32 proteins were determined by flow cytometry. Growth rates and viability were reduced in the majority of cell lines by culture in 2% O(2). Hsp expression was different in 2% compared to 20% O(2) and changes in Hsp90 expression correlated with cell line generation time (P<0.005) and viability (P<0.01). Greater total hsp expression correlated with improved viability in 2% but not 20% O(2) (P<0.05). Relative expression of the different hsps was consistent across cell lines and each correlated with the others (P = 0.0001) but not with Hsp32. Hsp expression was inversely correlated with cell line adhesion to laminin as well as collagen type IV and Breslow depth of the original primary tumour tissue (P<0.05), but not with Clark level or patient survival. All five hsps were identified on the cell surface. Culture in 2% O(2) variably altered hsp expression in a panel of melanoma cell lines. Hsp expression was associated with certain cell line characteristics and clinical parameters of the originating tumour.

  8. Abnormalities in structure and expression of the retinoblastoma gene in small cell lung cancer cell lines and xenografts in nude mice

    DEFF Research Database (Denmark)

    Rygaard, K; Sorenson, G D; Pettengill, O S

    1990-01-01

    the Rb protein, we investigated the expression of the Mr 110,000-116,000 Rb protein in SCLC tumors grown as xenografts in nude mice and/or as cell lines. Rb messenger RNA expression was determined by Northern blotting, and gross structural gene alterations were investigated by Southern blotting. Tumors...... small cell lung cancer (SCLC). Absence of the 4.7 kilobase mRNA has been found to be frequent in SCLC, and it has been reported that the Rb Mr 110,000-116,000 protein product is always absent, even in tumors expressing Rb mRNA. Using Western blotting technique with a monoclonal antibody directed against...

  9. Analysis of gene expression data from non-small cell lung carcinoma cell lines reveals distinct sub-classes from those identified at the phenotype level.

    Directory of Open Access Journals (Sweden)

    Andrew R Dalby

    Full Text Available Microarray data from cell lines of Non-Small Cell Lung Carcinoma (NSCLC can be used to look for differences in gene expression between the cell lines derived from different tumour samples, and to investigate if these differences can be used to cluster the cell lines into distinct groups. Dividing the cell lines into classes can help to improve diagnosis and the development of screens for new drug candidates. The micro-array data is first subjected to quality control analysis and then subsequently normalised using three alternate methods to reduce the chances of differences being artefacts resulting from the normalisation process. The final clustering into sub-classes was carried out in a conservative manner such that sub-classes were consistent across all three normalisation methods. If there is structure in the cell line population it was expected that this would agree with histological classifications, but this was not found to be the case. To check the biological consistency of the sub-classes the set of most strongly differentially expressed genes was be identified for each pair of clusters to check if the genes that most strongly define sub-classes have biological functions consistent with NSCLC.

  10. Optogenetic control of cell differentiation in channelrhodopsin-2-expressing OS3, a bipotential glial progenitor cell line.

    Science.gov (United States)

    Ono, Kenji; Suzuki, Hiromi; Yamamoto, Ryusei; Sahashi, Hideki; Takido, Yuhei; Sawada, Makoto

    2017-03-01

    Alterations in the intracellular ion environment have been identified as one of the signals playing a critical role in the control of cellular proliferation and differentiation; however, the mechanisms responsible for signal transduction remain unclear. Recent studies have reported that channelrhodopsin-2 (ChR2) is a rapidly gated blue light (BL)-sensitive cation channel suitable for the non-invasive control of ion influx. We herein examined the expression of differentiation-associated markers by photo-activation and its signal transduction in ChR2-expressing OS3 (OS3ChR2) cells, which are clonal bipotential glial progenitor cells. Increases were observed in intracellular Na + and Ca 2+ concentrations in OS3ChR2 cells with BL exposure. Alterations in the intracellular ion environment, particularly in Ca 2+ , led to increases in the expression of oligodendrocyte markers including galactocerebrosides (GalC) and decreases in that of astrocyte markers such as glial fibrillary acidic protein (GFAP). These alterations also triggered activation of the ERK1/2 signaling pathway, which is involved in cell survival, and PI3K/Akt/mTOR signaling pathway, which is involved in oligodendrocyte differentiation, characterized by GalC expression. Moreover, when photo-activated OS3ChR2 cells were injected into mice with lysophosphatidyl choline (LPC)-induced demyelination, deficits in motor function were reduced. Our results demonstrated that signal transduction by ChR2-expressing glial progenitor cells may be controlled through alterations induced in the intracellular ion environment by photo-activation and results in oligodendrocyte differentiation from glial progenitor cells. Our results also suggest that ChR2-expressing glial progenitor cells have potential as a useful tool for therapeutic approaches to brain and spinal cord disorders associated with oligodendrocyte dysfunctions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic.

    Science.gov (United States)

    Qi, Ben-Xiang; Chen, Ying-Jian; Su, Rui; Li, Yi-Fei; Zheng, Gui-Ling; Li, Chang-You

    2017-10-01

    Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.

  12. Qualitative and quantitative expression status of the human chromosome 20 genes in cancer tissues and the representative cell lines.

    Science.gov (United States)

    Wang, Quanhui; Wen, Bo; Yan, Guangrong; Wei, Junying; Xie, Liqi; Xu, Shaohang; Jiang, Dahai; Wang, Tingyou; Lin, Liang; Zi, Jin; Zhang, Ju; Zhou, Ruo; Zhao, Haiyi; Ren, Zhe; Qu, Nengrong; Lou, Xiaomin; Sun, Haidan; Du, Chaoqin; Chen, Chuangbin; Zhang, Shenyan; Tan, Fengji; Xian, Youqi; Gao, Zhibo; He, Minghui; Chen, Longyun; Zhao, Xiaohang; Xu, Ping; Zhu, Yunping; Yin, Xingfeng; Shen, Huali; Zhang, Yang; Jiang, Jing; Zhang, Chengpu; Li, Liwei; Chang, Cheng; Ma, Jie; Yan, Guoquan; Yao, Jun; Lu, Haojie; Ying, Wantao; Zhong, Fan; He, Qing-Yu; Liu, Siqi

    2013-01-04

    Under the guidance of the Chromosome-centric Human Proteome Project (C-HPP), (1, 2) we conducted a systematic survey of the expression status of genes located at human chromosome 20 (Chr.20) in three cancer tissues, gastric, colon, and liver carcinoma, and their representative cell lines. We have globally profiled proteomes in these samples with combined technology of LC-MS/MS and acquired the corresponding mRNA information upon RNA-seq and RNAchip. In total, 323 unique proteins were identified, covering 60% of the coding genes (323/547) in Chr.20. With regards to qualitative information of proteomics, we overall evaluated the correlation of the identified Chr.20 proteins with target genes of transcription factors or of microRNA, conserved genes and cancer-related genes. As for quantitative information, the expression abundances of Chr.20 genes were found to be almost consistent in both tissues and cell lines of mRNA in all individual chromosome regions, whereas those of Chr.20 proteins in cells are different from tissues, especially in the region of 20q13.33. Furthermore, the abundances of Chr.20 proteins were hierarchically evaluated according to tissue- or cancer-related distribution. The analysis revealed several cancer-related proteins in Chr.20 are tissue- or cell-type dependent. With integration of all the acquired data, for the first time we established a solid database of the Chr.20 proteome.

  13. Genome Wide Expression Profiling of Cancer Cell Lines Cultured in Microgravity Reveals Significant Dysregulation of Cell Cycle and MicroRNA Gene Networks.

    Directory of Open Access Journals (Sweden)

    Prasanna Vidyasekar

    Full Text Available Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated and 2542 (downregulated genes (>2 fold in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated and 444 (downregulated genes (>2 fold under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including MIR22HG, MIR17HG and MIR21HG. The MIR22HG, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, SP1, CDK6 and CCNA2.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel alpha(1A)-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human alpha(1A)-adrenoceptor splice variant. To determine a splice variant...... a cell line stably expressing a functional form of this splice variant. The expression of recombinant alpha(1A1)-adrenoceptor subtype was confirmed by Western immunoblot analysis, and its functionality demonstrated using a Fura-2 assay by a rise in intracellular calcium concentration ([Ca(2+)](i)) when...... human alpha(1A1)-adrenoceptors in competition binding studies confirmed much higher antagonist affinity of sertindole and risperidone than haloperidol for these receptors. In summary, it can be concluded that there is an approximately 10-fold higher adrenoceptor affinity of risperidone and sertindole...

  15. Growth suppression by transforming growth factor beta 1 of human small-cell lung cancer cell lines is associated with expression of the type II receptor

    DEFF Research Database (Denmark)

    Nørgaard, P; Damstrup, L; Rygaard, K

    1994-01-01

    the growth-suppressive effect of TGF-beta 1, the expression of functional pRb, as characterised by nuclear localisation, was examined by immunocytochemistry. Nuclear association of pRb was only seen in two of the five TGF-beta 1-responsive cell lines. These results indicate that in SCLC pRb is not required...

  16. Transepithelial resistance and claudin expression in trout RTgill-W1 cell line: effects of osmoregulatory hormones.

    Science.gov (United States)

    Trubitt, Rebecca T; Rabeneck, D Brett; Bujak, Joanna K; Bossus, Maryline C; Madsen, Steffen S; Tipsmark, Christian K

    2015-04-01

    In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80Ω⋅cm(2)), which was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to hormone (Gh). The effects of three osmoregulatory hormones, Gh, prolactin, and cortisol, on the mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30, and zonula occludens-1 (Zo-1). The expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) and also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments, and may provide a useful model for in vitro study of some in vivo gill phenomena. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Evaluation of the Selenotranscriptome Expression in Two Hepatocellular Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Stefano Guariniello

    2015-01-01

    Full Text Available Hepatocellular carcinoma (HCC is the most common type of liver cancer and is still one of the most fatal cancers. Hence, it needs to identify always new putative markers to improve its diagnosis and prognosis. Since the selenium is able to fight the oxidative damage which is one of the major origins of cell damage as well as cancer, we have recently focused our attention on selenoprotein family and their involvement in HCC. In the present paper we have carried out a global analysis of the selenotranscriptome expression in HepG2 and Huh7 cells compared to the normal human hepatocytes by reverse transcription-qPCR (RT-qPCR. Our data showed that in both cells there are three downregulated (DIO1, DIO2, and SELO and ten upregulated (GPX4, GPX7, SELK, SELM, SELN, SELT, SELV, SEP15, SEPW1, and TrxR1 genes. Additionally, interactomic studies were carried out to evaluate the ability of these down- and upregulated genes to interact between them as well as to identify putative HUB nodes representing the centers of correlation able to exercise a direct control over the coordinated genes.

  18. CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell lines HCT-116

    International Nuclear Information System (INIS)

    Dittfeld, Claudia; Dietrich, Antje; Peickert, Susann; Hering, Sandra; Baumann, Michael; Grade, Marian; Ried, Thomas; Kunz-Schughart, Leoni A.

    2009-01-01

    Background and purpose: CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133 +/- subpopulations of one cell line of interest. Materials and methods: Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133 + and CD133 - HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice. Results and conclusions: CRC cell lines could be classified into three groups: (i) CD133 - , (ii) CD133 + and (iii) those with two distinct CD133 + and CD133 - subpopulations. Isolated CD133 +/- HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133 - HCT-116 showed a striking enrichment in the CD133 + fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell lines. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.

  19. Genetic analysis of human traits in vitro: drug response and gene expression in lymphoblastoid cell lines.

    Directory of Open Access Journals (Sweden)

    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.

  20. A Sclerostin super-producer cell line derived from the human cell line SaOS-2: a new tool for the study of the molecular mechanisms driving Sclerostin expression.

    Science.gov (United States)

    Pérez-Campo, Flor M; Sañudo, Carolina; Delgado-Calle, Jesús; Arozamena, Jana; Zarrabeitia, María T; Riancho, José A

    2014-08-01

    Sclerostin, the product of the SOST gene, is a key regulator of bone homeostasis. Sclerostin interferes with the Wnt signalling pathway and, therefore, has a negative effect on bone formation. Although the importance of sclerostin in bone homeostasis is well established, many aspects of its biology are still unknown. Due to its restricted pattern of expression, in vitro studies of SOST gene regulation are technically challenging. Furthermore, a more profound investigation of the molecular mechanism controlling sclerostin expression has been hampered by the lack of a good human in vitro model. Here, we describe two cell lines derived from the human osteosarcoma cell line SaOS-2 that produce elevated levels of sclerostin. Analysis of the super-producer cell lines showed that sclerostin levels were still reduced in response to parathyroid hormone treatment or in response to mechanical loading, indicating that these regulatory mechanisms were not affected in the presented cell lines. In addition, we did not find differences between the promoter or ECR5 sequences of our clones and the SaOS-2 parental line. However, the methylation of the proximal CpG island located at the SOST promoter was lower in the super-producer clones, in agreement with a higher level of SOST transcription. Although the underlying biological causes of the elevated levels of sclerostin production in this cell line are not yet clear, we believe that it could be an extremely useful tool to study the molecular mechanisms driving sclerostin expression in humans.

  1. Altered expression of the IQGAP1 gene in human lung cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, C.E.; Palmisano, W.A.; Lechner, J.F. [and others

    1995-12-01

    IQGAP1 is a GTPase activation protein that accelerates GTP hydrolysis by normal p21 ras proteins. Therefore, IQGAP1 could act as an upstream affector of p21 ras activity by convert in excess amounts of active GTP-21 ras to inactive GDP-21 ras. IQGAP1 displays extensive sequence similarity to the catalytic domain of all previously reported ras GAPs, including the tumor suppressor gene protein neurofibromatosis type 1 (NF1). It has been shown that abnormal NF1 protein cannot negatively regulate the activity of ras proteins in neuroblast cells. This observation supports the hypothesis that NF1 is a tumor suppressor gene whose product acts upstream of ras. IQGAP1 is primarily expressed in lung, where it may play a role similar to NF1 in regulating the activity of H-ras or K-ras proteins. IQGAP1 functions as other GAPs by controlling the activity of ras.

  2. ENERGY-DEPENDENT PROCESSES INVOLVED IN REDUCED DRUG ACCUMULATION IN MULTIDRUG-RESISTANT HUMAN LUNG-CANCER CELL-LINES WITHOUT P-GLYCOPROTEIN EXPRESSION

    NARCIS (Netherlands)

    VERSANTVOORT, CHM; BROXTERMAN, HJ; PINEDO, HM; DEVRIES, EGE; FELLER, N; KUIPER, CM; LANKELMA, J

    1992-01-01

    Mechanisms contributing to reduced cytotoxic drug accumulation were studied in two multidrug-resistant (MDR) human lung cancer cell lines without P-glycoprotein expression. In these (non-small cell) SW-1573/ 2R120 and (small cell) GLC4/ADR MDR cells, the steady-state accumulation of

  3. Differential gene expression by 1,25(OH)2D3 in an endometriosis stromal cell line.

    Science.gov (United States)

    Ingles, Sue Ann; Wu, Liang; Liu, Benjamin T; Chen, Yibu; Wang, Chun-Yeh; Templeman, Claire; Brueggmann, Doerthe

    2017-10-01

    Endometriosis is a common female reproductive disease characterized by invasion of endometrial cells into other organs, frequently causing pelvic pain and infertility. Alterations of the vitamin D system have been linked to endometriosis incidence and severity. To shed light on the potential mechanism for these associations, we examined the effects of 1,25(OH) 2 D 3 on gene expression in endometriosis cells. Stromal cell lines derived from endometriosis tissue were treated with 1,25(OH) 2 D 3 , and RNA-seq was used to identify genes differentially expressed between treated and untreated cells. Gene ontology and pathway analyses were carried out using Partek Flow and Ingenuity software suites, respectively. We identified 1627 genes that were differentially expressed (886 down-regulated and 741 up-regulated) by 1,25(OH) 2 D 3 . Only one gene, CYP24A1, was strongly up-regulated (369-fold). Many genes were strongly down-regulated. 1,25(OH) 2 D 3 treatment down-regulated several genetic pathways related to neuroangiogenesis, cellular motility, and invasion, including pathways for axonal guidance, Rho GDP signaling, and matrix metalloprotease inhibition. These findings support a role for vitamin D in the pathophysiology of endometriosis, and provide new targets for investigation into possible causes and treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Characterization of the glycosylation profiles of Alzheimer's beta -secretase protein Asp-2 expressed in a variety of cell lines.

    Science.gov (United States)

    Charlwood, J; Dingwall, C; Matico, R; Hussain, I; Johanson, K; Moore, S; Powell, D J; Skehel, J M; Ratcliffe, S; Clarke, B; Trill, J; Sweitzer, S; Camilleri, P

    2001-05-18

    Amyloid 39-42 beta -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39-42 beta-peptide is formed from amyloid precursor protein by the sequential action of beta- and gamma-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have beta-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.

  5. Cloning, intracellular localization, and expression of the mammalian selenocysteine-containing protein SELENOI (SelI) in tumor cell lines.

    Science.gov (United States)

    Varlamova, E G; Goltyaev, M V; Novoselov, V I; Fesenko, E E

    2017-09-01

    The intracellular localization of human selenoprotein SelI and the degree of expression of its gene in different human tumor cell lines were determined. It was found that the SelI protein is present in the nucleus, cytoplasm, and endoplasmic reticulum and is absent in the nucleolus. Since the oxidative stress caused by a sharp increase in the content of free radicals in the body is one of the causes of malignant transformation, the study of the role of the trace element selenium and selenocysteine-containing proteins as antioxidants in carcinogenesis is of great scientific interest.

  6. Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2

    International Nuclear Information System (INIS)

    Vita, N.; Magazin, M.; Marchese, E.; Lupker, J.; Ferrara, P.

    1990-01-01

    We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and the structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2

  7. Taxol and LPS Modulation of c-kit and nm23 Expression in Macrophages and Normal vs. Malignant Breast Cancer Cell Lines.

    Science.gov (United States)

    1999-07-01

    AND SUBTITLE Taxol and LPS Modulation of c-kit and nm23 Expression in Macrophages and Normal vs. Malignant Breast Cancer Cell . Lines : — 3... cancer cells and AM has recently been implicated as an autocrine growth factor in malignant cells (6). For this reason, expression of the AM gene, and...i AD GRANT NUMBER DAMD17-96-1-6258 TITLE: Taxol and LPS Modulation of c-kit and nm23 Expression in Macrophages and Normal vs. Malignant Breast

  8. Thyroid stimulating hormone stimulates the expression of glucose transporter 2 via its receptor in pancreatic β cell line, INS-1 cells.

    Science.gov (United States)

    Lyu, Jingya; Imachi, Hitomi; Yoshimoto, Takuo; Fukunaga, Kensaku; Sato, Seisuke; Ibata, Tomohiro; Kobayashi, Toshihiro; Dong, Tao; Yonezaki, Kazuko; Yamaji, Nao; Kikuchi, Fumi; Iwama, Hisakazu; Ishikawa, Ryou; Haba, Reiji; Sugiyama, Yasunori; Zhang, Huanxiang; Murao, Koji

    2018-01-31

    Thyroid stimulating hormone (TSH) stimulates the secretion of thyroid hormones by binding the TSH receptor (TSHR). TSHR is well-known to be expressed in thyroid tissue, excepting it, TSHR has also been expressed in many other tissues. In this study, we have examined the expression of TSHR in rat pancreatic islets and evaluated the role of TSH in regulating pancreas-specific gene expression. TSHR was confirmed to be expressed in rodent pancreatic islets and its cell line, INS-1 cells. TSH directly affected the glucose uptake in INS cells by up-regulating the expression of GLUT2, and furthermore this process was blocked by SB203580, the specific inhibitor of the p38 MAPK signaling pathway. Similarly, TSH stimulated GLUT2 promoter activity, while both a dominant-negative p38MAPK α isoform (p38MAPK α-DN) and the specific inhibitor for p38MAPK α abolished the stimulatory effect of TSH on GLUT2 promoter activity. Finally, INS-1 cells treated with TSH showed increased protein level of glucokinase and enhanced glucose-stimulated insulin secretion. Together, these results confirm that TSHR is expressed in INS-1 cells and rat pancreatic islets, and suggest that activation of the p38MAPK α might be required for TSH-induced GLUT2 gene transcription in pancreatic β cells.

  9. Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus.

    Science.gov (United States)

    Garcia, J A; Ferreira, H L; Vieira, F V; Gameiro, R; Andrade, A L; Eugênio, F R; Flores, E F; Cardoso, T C

    2017-06-01

    Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy. © 2015 John Wiley & Sons Ltd.

  10. 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.

  11. Genome-wide gene copy number and expression analysis of primary gastric tumors and gastric cancer cell lines

    International Nuclear Information System (INIS)

    Junnila, Siina; Kokkola, Arto; Karjalainen-Lindsberg, Marja-Liisa; Puolakkainen, Pauli; Monni, Outi

    2010-01-01

    Gastric cancer is one of the most common malignancies worldwide and the second most common cause of cancer related death. Gene copy number alterations play an important role in the development of gastric cancer and a change in gene copy number is one of the main mechanisms for a cancer cell to control the expression of potential oncogenes and tumor suppressor genes. To highlight genes of potential biological and clinical relevance in gastric cancer, we carried out a systematic array-based survey of gene expression and copy number levels in primary gastric tumors and gastric cancer cell lines and validated the results using an affinity capture based transcript analysis (TRAC assay) and real-time qRT-PCR. Integrated microarray analysis revealed altogether 256 genes that were located in recurrent regions of gains or losses and had at least a 2-fold copy number- associated change in their gene expression. The expression levels of 13 of these genes, ALPK2, ASAP1, CEACAM5, CYP3A4, ENAH, ERBB2, HHIPL2, LTB4R, MMP9, PERLD1, PNMT, PTPRA, and OSMR, were validated in a total of 118 gastric samples using either the qRT-PCR or TRAC assay. All of these 13 genes were differentially expressed between cancerous samples and nonmalignant tissues (p < 0.05) and the association between copy number and gene expression changes was validated for nine (69.2%) of these genes (p < 0.05). In conclusion, integrated gene expression and copy number microarray analysis highlighted genes that may be critically important for gastric carcinogenesis. TRAC and qRT-PCR analyses validated the microarray results and therefore the role of these genes as potential biomarkers for gastric cancer

  12. Comparative 3'UTR analysis allows identification of regulatory clusters that drive Eph/ephrin expression in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Jennifer Winter

    Full Text Available Eph receptors are the largest family of receptor tyrosine kinases. Together with their ligands, the ephrins, they fulfill multiple biological functions. Aberrant expression of Ephs/ephrins leading to increased Eph receptor to ephrin ligand ratios is a critical factor in tumorigenesis, indicating that tight regulation of Eph and ephrin expression is essential for normal cell behavior. The 3'-untranslated regions (3'UTRs of transcripts play an important yet widely underappreciated role in the control of protein expression. Based on the assumption that paralogues of large gene families might exhibit a conserved organization of regulatory elements in their 3'UTRs we applied a novel bioinformatics/molecular biology approach to the 3'UTR sequences of Eph/ephrin transcripts. We identified clusters of motifs consisting of cytoplasmic polyadenylation elements (CPEs, AU-rich elements (AREs and HuR binding sites. These clusters bind multiple RNA-stabilizing and destabilizing factors, including HuR. Surprisingly, despite its widely accepted role as an mRNA-stabilizing protein, we further show that binding of HuR to these clusters actually destabilizes Eph/ephrin transcripts in tumor cell lines. Consequently, knockdown of HuR greatly modulates expression of multiple Ephs/ephrins at both the mRNA and protein levels. Together our studies suggest that overexpression of HuR as found in many progressive tumors could be causative for disarranged Eph receptor to ephrin ligand ratios leading to a higher degree of tissue invasiveness.

  13. Identification of genes differentially expressed in breast cancer cell line SKBR3: potential identification of new prognostic biomarkers.

    Science.gov (United States)

    Leirdal, Marianne; Shadidy, Mohsen; Røsok, Øystein; Sioud, Mouldy

    2004-08-01

    The identification of differentially expressed genes in tumour cells should have important implications in understanding carcinogenesis and developing new therapeutic and prognostic biomarkers. We have combined PCR-based cDNA subtraction and Northern blotting to identify truly differentially expressed genes in breast cancer cell line SKBR3 as compared to normal human mammary epithelial cells (HMEC). Hybridizing probe molecules were rescued from the Hybond N+ membranes and then PCR reamplified. The PCR reamplification is possible due to the fact that all probe molecules contain the same pair of adapter sequences on both ends. After cloning and sequencing three known genes, ribosomal protein L19 (RPL19), ADP/ATP carrier protein and ErbB-2 with high-elevated mRNA levels in SKBR3 were identified. In addition, two overexpressed genes with unknown functions, CXYorf1-related protein and hypothetical protein PRO2605, were found. High-titer andibodies against the recombinant RPL19 were detected in 5 patients out of 50 patients investigated. Thus, the present novel strategy based on the combination of PCR-based cDNA subtraction and Northern blotting should facilitate the identification of truly differentially expressed biomarkers, which may offer the potential to determine the proper drug for an individual patient at a given stage of disease or treatment.

  14. Elucidating the Mechanism of Regulation of Transforming Growth Factor β Type II Receptor Expression in Human Lung Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Sunil K. Halder

    2011-10-01

    Full Text Available Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor β (TGF-β-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-β in lung cancers occurs mostly through the loss of TGF-β type II receptor expression (TβRII. However, little is known about the mechanism of down-regulation of TβRII and how histone deacetylase (HDAC inhibitors (HDIs can restore TGF-β-induced tumor suppressor function. Here we show that HDIs restore TβRII expression and that DNA hypermethylation has no effect on TβRII promoter activity in lung cancer cell lines. TGF-β-induced tumor suppressor function is restored by HDIs in lung cancer cell lines that lack TβRII expression. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by either activated Ras or epidermal growth factor signaling is involved in the down-regulation of TβRII through histone deacetylation. We have immunoprecipitated the protein complexes by biotinylated oligonucleotides corresponding to the HDI-responsive element in the TβRII promoter (−127/(−75 and identified the proteins/factors using proteomics studies. The transcriptional repressor Meis1/2 is involved in repressing the TβRII promoter activity, possibly through its recruitment by Sp1 and NF-YA to the promoter. These results suggest a mechanism for the downregulation of TβRII in lung cancer and that TGF-β tumor suppressor functions may be restored by HDIs in lung cancer patients with the loss of TβRII expression.

  15. Gene expression profile in response to doxorubicin-rapamycin combined treatment of HER-2-overexpressing human mammary epithelial cell lines.

    Science.gov (United States)

    Trapé, Adriana Priscila; Katayama, Maria Lucia Hirata; Roela, Rosimeire Aparecida; Brentani, Helena; Ravacci, Graziela Rosa; de Araujo Lima, Leandro; Brentani, Maria Mitzi

    2012-02-01

    HER-2-positive breast cancers frequently sustain elevated AKT/mTOR signaling, which has been associated with resistance to doxorubicin treatment. Here, we investigated whether rapamycin, an mTOR inhibitor, increased the sensitivity to doxorubicin therapy in two HER-2-overexpressing cell lines: C5.2, which was derived from the parental HB4a by transfection with HER-2 and SKBR3, which exhibits HER-2 amplification. The epithelial mammary cell line HB4a was also analyzed. The combined treatment using 20 nmol/L of rapamycin and 30 nmol/L of doxorubicin arrested HB4a and C5.2 cells in S to G(2)-M, whereas SKBR3 cells showed an increase in the G(0)-G(1) phase. Rapamycin increased the sensitivity to doxorubicin in HER-2-overexpressing cells by approximately 2-fold, suggesting that the combination displayed a more effective antiproliferative action. Gene expression profiling showed that these results might reflect alterations in genes involved in canonical pathways related to purine metabolism, oxidative phosphorylation, protein ubiquitination, and mitochondrial dysfunction. A set of 122 genes modulated by the combined treatment and specifically related to HER-2 overexpression was determined by finding genes commonly regulated in both C5.2 and SKBR3 that were not affected in HB4a cells. Network analysis of this particular set showed a smaller subgroup of genes in which coexpression pattern in HB4a cells was disrupted in C5.2 and SKBR3. Altogether, our data showed a subset of genes that might be more robust than individual markers in predicting the response of HER-2-overexpressing breast cancers to doxorubicin and rapamycin combination.

  16. Establishment of a common acute lymphoblastic leukemia cell line (LC4-1) and effects of phorbol myristate acetate (PMA) on the surface antigen expression of the cell line.

    Science.gov (United States)

    Yoshimura, T; Mayumi, M; Yorifuji, T; Kim, K M; Heike, T; Miyanomae, T; Shinomiya, K; Mikawa, H

    1987-09-01

    A common acute lymphoblastic leukemia (ALL) cell line, designated LC4-1, was established from peripheral blood mononuclear cells of a patient with acute non-T-cell ALL. LC4-1 cells were characteristically positive for Ia, B4, and common ALL antigens (CALLA), but negative for B2, Tac, T3, T4, T8, T11, and M1 antigens and E-rosette formation. Approximately 30% of LC4-1 cells expressed detectable amounts of B1 antigens. LC4-1 cells expressed neither Epstein-Barr-virus-associated nuclear antigen (EBNA), cytoplasmic immunoglobulins (cIg) nor surface immunoglobulins (sIg). Gene rearrangements had already occurred in LC4-1 in the D-J region of immunoglobulin heavy chain genes, but not in T-cell receptor (beta-chain) genes, suggesting that LC4-1 is a progenitor cell line of B-cell lineage earlier than pre-B-cells. The expression of cell surface antigens of LC4-1 was changed by treatment with 4-phorbol 12-myristate 13-acetate (PMA) (0.1 ng/ml) for 2 days. Before treatment with PMA, about 98% of LC4-1 cells were positive for B4, CALLA, and Ia. However, following treatment they lost CALLA expression without any change in expression of Ia and B4. There was no change in B1-positive population. The change in surface antigens on LC4-1 cells seems to be due to differentiation induced in the cells by PMA. These results support the hypothesis that CALLA is a differentiation antigen and suggest one possible differentiation pathway for pre-B-cells.

  17. Up-modulation of the expression of functional keratinocyte growth factor receptors induced by high cell density in the human keratinocyte HaCaT cell line.

    Science.gov (United States)

    Capone, A; Visco, V; Belleudi, F; Marchese, C; Cardinali, G; Bellocci, M; Picardo, M; Frati, L; Torrisi, M R

    2000-11-01

    Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989-997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

  18. Real-time cell analysis and heat shock protein gene expression in the TcA Tribolium castaneum cell line in response to environmental stress conditions.

    Science.gov (United States)

    García-Reina, Andrés; Rodríguez-García, María Juliana; Ramis, Guillermo; Galián, José

    2017-06-01

    The rust red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae), is a pest of stored grain and one of the most studied insect model species. Some of the previous studies involved heat response studies in terms of survival and heat shock protein expression, which are regulated to protect other proteins against environmental stress conditions. In the present study, we characterize the impedance profile with the xCELLigence Real-Time Cell Analyzer and study the effect of increased temperature in cell growth and viability in the cell line BCIRL-TcA-CLG1 (TcA) of T. castaneum. This novel system measures cells behavior in real time and is applied for the first time to insect cells. Additionally, cells are exposed to heat shock, increased salinity, acidic pH and UV-A light with the aim of measuring the expression levels of Hsp27, Hsp68a, and Hsp83 genes. Results show a high thermotolerance of TcA in terms of cell growth and viability. This result is likely related to gene expression results in which a significant up-regulation of all studied Hsp genes is observed after 1 h of exposure to 40 °C and UV light. All 3 genes show similar expression patterns, but Hsp27 seems to be the most affected. The results of this study validate the RTCA method and reveal the utility of insect cell lines, real-time analysis and gene expression studies to better understand the physiological response of insect cells, with potential applications in different fields of biology such as conservation biology and pest management. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  19. Expression of the glycoprotein of viral haemorrhagic septicaemia virus (VHSV) on the surface of the fish cell line RTG-P1 induces type 1 interferon expression in neighbouring cells

    DEFF Research Database (Denmark)

    Acosta, F.; Collet, B.; Lorenzen, Niels

    2006-01-01

    In the present study using a luciferase/Mx promoter reporter system, it was shown that the rainbow trout gonad cell line (RTG-P1), a fibroblastic cell line, produces IFN when transfected with a plasmid encoding the glycoprotein of VHSV but not with plasmid vector alone. Only a small percentage...... of the cells expressed the G protein on the surface membrane as indicated by immunostaining of transfected cells. When transfection was performed in the presence of monoclonal antibodies (Mab) to the glycoprotein, the production of interferon mRNA transcripts was reduced by over 50%. This indicates...... that the surface expression of G protein was the major mechanism of interferon induction and that most of the interferon was being expressed by cells neighbouring the transfected cells. Crown...

  20. DNA methylation and histone acetylation regulate the expression of MGMT and chemosensitivity to temozolomide in malignant melanoma cell lines.

    Science.gov (United States)

    Chen, Ya-Ping; Hou, Xiao-Yang; Yang, Chun-Sheng; Jiang, Xiao-Xiao; Yang, Ming; Xu, Xi-Feng; Feng, Shou-Xin; Liu, Yan-Qun; Jiang, Guan

    2016-08-01

    Malignant melanoma is an aggressive, highly lethal dermatological malignancy. Chemoresistance and rapid metastasis limit the curative effect of multimodal therapies like surgery or chemotherapy. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes adducts from the O6-position of guanine to repair DNA damage. High MGMT expression is associated with resistance to therapy in melanoma. However, it is unknown if MGMT is regulated by DNA methylation or histone acetylation in melanoma. We examined the effects of the DNA methylation inhibitor 5-Aza-2'-deoxycytidine and histone deacetylase inhibitor Trichostatin A alone or in combination on MGMT expression and promoter methylation and histone acetylation in A375, MV3, and M14 melanoma cells. This study demonstrates that MGMT expression, CpG island methylation, and histone acetylation vary between melanoma cell lines. Combined treatment with 5-Aza-2'-deoxycytidine and Trichostatin A led to reexpression of MGMT, indicating that DNA methylation and histone deacetylation are associated with silencing of MGMT in melanoma. This study provides information on the role of epigenetic modifications in malignant melanoma that may enable the development of new strategies for treating malignant melanoma.

  1. A novel cell line derived from pleomorphic adenoma expresses MMP2, MMP9, TIMP1, TIMP2, and shows numeric chromosomal anomalies.

    Directory of Open Access Journals (Sweden)

    Aline Semblano Carreira Falcão

    Full Text Available Pleomorphic adenoma is the most common salivary gland neoplasm, and it can be locally invasive, despite its slow growth. This study aimed to establish a novel cell line (AP-1 derived from a human pleomorphic adenoma sample to better understand local invasiveness of this tumor. AP-1 cell line was characterized by cell growth analysis, expression of epithelial and myoepithelial markers by immunofluorescence, electron microscopy, 3D cell culture assays, cytogenetic features and transcriptomic study. Expression of matrix metalloproteinases (MMPs and their tissue inhibitors (TIMPs was also analyzed by immunofluorescence and zymography. Furthermore, epithelial and myoepithelial markers, MMPs and TIMPs were studied in the tumor that originated the cell line. AP-1 cells showed neoplastic epithelial and myoepithelial markers, such as cytokeratins, vimentin, S100 protein and smooth-muscle actin. These molecules were also found in vivo, in the tumor that originated the cell line. MMPs and TIMPs were observed in vivo and in AP-1 cells. Growth curve showed that AP-1 exhibited a doubling time of 3.342 days. AP-1 cells grown inside Matrigel recapitulated tumor architecture. Different numerical and structural chromosomal anomalies were visualized in cytogenetic analysis. Transcriptomic analysis addressed expression of 7 target genes (VIM, TIMP2, MMP2, MMP9, TIMP1, ACTA2 e PLAG1. Results were compared to transcriptomic profile of non-neoplastic salivary gland cells (HSG. Only MMP9 was not expressed in both libraries, and VIM was expressed solely in AP-1 library. The major difference regarding gene expression level between AP-1 and HSG samples occurred for MMP2. This gene was 184 times more expressed in AP-1 cells. Our findings suggest that AP-1 cell line could be a useful model for further studies on pleomorphic adenoma biology.

  2. Regulation of Programmed Death Ligand 1 (PD-L1) Expression in Breast Cancer Cell Lines In Vitro and in Immunodeficient and Humanized Tumor Mice.

    Science.gov (United States)

    Rom-Jurek, Eva-Maria; Kirchhammer, Nicole; Ugocsai, Peter; Ortmann, Olaf; Wege, Anja K; Brockhoff, Gero

    2018-02-13

    Programmed death ligand 1 (PD-L1) expression is an efficient strategy of tumor cells to escape immunological eradiation. However, only little is known about the factors that affect the cellular expression levels. Here we assessed the PD-L1 expression on different breast cancer cell lines under standard in vitro culture conditions and as a function of Epirubicin or Paclitaxel treatment. Moreover, we evaluated the expression in immunodeficient tumor mice as well as in humanized tumor mice (i.e., in the presence of a human immune system). We found highest PD-L1 levels in JIMT-1 and MDA-MB-231 cells. Epirubicin treatment caused a decrease and Paclitaxel treatment an increased PD-L1 expression in MDA-MB-231 cells. In addition, we identified nuclear PD-L1 in MDA-MB-231 cells. All in vivo transplanted breast cancer cell lines downregulated PD-L1 expression compared to their in vitro counterpart. Neither the gene copy number nor the presence of human immune system in humanized tumor mice had an effect on the PD-L1 content. We demonstrate that the degree of PD-L1 expression amongst breast cancer cell lines varies considerably. In addition, cytotoxic treatments and other extrinsic parameters differentially affect the expression. Hence, further investigations including in vivo evaluations are necessary to understand PD-L1 regulation for advanced breast cancer stratification.

  3. mRNA sequencing of novel cell lines from human papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5.

    Science.gov (United States)

    Bryant, Dean; Onions, Tiffany; Raybould, Rachel; Flynn, Áine; Tristram, Amanda; Meyrick, Sian; Giles, Peter; Ashelford, Kevin; Hibbitts, Samantha; Fiander, Alison; Powell, Ned

    2014-09-01

    Vulval intraepithelial neoplasia is a precursor of vulval cancer and is commonly caused by infection with Human Papillomavirus (HPV). Development of topical treatments for vulval intraepithelial neoplasia requires appropriate in vitro models. This study evaluated the feasibility of primary culture of vulval intraepithelial neoplasia biopsy tissue to produce cell lines for use as in vitro models. A potentially immortal cell line was produced which gave rise to three monoclonal lines. These lines were characterized for HPV genomic integration and for viral gene expression using ligation-mediated PCR and quantitative PCR. Distinct patterns of viral integration and gene expression were observed among the three lines. Integration and expression data were validated using deep sequencing of mRNA. Gene ontology analyses of these data also demonstrated that expression of the HPV16 E4 and E5 proteins resulted in substantial changes in the composition of the cell membrane and extracellular space, associated with alterations in cell adhesion and differentiation. These data illustrate the diverse patterns of HPV gene expression potentially present within a single lesion. The derived cell lines provide useful models to investigate the biology of vulval intraepithelial neoplasia and the interactions between different HPV gene products and potential therapeutic agents. © 2014 Wiley Periodicals, Inc.

  4. Expression and purification of biologically active recombinant human paraoxonase 1 from a Drosophila S2 stable cell line.

    Science.gov (United States)

    Yun, Hyeongseok; Yu, Jiyeon; Kim, Sumi; Lee, Nari; Lee, Jinhee; Lee, Sungrae; Kim, Nam Doo; Yu, Chiho; Rho, Jaerang

    2017-03-01

    Many pesticides and chemical warfare nerve agents are highly toxic organophosphorus compounds (OPs), which inhibit acetylcholinesterase activity. Human paraoxonase 1 (PON1) has demonstrated significant potential for use as a catalytic bioscavenger capable of hydrolyzing a broad range of OPs. However, there are several limitations to the use of human PON1 as a catalytic bioscavenger, including the relatively difficult purification of PON1 from human plasma and its dependence on the presence of hydrophobic binding partners to maintain stability. Therefore, research efforts to efficiently produce recombinant human PON1 are necessary. In this study, we developed a Drosophila S2 stable cell line expressing recombinant human PON1. The recombinant human PON1 was fused with the human immunoglobulin Fc domain (PON1-hFc) to improve protein stability and purification efficiency. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis. We purified the recombinant human PON1-hFc from the S2 stable cell line and characterized its enzymatic properties for OP hydrolysis compared with those of the recombinant human PON1 derived from E. coli. We observed that the recombinant human PON1-hFc is functionally more stable for OP hydrolyzing activities compared to the recombinant human PON1. The catalytic efficiency of the recombinant PON1-hFc towards diisopropyl fluorophosphate (DFP, 0.26 × 10 6  M -1  min -1 ) and paraoxon hydrolysis (0.015 × 10 6  M -1  min -1 ) was 1.63- and 1.24-fold higher, respectively, than the recombinant human PON1. Thus, we report that the recombinant PON1-hFc exerts hydrolytic activity against paraoxon and DFP. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Systematic assessment of multi-gene predictors of pan-cancer cell line sensitivity to drugs exploiting gene expression data.

    Science.gov (United States)

    Nguyen, Linh; Dang, Cuong C; Ballester, Pedro J

    2016-01-01

    Background: Selected gene mutations are routinely used to guide the selection of cancer drugs for a given patient tumour. Large pharmacogenomic data sets, such as those by Genomics of Drug Sensitivity in Cancer (GDSC) consortium, were introduced to discover more of these single-gene markers of drug sensitivity. Very recently, machine learning regression has been used to investigate how well cancer cell line sensitivity to drugs is predicted depending on the type of molecular profile. The latter has revealed that gene expression data is the most predictive profile in the pan-cancer setting. However, no study to date has exploited GDSC data to systematically compare the performance of machine learning models based on multi-gene expression data against that of widely-used single-gene markers based on genomics data. Methods: Here we present this systematic comparison using Random Forest (RF) classifiers exploiting the expression levels of 13,321 genes and an average of 501 tested cell lines per drug. To account for time-dependent batch effects in IC 50 measurements, we employ independent test sets generated with more recent GDSC data than that used to train the predictors and show that this is a more realistic validation than standard k-fold cross-validation. Results and Discussion: Across 127 GDSC drugs, our results show that the single-gene markers unveiled by the MANOVA analysis tend to achieve higher precision than these RF-based multi-gene models, at the cost of generally having a poor recall (i.e. correctly detecting only a small part of the cell lines sensitive to the drug). Regarding overall classification performance, about two thirds of the drugs are better predicted by the multi-gene RF classifiers. Among the drugs with the most predictive of these models, we found pyrimethamine, sunitinib and 17-AAG. Conclusions: Thanks to this unbiased validation, we now know that this type of models can predict in vitro tumour response to some of these drugs. These models

  6. Sonicated Protein Fractions of Mycoplasma hyopneumoniae Induce Inflammatory Responses and Differential Gene Expression in a Murine Alveolar Macrophage Cell Line.

    Science.gov (United States)

    Damte, Dereje; Lee, Seung-Jin; Birhanu, Biruk Tesfaye; Suh, Joo-Won; Park, Seung-Chun

    2015-12-28

    Mycoplasma hyopneumoniae is known to cause porcine enzootic pneumonia (EP), an important disease in swine production. The objective of this study was to examine the effects of sonicated protein fractions of M. hyopneumoniae on inflammatory response and gene expression in the murine alveolar macrophage MH-S cell line. The effects of sonicated protein fractions and intact M. hyopneumoniae on the gene expression of cytokines and iNOS were assessed using RT-PCR. The Annealing Control Primer (ACP)-based PCR method was used to screen differentially expressed genes. Increased transcription of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, COX-2, and iNOS mRNA was observed after exposure to the supernatant (SPT), precipitant (PPT), and intact M. hyopneumoniae protein. A time-dependent analysis of the mRNA expression revealed an upregulation after 4 h for IL-6 and iNOS and after 12 h for IL-1β and TNF-α, for both SPT and PPT; the fold change in COX-2 expression was less. A dose- and time-dependent correlation was observed in nitrite (NO) production for both protein fractions; however, there was no significant difference between the effects of the two protein fractions. In a differential gene analysis, PCR revealed differential expression for nine gene bands after 3 h of stimulation - only one gene was downregulated, while the remaining eight were upregulated. The results of this study provide insights that help improve our understanding of the mechanisms underlying the pathogenesis of and macrophage defenses against M. hyopneumoniae assault, and suggest targets for future studies on therapeutic interventions for M. hyopneumoniae infections.

  7. Modulation of Epidermal Growth Factor Receptor Expression by Chemotherapeutic Agents in Breast Cancer Cell Lines

    National Research Council Canada - National Science Library

    Welch, James

    2002-01-01

    ... this protein a potentially powerful tumor promoter. The association between higher EOFR expression and poorer prognosis in breast cancer and the frequency of higher EGFR levels in more aggressive/metastatic breast tumors reinforce this possibility...

  8. Modulation of Epidermal Growth Factor Receptor Expression by Chemotherapeutic Agents in Breast Cancer Cell Lines

    National Research Council Canada - National Science Library

    Welch, James

    2001-01-01

    ... this protein a potentially powerful tumor promoter. The association between higher EGFR expression and poorer prognosis in breast cancer and the frequency of higher EGFR levels in more aggressive/metastatic breast tumors reinforce this possibility...

  9. The influence of arsenic trioxide on the cell cycle, apoptosis and expression of cyclin D1 in the Jurkat cell line.

    Science.gov (United States)

    Zuryń, Agnieszka; Litwiniec, Anna; Gagat, Maciej; Drzewucka, Joanna; Gackowska, Lidia; Grzanka, Alina

    2014-10-01

    Cyclin D1 drives cell cycle progression at the G1/S transition and is believed to play a significant role in tumorigenesis, contributing to efficient proliferation of many cancer cells. Consequently, it is also recognized as an end-point biomarker of therapeutic outcome for different treatment modalities in cancer. In this study we aimed to evaluate the expression and localization of cyclin D1 in arsenic trioxide (ATO) treated Jurkat cells (lymphoblastic leukemia cell line) and to correlate these results with the extent of cell death and/or cell cycle alterations. Jurkat cells were incubated with increasing concentrations of ATO (0.2, 0.6 and 1.0μM) for 24h in standard cell culture conditions. To reach our goal we performed annexin V/PI labeling for detection of cell death and RNase/PI labeling for evaluation of cell cycle distribution, which were followed by the respective flow cytometric analyses of ATO-treated Jurkat cells. Transmission electron microscopy was applied for visualization of the cell ultrastructure. For cyclin D1 estimation a biparametric cyclinD1/cell cycle assay was done and localization of the protein was shown after immuno-labeling using light microscopy (ABC procedure) and confocal fluorescence microscopy. We found that there were no significant changes in the percentages of cyclin D1-positive cells after the treatment with ATO, but at the same time mean fluorescence intensity reflecting cyclin D1 content was gradually increasing along with the cell cycle progression, irrespective of the applied dose of the drug. On the other hand, we found a nuclear-cytoplasmic shift of this protein as a major treatment-related response, which was in good accord with an increased rate of cell death and suggested that cyclin D1 cytoplasmic degradation is an important determinant of the therapeutic efficiency of ATO in the Jurkat cell line. Copyright © 2014 Elsevier GmbH. All rights reserved.

  10. Evaluation of HSP70 expression and DNA damage in cells of a human trophoblast cell line exposed to 1.8 GHz amplitude-modulated radiofrequency fields.

    Science.gov (United States)

    Valbonesi, Paola; Franzellitti, Silvia; Piano, Annamaria; Contin, Andrea; Biondi, Carla; Fabbri, Elena

    2008-03-01

    The aim of this study was to determine whether high-frequency electromagnetic fields (EMFs) could induce cellular effects. The human trophoblast cell line HTR-8/SVneo was used as a model to evaluate the expression of proteins (HSP70 and HSC70) and genes (HSP70A, B, C and HSC70) of the HSP70 family and the primary DNA damage response after nonthermal exposure to pulse-modulated 1817 MHz sinusoidal waves (GSM-217 Hz; 1 h; SAR of 2 W/kg). HSP70 expression was significantly enhanced by heat, which was applied as the prototypical stimulus. The HSP70A, B and C transcripts were differentially expressed under basal conditions, and they were all significantly induced above basal levels by thermal stress. Conversely, HSC70 protein and gene expression was not influenced by heat. Exposing HTR-8/SVneo cells to high-frequency EMFs did not change either HSP70 or HSC70 protein or gene expression. A significant increase in DNA strand breaks was caused by exposure to H(2)O(2), which was used as a positive stimulus; however, no effect was observed after exposure of cells to high-frequency EMFs. Overall, no evidence was found that a 1-h exposure to GSM-217 Hz induced a HSP70-mediated stress response or primary DNA damage in HTR-8/SVneo cells. Nevertheless, further investigations on trophoblast cell responses after exposure to GSM signals of different types and durations are needed.

  11. Induction of tumor necrosis factor expression and resistance in a human breast tumor cell line.

    OpenAIRE

    Spriggs, D; Imamura, K; Rodriguez, C; Horiguchi, J; Kufe, D W

    1987-01-01

    Tumor necrosis factor (TNF) is a polypeptide cytokine that is cytotoxic to some but not all tumor cells. The basis for resistance to the cytotoxic effects of this agent remains unclear. We have studied the development of TNF resistance in human ZR-75-1 breast carcinoma cells. ZR-75-1 cells have undetectable levels of TNF RNA and protein. However, TNF transcripts are transiently induced in these cells by exposure to recombinant human TNF. This induction of TNF RNA is associated with production...

  12. MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

    Directory of Open Access Journals (Sweden)

    Fiorella Faggi

    Full Text Available The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3 in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS, an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

  13. Profiling of gene expression regulated by 17β-estradiol and tamoxifen in estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Rangel N

    2017-09-01

    Full Text Available Nelson Rangel,1,2 Victoria E Villegas,2 Milena Rondón-Lagos3 1Department of Medical Sciences, University of Turin, Turin, Italy; 2Faculty of Natural Sciences and Mathematics, Universidad del Rosario, Bogotá, Colombia; 3School of Biological Sciences, Universidad Pedagógica y Tecnológica de Colombia, Tunja, Colombia Abstract: One area of great importance in breast cancer (BC research is the study of gene expression regulated by both estrogenic and antiestrogenic agents. Although many studies have been performed in this area, most of them have only addressed the effects of 17β-estradiol (E2 and tamoxifen (TAM on MCF7 cells. This study aimed to determine the effect of low doses of E2 and TAM on the expression levels of 84 key genes, which are commonly involved in breast carcinogenesis, in four BC cell lines differentially expressing estrogen receptor (ER α and HER2 (MCF7, T47D, BT474, and SKBR3. The results allowed us to determine the expression patterns modulated by E2 and TAM in ERα+ and ERα− cell lines, as well as to identify differences in expression patterns. Although the MCF7 cell line is the most frequently used model to determine gene expression profiles in response to E2 and TAM, the changes in gene expression patterns identified in ERα+ and ERα− cell lines could reflect distinctive properties of these cells. Our results could provide important markers to be validated in BC patient samples, and subsequently used for predicting the outcome in ERα+ and ERα− tumors after TAM or hormonal therapy. Considering that BC is a molecularly heterogeneous disease, it is important to understand how well, and which cell lines, best model that diversity. Keywords: breast cancer, cell lines, 17β-estradiol, tamoxifen, ERα+, ERα−, qPCR

  14. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

    Science.gov (United States)

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-01

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  15. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia.

    Science.gov (United States)

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-10

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  16. Effects of schizandrin on the expression of thymic stromal lymphopoietin in human mast cell line HMC-1.

    Science.gov (United States)

    Moon, Phil-Dong; Jeong, Hyun-Ja; Kim, Hyung-Min

    2012-10-05

    Thymic stromal lymphopoietin (TSLP) plays an important role in allergic diseases such as asthma and atopic dermatitis. Schizandrin has various effects such as anti-asthmatic, anti-cancer and anti-inflammatory effects. However, the effect of schizandrin on the production of TSLP has not been clarified. Thus, we investigated how schizandrin inhibits the production of TSLP in the human mast cell line HMC-1 cells. We used enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, luciferase assay, and Western blot analysis to investigate the effects of schizandrin. Schizandrin inhibited the production and mRNA expression of TSLP in HMC-1 cells. The maximal inhibition rate of TSLP production by schizandrin (10 μM) was 68.62 ± 3.47%. Schizandrin inhibited the translocation and luciferase activity of nuclear factor-κB induced by phorbol myristate acetate plus A23187. In the activated HMC-1 cells, the activation of caspase-1 was increased, whereas the activation of caspase-1 was decreased by pretreatment with schizandrin. These results suggest that schizandrin can be used to treat inflammatory and atopic diseases through the inhibition of TSLP. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Cell line provenance.

    Science.gov (United States)

    Freshney, R Ian

    2002-07-01

    Cultured cell lines have become an extremely valuable resource, both in academic research and in industrial biotechnology. However, their value is frequently compromised by misidentification and undetected microbial contamination. As detailed elsewhere in this volume, the technology, both simple and sophisticated, is available to remedy the problems of misidentification and contamination, given the will to apply it. Combined with proper records of the origin and history of the cell line, assays for authentication and contamination contribute to the provenance of the cell line. Detailed records should start from the initiation or receipt of the cell line, and should incorporate data on the donor as well as the tissue from which the cell line was derived, should continue with details of maintenance, and include any accidental as well as deliberate deviations from normal maintenance. Records should also contain details of authentication and regular checks for contamination. With this information, preferably stored in a database, and suitable backed up, the provenance of the cell line so created makes the cell line a much more valuable resource, fit for validation in industrial applications and more likely to provide reproducible experimental results when disseminated for research in other laboratories.

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

    OpenAIRE

    ITO, TOMOAKI; KAJINO, KAZUNORI; ABE, MASAAKI; SATO, KOICHI; MAEKAWA, HIROSHI; SAKURADA, MUTSUMI; ORITA, HAJIME; WADA, RYO; KAJIYAMA, YOSHIAKI; HINO, OKIO

    2013-01-01

    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...

  19. Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data

    Directory of Open Access Journals (Sweden)

    Ryan Aideen E

    2006-02-01

    Full Text Available Abstract Background During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period. Methods RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat and three mouse (CMT93, CT26, B16F10 cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody. Results Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent. Conclusion These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.

  20. [Construction of γ-synuclein eukaryotic expression vector and its effect on invasion and metastasis of colon cancer cell line SW1116 in vitro].

    Science.gov (United States)

    Ye, Qing; Huang, Feng; Zheng, Qiuhong; Wang, Xiaoying; Xu, Yangmei; Gong, Fusheng; Huang, Lijie

    2014-01-01

    To construct γ-synuclein gene eukaryotic expression vector, and to study its effect on the invasion of colon cancer cell line SW1116 and the adhesion between SW1116 and human umbilical vein endothelial cells(HUVECs) in vitro. Total RNA was extracted from colon cancer cell line HT29 and the cDNA of γ-synuclein was amplified using RT-PCR. The digested fragment of cDNA coding sequence was linked to the eukaryotic expression vector pEGFP-C1 containing the GFP gene. After identification by sequence analysis, the recombinant plasmid was transfected into colon cancer cell line SW1116 via lipofectamine. The stable cell line was selected with G-418. The invasion in vitro was tested by Transwell invasion chamber assay. HUVECs were previously seeded onto 96-well plates before SW1116 cells seeded, and fluorescence intensity of GFP was detected to represent the amount of adhesion cells by ELISA. Human γ-synuclein eukaryotic expression vector was successfully constructed, which was stably expressed in SW1116 cells and could translate the GFP-γ-synuclein protein in vitro. γ-synuclein facilitated SW1116 cell passing through matrigel and filter membrane(198.4±20.7 vs. 98.8±13.2, Pcells to HUVECs(3.08±0.36 vs. 1.22±0.21, Pcell SW1116 potentiality of invasion and metastasis in vitro.

  1. Expression of glutathione transferases in corneal cell lines, corneal tissues and a human cornea construct.

    Science.gov (United States)

    Kölln, Christian; Reichl, Stephan

    2016-06-15

    Glutathione transferase (GST) expression and activity were examined in a three-dimensional human cornea construct and were compared to those of excised animal corneas. The objective of this study was to characterize phase II enzyme expression in the cornea construct with respect to its utility as an alternative to animal cornea models. The expression of the GSTO1-1 and GSTP1-1 enzymes was investigated using immunofluorescence staining and western blotting. The level of total glutathione transferase activity was determined using 1-chloro-2,4- dinitrobenzene as the substrate. Furthermore, the levels of GSTO1-1 and GSTP1-1 activity were examined using S-(4-nitrophenacyl)glutathione and ethacrynic acid, respectively, as the specific substrates. The expression and activity levels of these enzymes were examined in the epithelium, stroma and endothelium, the three main cellular layers of the cornea. In summary, the investigated enzymes were detected at both the protein and functional levels in the cornea construct and the excised animal corneas. However, the enzymatic activity levels of the human cornea construct were lower than those of the animal corneas. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Regulation of type III iodothyronine deiodinase expression in human cell lines

    NARCIS (Netherlands)

    Kester, Monique H. A.; Kuiper, George G. J. M.; Versteeg, Rogier; Visser, Theo J.

    2006-01-01

    Type I iodothyronine deiodinase (D1) and type II iodothyronine deiodinase (D2) catalyze the activation of the prohormone T4 to the active hormone T3; type III iodothyronine deiodinase (D3) catalyzes the inactivation of T4 and T3. D3 is highly expressed in brain, placenta, pregnant uterus, and fetal

  3. Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

    Directory of Open Access Journals (Sweden)

    Emma J Mead

    Full Text Available Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based and engineering (nonlinear models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.

  4. Calcium regulation of androgen receptor expression in the human prostate cancer cell line LNCaP

    NARCIS (Netherlands)

    L.J. Blok (Leen); J.E. Perry; J.K. Lindzey; D.J. Tindall; Y. Gong (Yuewen)

    1995-01-01

    textabstractElevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of

  5. Effect of radiation on the expression of E-cadherin and α-catenin and invasive capacity in human lung cancer cell line in vitro

    International Nuclear Information System (INIS)

    Akimoto, Tetsuo; Mitsuhashi, Norio; Saito, Yoshihiro; Ebara, Takeshi; Niibe, Hideo

    1998-01-01

    Purpose: To investigate the effect of radiation on E-cadherin and α-catenin expression in a human lung cancer cell line, and also evaluate invasive capacity in the membrane invasion culture system using the Boyden Chamber. Materials and Methods: The immunoblot and immunofluorescence analyses were performed using the human lung cancer cell line A549 to examine altered expression of E-cadherin and α-catenin after irradiation. We also compared invasive capacity of untreated cells with that of irradiated cells. Results: Immunoblot analysis revealed that the expression of E-cadherin increased after irradiation. In a time-course analysis, the expression was increased 6 h after irradiation with 10 Gy and reached its peak level at 24 h, being 2.3 times the control value, whereas expression at 1 and 3 h after irradiation was almost equivalent to that of the control. A slight increase in expression was observed after irradiation of 2 Gy and the expression reached peak levels after 5 Gy. After fractionated irradiation, the increase in expression of both E-cadherin and α-catenin was observed, and the alteration of α-catenin was more prominent than that after a single irradiation of the same total dose. In the immunofluorescence study for E-cadherin antibody analyzed by confocal laser scanning microscopy, increased intensity in irradiated cells produced as a nondisrupted and continuous line at cell-cell contact sites. In an invasive assay, the number of migrated cells in irradiated cells after a dose of 5 and 10 Gy was reduced significantly compared to untreated cells. Conclusion: The results indicate that irradiation of A549 increased the expression of E-cadherin, possibly preserving their functional property

  6. Apium graveolens Extract Inhibits Cell Proliferation and Expression of Vascular Endothelial Growth Factor and Induces Apoptosis in the Human Prostatic Carcinoma Cell Line LNCaP.

    Science.gov (United States)

    Köken, Tülay; Koca, Buğra; Özkurt, Mete; Erkasap, Nilüfer; Kuş, Gökhan; Karalar, Mustafa

    2016-12-01

    Apium graveolens has been shown to inhibit the growth of a variety of cancer tissues. In this study, we investigated the anticancer effect of A. graveolens on the human prostatic carcinoma cell line LNCaP. LNCaP cells were treated with increasing concentrations of an ethanolic extract of A. graveolens ranging from 1000 to 3000 μg/mL, and viability was determined after 24 and 48 h using the XTT cell proliferation assay. The levels of cleaved poly (ADP-ribose) polymerase (PARP), one of the best biomarkers of apoptosis, were analyzed. Finally, quantitative gene expression analysis of vascular endothelial growth factor (VEGF), a critical mediator of angiogenesis, was performed using real-time reverse transcription-polymerase chain reaction. A. graveolens extract inhibited cell viability in both a time- and dose-dependent manner. Data from cleaved PARP assays suggested that A. graveolens caused induction of apoptosis in these cells. Treatment of cells with A. graveolens also resulted in downregulation of VEGF expression. This study showed that the antiproliferative effect exerted by an ethanolic extract of A. graveolens is triggered by induction of apoptosis. We also demonstrated that VEGF expression was downregulated by treatment with A. graveolens extract.

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

    Directory of Open Access Journals (Sweden)

    Lin-Lin Liu

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

  8. Expression of TP53 isoforms p53β or p53γ enhances chemosensitivity in TP53(null cell lines.

    Directory of Open Access Journals (Sweden)

    Elisabeth Silden

    Full Text Available The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1, Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(PH quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level.

  9. Epithelium-Specific Ets-Like Transcription Factor 1, ESE-1, Regulates ICAM-1 Expression in Cultured Lung Epithelial Cell Lines

    Directory of Open Access Journals (Sweden)

    Zhiqi Yu

    2015-01-01

    Full Text Available Cystic fibrosis (CF patients suffer from chronic airway inflammation with excessive neutrophil infiltration. Migration of neutrophils to the lung requires chemokine and cytokine signaling as well as cell adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1, which plays an important role in mediating adhesive interactions between effector and target cells in the immune system. In this study, we investigated the relationship between ICAM-1 and epithelium-specific ETS-like transcription factor 1 (ESE-1 and found that ICAM-1 expression is upregulated in cell lines of CF (IB3-1 as well as non-CF (BEAS-2B and A549 epithelial origin in response to inflammatory cytokine stimulation. Since ESE-1 is highly expressed in A549 cells without stimulation, we examined the effect of ESE-1 knockdown on ICAM-1 expression in these cells. We found that ICAM-1 expression was downregulated when ESE-1 was knocked down in A549 cells. We also tested the effect of ESE-1 knockdown on cell-cell interactions and demonstrate that the knocking down ESE-1 in A549 cells reduce their interactions with HL-60 cells (human promyelocytic leukemia cell line. These results suggest that ESE-1 may play a role in regulating airway inflammation by regulating ICAM-1 expression.

  10. Differential protein expression profile in the hypothalamic GT1-7 cell line after exposure to anabolic androgenic steroids.

    Science.gov (United States)

    Martínez-Rivera, Freddyson J; Pérez-Laspiur, Juliana; Santiago-Gascot, María E; Alemán-Reyes, Abner G; García-Santiago, Emanuel; Rodríguez-Pérez, Yolanda; Calo-Guadalupe, Cristhian; Otero-Pagán, Inelia; Ayala-Pagán, Roxsana N; Martínez, Magdiel; Cantres-Rosario, Yisel M; Meléndez, Loyda M; Barreto-Estrada, Jennifer L

    2017-01-01

    The abuse of anabolic androgenic steroids (AAS) has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG) axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH). In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 μM). These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH in vivo. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.

  11. Differential protein expression profile in the hypothalamic GT1-7 cell line after exposure to anabolic androgenic steroids.

    Directory of Open Access Journals (Sweden)

    Freddyson J Martínez-Rivera

    Full Text Available The abuse of anabolic androgenic steroids (AAS has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH. In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 μM. These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH in vivo. Two-dimensional difference in gel electrophoresis (2D-DIGE and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.

  12. Expression profiling indicating low selenium-sensitive microRNA levels linked to cell cycle and cell stress response pathways in the CaCo-2 cell line.

    Science.gov (United States)

    McCann, Mark J; Rotjanapun, Kunjana; Hesketh, John E; Roy, Nicole C

    2017-05-01

    Se is an essential micronutrient for human health, and fluctuations in Se levels and the potential cellular dysfunction associated with it may increase the risk for disease. Although Se has been shown to influence several biological pathways important in health, little is known about the effect of Se on the expression of microRNA (miRNA) molecules regulating these pathways. To explore the potential role of Se-sensitive miRNA in regulating pathways linked with colon cancer, we profiled the expression of 800 miRNA in the CaCo-2 human adenocarcinoma cell line in response to a low-Se (72 h at <40 nm) environment using nCounter direct quantification. These data were then examined using a range of in silico databases to identify experimentally validated miRNA-mRNA interactions and the biological pathways involved. We identified ten Se-sensitive miRNA (hsa-miR-93-5p, hsa-miR-106a-5p, hsa-miR-205-5p, hsa-miR-200c-3p, hsa-miR-99b-5p, hsa-miR-302d-3p, hsa-miR-373-3p, hsa-miR-483-3p, hsa-miR-512-5p and hsa-miR-4454), which regulate 3588 mRNA in key pathways such as the cell cycle, the cellular response to stress, and the canonical Wnt/β-catenin, p53 and ERK/MAPK signalling pathways. Our data show that the effects of low Se on biological pathways may, in part, be due to these ten Se-sensitive miRNA. Dysregulation of the cell cycle and of the stress response pathways due to low Se may influence key genes involved in carcinogenesis.

  13. A gene encoding 22 highly related zinc fingers is expressed in lymphoid cell lines.

    OpenAIRE

    Lovering, R; Trowsdale, J

    1991-01-01

    A cDNA was isolated from a T cell library using an oligonucleotide probe corresponding to a sequence conserved in proteins with multiple zinc fingers of the C2H2 type. The predicted protein structure of this cDNA (ZNF43) showed that it contained 22 of the Krüppel type of zinc finger motifs in tandem. The amino acid sequence was strongly conserved between each of the finger domains of this cDNA, except for variable residue positions within the putative DNA binding site. Within the zinc finger ...

  14. Global analysis of gene expression mediated by OX1 orexin receptor signaling in a hypothalamic cell line.

    Science.gov (United States)

    Koesema, Eric; Kodadek, Thomas

    2017-01-01

    The orexins and their cognate G-protein coupled receptors have been widely studied due to their associations with various behaviors and cellular processes. However, the detailed downstream signaling cascades that mediate these effects are not completely understood. We report the generation of a neuronal model cell line that stably expresses the OX1 orexin receptor (OX1) and an RNA-Seq analysis of changes in gene expression seen upon receptor activation. Upon treatment with orexin, several families of related transcription factors are transcriptionally regulated, including the early growth response genes (Egr), the Kruppel-like factors (Klf), and the Nr4a subgroup of nuclear hormone receptors. Furthermore, some of the transcriptional effects observed have also been seen in data from in vivo sleep deprivation microarray studies, supporting the physiological relevance of the data set. Additionally, inhibition of one of the most highly regulated genes, serum and glucocorticoid-regulated kinase 1 (Sgk1), resulted in the diminished orexin-dependent induction of a subset of genes. These results provide new insight into the molecular signaling events that occur during OX1 signaling and support a role for orexin signaling in the stimulation of wakefulness during sleep deprivation studies.

  15. Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea).

    Science.gov (United States)

    Parton, Angela; Bayne, Christopher J; Barnes, David W

    2010-09-01

    Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories "envelope" and "oxidoreductase activity" but the SAE transcripts did not. GO analysis of SAE transcripts identified the category "anatomical structure formation" that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes. Copyright 2010 Elsevier Inc. All rights reserved.

  16. Effects of Urtica dioica dichloromethane extract on cell apoptosis and related gene expression in human breast cancer cell line (MDA-MB-468).

    Science.gov (United States)

    Mohammadi, A; Mansoori, B; Goldar, S; Shanehbandi, D; Khaze, V; Mohammadnejad, L; Baghbani, E; Baradaran, B

    2016-02-29

    Breast cancer is the most common cancer among women in worldwide, especially in developing countries. Therefore, a large number of anticancer agents with herbal origins have been reported against this deadly disease. This study is the first to examine the cytotoxic and apoptotic effects of Urtica dioica in MDA-MB-468, human breast adenocarcinoma cells. The 3-(4,5-dimethylethiazol-2 yl)-2,5- diphenyltetrazolium (MTT) reduction and trypan-blue exclusion assay were performed in MDA-MB-468 cells as well as control cell line L929 to analyze the cytotoxic activity of the dichloromethane extract. In addition, Apoptosis induction of Urtica dioica on the MDA-MB-468 cells was assessed using TUNEL (terminal deoxy transferase (TdT)-mediated dUTP nick- end labeling) assay and DNA fragmentation analysis and real-time polymerase chain reaction (PCR). The results showed that the extract significantly inhibited cell growth and viability without inducing damage to normal control cells. Nuclei Staining in TUNEL and DNA fragments in DNA fragmentation assay and increase in the mRNA expression levels of caspase-3, caspase-9, decrease in the bcl2 and no significant change in the caspase-8 mRNA expression level, showed that the induction of apoptosis was the main mechanism of cell death that induce by Urtica dioica extract. Our results suggest that urtica dioica dichloromethane extract may contain potential bioactive compound(s) for the treatment of breast adenocarcinoma.

  17. Effect of Flavonoids on Glutathione Level, Lipid Peroxidation and Cytochrome P450 CYP1A1 Expression in Human Laryngeal Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Lidija Vuković

    2007-01-01

    Full Text Available Flavonoids are phytochemicals exhibiting a wide range of biological activities, among which are antioxidant activity, the ability to modulate activity of several enzymes or cell receptors and possibility to interfere with essential biochemical pathways. Using human laryngeal carcinoma HEp2 cells and their drug-resistant CK2 subline, we examined the effect of five flavonoids, three structurally related flavons (quercetin, fisetin, and myricetin, one flavonol (luteolin and one glycosilated flavanone (naringin for: (i their ability to inhibit mitochondrial dehydrogenases as an indicator of cytotoxic effect, (ii their influence on glutathione level, (iii antioxidant/prooxidant effects and influence on cell membrane permeability, and (iv effect on expression of cytochrome CYP1A1. Cytotoxic action of the investigated flavonoids after 72 hours of treatment follows this order: luteolin>quercetin>fisetin>naringin>myricetin. Our results show that CK2 were more resistant to toxic concentrations of flavonoids as compared to parental cells. Quercetin increased the total GSH level in both cell lines. CK2 cells are less perceptible to lipid peroxidation and damage caused by free radicals. Quercetin showed prooxidant effect in both cell lines, luteolin only in HEp2 cells, whereas other tested flavonoids did not cause lipid peroxidation in the tested cell lines. These data suggest that the same compound, quercetin, can act as a prooxidant, but also, it may prevent damage in cells caused by free radicals, due to the induction of GSH, by forming less harmful complex. Quercetin treatment damaged cell membranes in both cell lines. Fisetin caused higher cell membrane permeability only in HEp2 cells. However, these two compounds did not enhance the damage caused by hydrogen peroxide. Quercetin, naringin, myricetin and fisetin increased the expression of CYP1A1 in both cell lines, while luteolin decreased basal level of CYP1A1 only in HEp2 cells. In conclusion, small

  18. Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment.

    Science.gov (United States)

    Kubala, Marta H; DeClerck, Yves A

    2017-11-23

    siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system 1 , and the tetracycline regulated gene expression systems. The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

  19. Correlation between expression of major histocompatibility complex class I and that of antigen presenting machineries in carcinoma cell lines of the pancreas, biliary tract and colon

    OpenAIRE

    Imanishi, Tatsuya; Kamigaki, Takashi; Nakamura, Tetsu; Hayashi, Shun; Yasuda, Takashi; Kawasaki, Kentaro; Takase, Shiro; Ajiki, Tetsuo; Kuroda, Yoshikazu

    2006-01-01

    To elicit a tumor immune response, tumor antigens represented by majorhistocompatibility (MHC) class I complex on the cell surface is indispensable. Someinvestigators demonstrated that many cancer cells reduce expression ofβ2-microglobulin, a transporter of antigen presenting (TAP) or low molecular protein(LMP), due to the deletion mutant or point mutation. We investigated gene expressionlevels of antigen presenting machineries in 13 cell lines of the pancreas, biliary tractand colon cancer b...

  20. High value of the radiobiological parameter Dq correlates to expression of the transforming growth factor beta type II receptor in a panel of small cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Hougaard, S; Krarup, M; Nørgaard, P

    1998-01-01

    Our panel of SCLC cell lines have previously been examined for their radiobiological characteristics and sensitivity to treatment with TGF beta 1. In this study we examined the possible correlations between radiobiological parameters and the expression of the TGF beta type II receptor (TGF beta......-rII). We have, in other studies, shown that the presence of TGF beta-rII was mandatory for transmitting the growth inhibitory effect of TGF beta. The results showed a statistically significant difference in Dq, i.e. the shoulder width of the survival curve, between cell lines expressing TGF beta......-rII and cell lines which did not express the receptor (P = 0.01). Cell lines expressing TGF beta-rII had a high Dq-value. TGF beta-rII expression did not correlate with any other radiobiological parameters. We suggest that an intact growth inhibitory pathway mediated by the TGF beta-rII may have a significant...

  1. Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2

    International Nuclear Information System (INIS)

    Nishida, Shozo; Tsubaki, Masanobu; Hoshino, Mayumi; Namimatsu, Ayumi; Uji, Hiromi; Yoshioka, Shohei; Tanimori, Yoshihiro; Yanae, Masashi; Iwaki, Masahiro; Irimajiri, Kiyohiro

    2005-01-01

    Increase in bone resorption by osteoclasts can cause metabolic bone diseases, such as osteoporosis. Recent attention has been paid to the receptor activator of the NF-κB ligand (RANKL), an accelerator of osteoclast differentiation. RANKL is expressed on the bone marrow-derived stromal cell membrane and induces the differentiation of osteoclasts by binding to RANK expressed on the osteoclast precursor cell membrane. Since the inhibition of RANKL expression can lead to the inhibition of osteoclastic bone resorption, the clinical application of RANKL inhibition could be expected to have a major effect on metabolic bone disease therapy. In this study, we investigated whether or not YM529/ONO-5920, a nitrogen-containing bisphosphonate (a novel minodronic acid), inhibits RANKL expression in a bone marrow-derived stromal cell line (ST2 cells). Reverse transcription-polymerase chain reaction revealed that the administration of YM529/ONO-5920 to ST2 cells inhibited RANKL mRNA expression and reduced RANKL proteins as assessed by Western blot analysis. The inhibition of RANKL mRNA expression was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination. Furthermore, YM529/ONO-5920 reduced phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and similarly, U0126, a mitogen-activated protein kinase kinase 1/2 inhibitor, inhibited RANKL expression. Pretreatment with GGPP reversed the YM529/ONO-5920-induced decrease in phosphorylation of ERK. Furthermore, YM529/ONO-5920 decreased TRAP-positive cells in co-culture of ST2 cells and an osteoclast cell line, C7 cells, and this decrease was inhibited by pretreatment with GGPP. This indicates that YM529/ONO-5920 inhibits GGPP biosynthesis in the mevalonate pathway and then signal transduction in the Ras-mitogen-activated protein kinase pathway, thereby inhibiting RANKL expression on ST2 cells. These results suggest a newly elucidated action of bisphosphonates in

  2. Identification of the thiamin pyrophosphokinase gene in rainbow trout: Characteristic structure and expression of seven splice variants in tissues and cell lines and during embryo development

    Science.gov (United States)

    Yuge, Shinya; Richter, Catherine A.; Wright-Osment, Maureen K.; Nicks, Diane; Saloka, Stephanie K.; Tillitt, Donald E.; Li, Weiming

    2012-01-01

    Thiamin pyrophosphokinase (TPK) converts thiamin to its active form, thiamin diphosphate. In humans, TPK expression is down-regulated in some thiamin deficiency related syndrome, and enhanced during pregnancy. Rainbow trout are also vulnerable to thiamin deficiency in wild life and are useful models for thiamin metabolism research. We identified the tpk gene transcript including seven splice variants in the rainbow trout. Almost all cell lines and tissues examined showed co-expression of several tpk splice variants including a potentially major one at both mRNA and protein levels. However, relative to other tissues, the longest variant mRNA expression was predominant in the ovary and abundant in embryos. During embryogenesis, total tpk transcripts increased abruptly in early development, and decreased to about half of the peak shortly after hatching. In rainbow trout, the tpk transcript complex is ubiquitously expressed for all tissues and cells examined, and its increase in expression could be important in the early-middle embryonic stages. Moreover, decimated tpk expression in a hepatoma cell line relative to hepatic and gonadal cell lines appears to be consistent with previously reported down-regulation of thiamin metabolism in cancer.

  3. 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.

  4. Real-Time Expression Analysis of Selected Anticarsia gemmatalis multiple nucleopolyhedrovirus Gene Promoters during Infection of Permissive, Semipermissive and Nonpermissive Cell Lines.

    Science.gov (United States)

    Morgado, Fabricio da Silva; Ardisson-Araújo, Daniel Mendes Pereira; Ribeiro, Bergmann Morais

    2017-06-01

    Baculovirus infection follows a transcriptionally controlled sequence of gene expression that occurs by activation of different viral gene promoter sequences during infection. This sequence of promoter activation may be disrupted by cellular defenses against viral infection, which might interfere with viral progeny formation. In this work, the activity of the ie1 , gp64 , lef-1 , vp39 , p6.9 and polh promoters of the Anticarsia gemmatalis multiple nucleopolyhedrovirus was assessed during infection of permissive, semipermissive and nonpermissive cell lines by a novel methodology that detects reporter protein luminescence in real-time. This technique allowed us to characterize in rich detail the AgMNPV promoters in permissive cell lines and revealed differential profiles of expression in cells with limited permissivity that correlate well with limitations in viral DNA replication. Semipermissive and nonpermissive cell lines presented delays and restrictions in late and very late promoter expression. Cells undergoing apoptosis did not inhibit late gene expression; however, viral progeny formation is severely affected. This work demonstrates the application of the real-time luminescence detection methodology and how the promoter expression profile may be used to diagnose cellular permissivity to baculovirus infection.

  5. Fenofibrate down-regulates the expressions of androgen receptor (AR) and AR target genes and induces oxidative stress in the prostate cancer cell line LNCaP

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Hu; Zhu, Chen; Qin, Chao [State Key Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Tao, Tao [Department of Neurosurgery, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Li, Jie; Cheng, Gong; Li, Pu; Cao, Qiang; Meng, Xiaoxin; Ju, Xiaobing; Shao, Pengfei; Hua, Lixin [State Key Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Gu, Min, E-mail: medzhao1980@163.com [State Key Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China); Yin, Changjun, E-mail: drcjyin@gmail.com [State Key Laboratory of Reproductive Medicine, Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing (China)

    2013-03-08

    Highlights: ► Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells. ► Fenofibrate reduces the expressions of androgen receptor in LNCaP cells. ► Fenofibrate induces oxidative stress in the prostate cancer cell line LNCaP. -- Abstract: Fenofibrate, a peroxisome proliferator-androgen receptor-alpha agonist, is widely used in treating different forms of hyperlipidemia and hypercholesterolemia. Recent reports have indicated that fenofibrate exerts anti-proliferative and pro-apoptotic properties. This study aims to investigate the effects of fenofibrate on the prostate cancer (PCa) cell line LNCaP. The effects of fenofibrate on LNCaP cells were evaluated by flow cytometry, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assays, Western blot analysis, and dual-luciferase reporter assay. Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells, reduces the expressions of androgen receptor (AR) and AR target genes (prostate-specific antigen and TMPRSS2), and inhibits Akt phosphorylation. Fenofibrate can induce the accumulation of intracellular reactive oxygen species and malondialdehyde, and decrease the activities of total anti-oxidant and superoxide dismutase in LNCaP cells. Fenofibrate exerts an anti-proliferative property by inhibiting the expression of AR and induces apoptosis by causing oxidative stress. Therefore, our data suggest fenofibrate may have beneficial effects in fenofibrate users by preventing prostate cancer growth through inhibition of androgen activation and expression.

  6. Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes

    Directory of Open Access Journals (Sweden)

    Schulte Ina

    2012-12-01

    Full Text Available Abstract Background It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements. Results We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing—55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads. From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer—in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator. Conclusions This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports

  7. Expression of P-gp, MRP, LRP, GST-π and TopoIIα and intrinsic resistance in human lung cancer cell lines.

    Science.gov (United States)

    Wang, Jiarui; Zhang, Jinhui; Zhang, Lichuan; Zhao, Long; Fan, Sufang; Yang, Zhonghai; Gao, Fei; Kong, Ying; Xiao, Gary Guishan; Wang, Qi

    2011-11-01

    This study aimed to determine the relationship between the endogenous levels of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione-s-transferase-π (GST‑π) and topoisomerase IIα (TopoIIα) and intrinsic drug resistance in four human lung cancer cell lines, SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446, of different histological types. The expression of P-gp, MRP, LRP, GST-π and TopoIIα was measured by immunofluorescence, Western blotting and RT-PCR. Drug resistance to cisplatin, doxorubicin and VP-16 was determined using MTT assays. The correlation between expression of the resistance-related proteins and their roles in the resistance to drugs in these cancer cell lines was analyzed. We found that the endogenous levels of P-gp, MRP, LRP, GST-π and TopoIIα in the four cell lines varied. The level of GST-π in the SK-MES-1 cells was the highest, whereas the level of P-gp in the SPCA-1 cells was the lowest. The chemoresistance to cisplatin, doxorubicin and VP-16 in the four cell lines was different. The SPCA-1 cell line was most resistance to cisplatin; SK-MES-1 was most resistance to VP-16; whereas SK-MES-1 was most sensitive to doxorubicin. There was a positive correlation between GST-π expression and resistance to cisplatin, between TopoIIα expression and resistance to VP-16; and a negative correlation was noted between TopoIIα expression and resistance to doxorubicin. In summary, the endogenous expression of P-gp, MRP, LRP, GST-π and TopoIIα was different in the four human lung cancer cell lines of different histological types, and this variance may be associated with the variation in chemosensitivity to cisplatin, doxorubicin and VP-16. Among the related proteins, GST-π may be useful for the prediction of the intrinsic resistance to cisplatin, whereas TopoIIα may be useful to predict resistance to doxorubicin and VP-16 in human lung cancer cell lines.

  8. B7.1 expression on tumor cells circumvents the need of professional antigen presentation for in vitro propagation of cytotoxic T cell lines.

    Science.gov (United States)

    Iezzi, G; Protti, M P; Rugarli, C; Bellone, M

    1996-01-01

    In vitro propagation of tumor-specific CTLs, to be used for identification of tumor antigens (Ag) and/or adoptive immunotherapy, is hampered by the need of large amounts of professional antigen-presenting cells (APC) used for periodical cycles of restimulation. We evaluated whether RMA T lymphoma cells, stably transfected with the cDNA encoding for the B7.1 costimulatory molecule, provided the activation signals to CD8+ T lymphocytes in the absence of professional APC and CD4+ helper cells. We demonstrate here that long-term CD8+ cell lines can be efficiently propagated in vitro by repeated cycles of stimulation with tumor cells stably expressing B7.1. Professional APC and CD4+ helper cells are not required as far as interleukin 2 is exogenously provided. Furthermore, CD8+ blasts needed both signal 1 (Ag in the contest of the MHC molecule) and signal 2 (interaction of costimulatory molecules) for restimulation. T cell blasts in the presence of signal 1 or 2 only still retained their effector potential but did not undergo clonal expansion. These results are very promising for further applications of specific immunotherapies in humans.

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  10. Increased expression of heparin binding EGF (HB-EGF), amphiregulin, TGF alpha and epiregulin in androgen-independent prostate cancer cell lines.

    DEFF Research Database (Denmark)

    Tørring, Niels; Sørensen, Boe Sandahl; Nexø, Ebba

    2000-01-01

    BACKGROUND: The proliferation of androgen-independent prostate cancer cell lines has previously been shown to be influenced by an autocrine loop of the epidermal growth factor (EGF) system. This observation has alerted us to study the expression of ligands and receptors from the EGF-system in pro...

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

    NARCIS (Netherlands)

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

    2006-01-01

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

  12. High expression of lnc-CRNDE presents as a biomarker for acute myeloid leukemia and promotes the malignant progression in acute myeloid leukemia cell line U937.

    Science.gov (United States)

    Wang, Y; Zhou, Q; Ma, J-J

    2018-02-01

    To detect the expression of long non-coding RNA-CRNDE in patients with acute myeloid leukemia and its effect on proliferation and apoptosis in acute myeloid leukemia cell line U937. 81 cases of newly diagnosed acute myeloid leukemia (AML) were enrolled, and 35 non-malignant hematological patients were selected as controls. Quantitative RT-PCR (qRT-PCR) was performed to detect the expression of lncRNA-CRNDE in the bone marrow specimens of the subjects, and the difference between the two groups was also compared. The correlation between the expression of lncRNA-CRNDE and the sex, age, classification and total survival of clinical patients was analyzed according to the clinical data. U937 cells and monocytes isolated from normal people were cultured, and the expression of lncRNA-CRNDE in acute myeloid leukemia cell line U937 and normal monocytes was compared. SiRNA-CRNDE and pcDNA-CRNDE were transfected into U937 cells, and cell counting kit-8 (CCK-8) assay was performed to detect proliferation of U937 cells, Annexin V/PI flow cytometry was carried out to detect cell apoptosis. Cell cycle was measured by flow cytometry. The expression of lncRNA-CRNDE in patients with AML and U937 cells was significantly higher than that in non-malignant hematological controls. Results of clinical data showed that the expression of lncRNA-CRNDE was associated with the classification and total survival of myeloid leukemia in clinical patients. After transfection of siRNA-CRNDE, the proliferation and cloning ability of U937 cells decreased, while the apoptosis increased (p < 0.01) and cells were arrested in G0-G1 phase. Meanwhile, after transfection of pcDNA-CRNDE, the proliferation ability of U937 cells increased significantly, which indicated that the expression of lncRNA-CRNDE might play an essential role in promoting the proliferation of U937 cells. LncRNA-CRNDE is highly expressed in the bone marrow tissues of AML patients, and the expression level is negatively correlated with the

  13. High expression of CD109 antigen regulates the phenotype of cancer stem-like cells/cancer-initiating cells in the novel epithelioid sarcoma cell line ESX and is related to poor prognosis of soft tissue sarcoma.

    Directory of Open Access Journals (Sweden)

    Makoto Emori

    Full Text Available Epithelioid sarcoma (ES is a relatively rare, highly malignant soft tissue sarcoma. The mainstay of treatment is resection or amputation. Currently other therapeutic options available for this disease are limited. Therefore, a novel therapeutic option needs to be developed. In the present study, we established a new human ES cell line (ESX and analyzed the characteristics of its cancer stem-like cells/cancer-initiating cells (CSCs/CICs based on ALDH1 activity. We demonstrated that a subpopulation of ESX cells with high ALDH1 activity (ALDH(high cells correlated with enhanced clonogenic ability, sphere-formation ability, and invasiveness in vitro and showed higher tumorigenicity in vivo. Next, using gene expression profiling, we identified CD109, a GPI-anchored protein upregulated in the ALDH(high cells. CD109 mRNA was highly expressed in various sarcoma cell lines, but weakly expressed in normal adult tissues. CD109-positive cells in ESX predominantly formed spheres in culture, whereas siCD109 reduced ALDH1 expression and inhibited the cell proliferation in vitro. Subsequently, we evaluated the expression of CD109 protein in 80 clinical specimens of soft tissue sarcoma. We found a strong correlation between CD109 protein expression and the prognosis (P = 0.009. In conclusion, CD109 might be a CSC/CIC marker in epithelioid sarcoma. Moreover, CD109 is a promising prognostic biomarker and a molecular target of cancer therapy for sarcomas including ES.

  14. Epithelial-mesenchymal transition markers and HER3 expression are predictors of elisidepsin treatment response in breast and pancreatic cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Cristina Teixidó

    Full Text Available Elisidepsin (elisidepsin trifluoroacetate, Irvalec®, PM02734 is a new synthetic depsipeptide, a result of the PharmaMar Development Program that seeks synthetic products of marine origin-derived compounds. Elisidepsin is a drug with antiproliferative activity in a wide range of tumors. In the present work we studied and characterized the mechanisms associated with sensitivity and resistance to elisidepsin treatment in a broad panel of tumor cell lines from breast and pancreas carcinomas, focusing on different factors involved in epithelial-mesenchymal transition (EMT and the use of HER family receptors in predicting the in vitro drug response. Interestingly, we observed that the basal protein expression levels of EMT markers show a significant correlation with cell viability in response to elisidepsin treatment in a panel of 12 different breast and pancreatic cancer cell lines. In addition, we generated three elisidepsin treatment-resistant cell lines (MCF-7, HPAC and AsPC-1 and analyzed the pattern of expression of different EMT markers in these cells, confirming that acquired resistance to elisidepsin is associated with a switch to the EMT state. Furthermore, a direct correlation between basal HER3 expression and sensitivity to elisidepsin was observed; moreover, modulation of HER3 expression levels in different cancer cell lines alter their sensitivities to the drug, making them more resistant when HER3 expression is downregulated by a HER3-specific short hairpin RNA and more sensitive when the receptor is overexpressed. These results show that HER3 expression is an important marker of sensitivity to elisidepsin treatment.

  15. Expression of matrix metalloproteinase-2 and -9 and membrane-type 1 matrix metalloproteinase in melanocytic tumors of dogs and canine melanoma cell lines.

    Science.gov (United States)

    Docampo, María-José; Cabrera, Jennifer; Rabanal, Rosa M; Bassols, Anna

    2011-08-01

    To evaluate expression of matrix metalloproteinase (MMP)-2 and -9 and membrane-type 1 MMP (MT1-MMP) in melanocytomas and malignant melanomas of dogs, analyze in vitro production of MMPs by canine melanoma cell lines and primary dermal fibroblasts, and investigate mutual communication between tumor cells and fibroblasts and the influence of collagen on MMP regulation. 35 biopsy specimens from melanocytic tumors and primary dermal fibroblasts of dogs and 3 canine melanoma cell lines (CML-1, CML-10c2, and CML-6M). MMP-2, MMP-9, and MT1-MMP were detected in tumor samples by use of immunohistochemical analysis. In vitro production was analyzed via reverse transcriptase-PCR assay, immunocytochemical analysis, zymography, and immunoblotting. MMP-9 was overexpressed in malignant melanomas, compared with expression in melanocytomas, whereas no significant differences in MMP-2 and MT1-MMP immunostaining were detected. Stromal cells also often had positive staining results. In vitro, all 3 melanoma cell lines and dermal fibroblasts had evidence of MMP-2 and MT1-MMP, but only melanoma cells had evidence of MMP-9. Coculture of CML-1 or CML-10c2 cells and dermal fibroblasts induced an increase in expression of the active form of MMP-2. Culture of melanoma cells on type I collagen increased the activation state of MT1-MMP. MMP-9 expression was increased in malignant melanomas of dogs. Stromal cells were a source for MMPs. Stromal cells, in combination with matrix components such as type I collagen, can interact with tumor cells to regulate MMP production. Information about MMP production and regulation could help in the development of new treatments.

  16. Gene expression profiling of breast tumor cell lines to predict for therapeutic response to microtubule-stabilizing agents.

    Science.gov (United States)

    Kadra, Gais; Finetti, Pascal; Toiron, Yves; Viens, Patrice; Birnbaum, Daniel; Borg, Jean-Paul; Bertucci, François; Gonçalves, Anthony

    2012-04-01

    Microtubule-targeting agents, including taxanes (Tax) and ixabepilone (Ixa), are important components of modern breast cancer chemotherapy regimens, but no molecular parameter is currently available that can predict for their efficiency. We sought to develop pharmacogenomic predictors of Tax- and Ixa-response from a large panel of human breast tumor cell lines (BTCL), then to evaluate their performance in clinical samples. Thirty-two BTCL, representative of the molecular diversity of breast cancers (BC), were treated in vitro with Tax (paclitaxel (Pac), docetaxel (Doc)), and ixabepilone (Ixa), then classified as drug-sensitive or resistant according to their 50% inhibitory concentrations (IC50s). Baseline gene expression data were obtained using Affymetrix U133 Plus 2.0 human oligonucleotide microarrays. Gene expression set (GES) predictors of response to taxanes were derived, then tested for validation internally and in publicly available gene expression datasets. In vitro IC50s of Pac and Doc were almost identical, whereas some Tax-resistant BTCL retained sensitivity to Ixa. GES predictors for Tax-sensitivity (333 genes) and Ixa-sensitivity (79 genes) were defined. They displayed a limited number of overlapping genes. Both were validated by leave-n-out cross-validation (n = 4; for overall accuracy (OA), P = 0.028 for Tax, and P = 0.0005 for Ixa). The GES predictor of Tax-sensitivity was tested on publicly available external datasets and significantly predicted Pac-sensitivity in 16 BTCL (P = 0.04 for OA), and pathological complete response to Pac-based neoadjuvant chemotherapy in BC patients (P = 0.0045 for OA). Applying Tax and Ixa-GES to a dataset of clinically annotated early BC patients identified subsets of tumors with potentially distinct phenotypes of drug sensitivity: predicted Ixa-sensitive/Tax-resistant BC were significantly (P Tax-sensitive BC. Genomic predictors for Tax- and Ixa-sensitivity can be derived from BTCL and may be helpful for better

  17. Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line

    DEFF Research Database (Denmark)

    Nielsen, C U; Amstrup, J; Steffansen, B

    2001-01-01

    The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [(14)C]glycylsarcosine ([(14)C]Gly-Sar) up to 50.2 +/- 3.6% (n = 6) of control values. Kinetic analysis......) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane....

  18. Effect of taurine on expressions of MMP-2 in K562 leukemia cell line exposed to γ-rays

    International Nuclear Information System (INIS)

    Fan Yan; Wu Shiliang; Xu Lan; Chou Hao; Zhou Yinghui

    2003-01-01

    Objective: To study the effect of γ-irradiation on expressions of MMP-2 in leukaemia cells and the suppressive effect of taurine(Tau) on irradiated tumour cells in terms of cellular level. Methods: The cells in the control group and Tau (50 mg/L, 100 mg/L, 200 mg/L) groups were irradiated with 15 Gy γ-rays. The expressions of MMP-2 were examined through Western-blotting after handled with gel-loading buffer within 12 h. Results: The expressions of MMP-2 were enhanced evidently in the positive control group, while they were less in the negative control group. In the Tau(50 mg/L, 100 mg/L, 200 mg/L) groups, the expressions of MMP-2 were diminished in turns, and they were almost identical between the negative control group and the Tau 200 mg/L group. Conclusion: Irradiation with γ-rays at a dose of 15 Gy can significantly stimulate the expressions of MMP-2 in K562 cells; Tau can inhibit the expressions of MMP-2 and its effect depends on to its dosage; Tau can inhabit the invasiveness and migration of irradiated tumour cells, so it has the biologic protective and therapeutic effects

  19. Expression and differential regulation of HLA-G isoforms in the retinal pigment epithelial cell line, ARPE-19

    DEFF Research Database (Denmark)

    Svendsen, Signe Goul; Udsen, Maja Søberg; Daouya, Marina

    2017-01-01

    by digital droplet PCR, measuring the gene expression of HLA-G in total RNA. The protein expression was analysed by immunohistochemistry and by immunofluorescence followed by confocal microscopy and the expression of the HLA-G isoforms was explored by fragment analysis. In the current study, we show that HLA......-G is expressed by ARPE-19 cells and is upregulated as a response to pro-inflammatory cytokines. Moreover, we are the first to describe a differential regulation of the HLA-G isoforms as a direct response to stimulation. These results might indicate that HLA-G can be part of the immune privilege of the posterior...

  20. Molecular analysis of Bcl-2 and cyclin D1 expression in differentially expressing estrogen receptor breast cancer MCF7, T47D and MDA-MB-468 cell lines treated with adriamycin

    Directory of Open Access Journals (Sweden)

    2008-08-01

    Full Text Available Background and purpose of the study: Bcl-2 and Cyclin D1 (CCND1 are key elements in cancer development and progression. Bcl-2 acts as a cell death suppressor and is involved in apoptosis regulation. Cyclin D1 is an important regulator of G1/S phase of the cell cycle progression. In addition, estrogen receptor (ER is an important prognostic factor in breast cancer cells. Therefore it is important to determine the Bcl-2 and CCND1 expression in MCF7, T47D and MDA-MB-468 breast cancer cell lines with different ER status following Adriamycin (ADR treatment. Methods: Cytotoxicity of ADR (250 and 500nM after 1-5 days exposure of the cell lines was evaluated by MTT assay. The mRNA and protein levels of Bcl-2 and cyclin D1 in tested cell lines were also analyzed by RT-PCR and immunocytochemistry (ICC methods. Results: ADR cytotoxicity was highest in MDA-MB-468 and lowest in MCF7 cells in a time-dependent manner. Bcl-2 mRNA increased in MCF7 and decreased in MDA-MB-468 after exposure to ADR but it was less detectable in T47D cells. The expression of CCND1 in MCF7 with high level of ER expression was higher than the other two cell lines in untreated conditions. However, CCND1 mRNA did not show significant changes after ADR treatment. Immunocytochemical analysis did not show significant differences between Bcl-2 protein expression in the presence or absence of ADR in MDA-MB-468 cell line while in T47D and MCF7 cells its expression decreased after exposure to ADR. In addition to nuclear expression of cyclin D1 in all cell lines, strong cytoplasmic expression of cyclin D1 protein was observed only in MCF7 and T47D cells. Conclusion: The tested cell lines with different levels of ER expression showed differential molecular responses to ADR that is important in tumor-targeted cancer therapy.

  1. Anti-apoptotic signature in thymic squamous cell carcinomas - functional relevance of anti-apoptotic BIRC3 expression in the thymic carcinoma cell line 1889c

    Directory of Open Access Journals (Sweden)

    Bei eHuang

    2013-12-01

    Full Text Available The molecular pathogenesis of thymomas and thymic carcinomas (TCs is poorly understood and results of adjuvant therapy are unsatisfactory in case of metastatic disease and tumor recurrence. For these clinical settings, novel therapeutic strategies are urgently needed. Recently, limited sequencing efforts revealed that a broad spectrum of genes that play key roles in various common cancers are rarely affected in thymomas and thymic carcinomas, suggesting that other oncogenic principles might be important. This made us re-analyze historic expression data obtained in a spectrum of thymomas and thymic squamous cell carcinomas (TSCC with a custom made cDNA microarray. By cluster analysis, different anti-apoptotic signatures were detected in type B3 thymoma and TSCC, including overexpression of BIRC3 in TSCCs. This was confirmed by qRT-PCR in the original and an independent validation set of tumors. In contrast to several other cancer cell lines, the BIRC3-positive TSCC cell line, 1889c showed spontaneous apoptosis after BIRC3 knock-down. Targeting apoptosis genes is worth testing as therapeutic principle in TSCC.

  2. IPEC-J2 MDR1, a Novel High-Resistance Cell Line with Functional Expression of Human P-glycoprotein (ABCB1) for Drug Screening Studies

    DEFF Research Database (Denmark)

    Saaby, Lasse; Helms, Hans Christian Cederberg; Brodin, Birger

    2016-01-01

    The P-glycoprotein (P-gp) efflux pump has been shown to affect drug distribution and absorption in various organs and to cause drug resistance in cancer therapy. The aim of this work was to develop a cell line to serve as a screening system for potential substrates of P-gp. This requires a cell...... line with high paracellular tightness, low expression of nonhuman ABC transporters, and high expression of functional human P-gp (ABCB1). The porcine intestinal epithelial cell line, IPEC-J2, was selected as a transfection host, due to its ability to form extremely high-resistance monolayers (>10,000 Ω......·cm(2)) and its low endogenous expression of ABC-type efflux transporters. The IPEC-J2 cells were transfected with a plasmid that contained the sequence of the human MDR1 gene, which encodes P-gp, followed by a selection of successfully transfected cells with geneticin and puromycin. The resulting cell...

  3. The Comparison of The Effects of Silybin and Silybin-Phosphatidylcholine on Viability and ESR Expression in Human Breast Cancer T47D Cell Line.

    Science.gov (United States)

    Mahmoodi, Narges; Motamed, Nasrin; Paylakhi, Seyed Hassan

    2014-01-01

    Silybin is a polyphenol with anti-oxidant and anti-cancer properties. The poor bioavailability of some polyphenols can be improved by binding to phosphatidylcholine. In recent years, studies have been conducted to evaluate the anti-cancer effect of silybin. We studied the effect of silybin and silybin-phosphatidylcholine on ESR1 and ESR2 gene expression and viability in the T47D breast cancer cell line. In this experimental study, a 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide test (MTT test) was used to determine doses for cell treatment, and the gene expression was analyzed by real-time reverse transcriptase-polymerase chain reaction (real-time RT- PCR). Significant dose- and time-dependent cell growth inhibitory effects of silybin and silybin-phosphatidylcholine along with ESR1 down-regulation were observed in T47D cells. In contrast to ESR1, the T47D cell line showed negligible ESR2 expression. This study suggests that silybin and silybin-phosphatidylcholine down-regulate ESR1 in ER(+)breast cancers. Results also show that in the T47D cell line, silybindown-regulation of ESR1 compared with silybin.

  4. Pitaya Extracts Induce Growth Inhibition and Proapoptotic Effects on Human Cell Lines of Breast Cancer via Downregulation of Estrogen Receptor Gene Expression

    Directory of Open Access Journals (Sweden)

    Deborah de Almeida Bauer Guimarães

    2017-01-01

    Full Text Available Breast cancer is one of the most prevalent cancers in the world and is also the leading cause of cancer death in women. The use of bioactive compounds of functional foods contributes to reduce the risk of chronic diseases, such as cancer and vascular disorders. In this study, we evaluated the antioxidant potential and the influence of pitaya extract (PE on cell viability, colony formation, cell cycle, apoptosis, and expression of BRCA1, BRCA2, PRAB, and Erα in breast cancer cell lines (MCF-7 and MDA-MB-435. PE showed high antioxidant activity and high values of anthocyanins (74.65 ± 2.18. We observed a selective decrease in cell proliferation caused by PE in MCF-7 (ER+ cell line. Cell cycle analysis revealed that PE induced an increase in G0/G1 phase followed by a decrease in G2/M phase. Also, PE induced apoptosis in MCF-7 (ER+ cell line and suppressed BRCA1, BRCA2, PRAB, and Erα gene expression. Finally, we also demonstrate that no effect was observed with MDA-MB-435 cells (ER− after PE treatment. Taken together, the present study suggests that pitaya may have a protective effect against breast cancer.

  5. Pitaya Extracts Induce Growth Inhibition and Proapoptotic Effects on Human Cell Lines of Breast Cancer via Downregulation of Estrogen Receptor Gene Expression.

    Science.gov (United States)

    Guimarães, Deborah de Almeida Bauer; De Castro, Danielle Dos Santos Bonfim; de Oliveira, Felipe Leite; Nogueira, Eduardo Matos; da Silva, Marco Antônio Mota; Teodoro, Anderson Junger

    2017-01-01

    Breast cancer is one of the most prevalent cancers in the world and is also the leading cause of cancer death in women. The use of bioactive compounds of functional foods contributes to reduce the risk of chronic diseases, such as cancer and vascular disorders. In this study, we evaluated the antioxidant potential and the influence of pitaya extract (PE) on cell viability, colony formation, cell cycle, apoptosis, and expression of BRCA 1 , BRCA 2 , PRAB, and Er α in breast cancer cell lines (MCF-7 and MDA-MB-435). PE showed high antioxidant activity and high values of anthocyanins (74.65 ± 2.18). We observed a selective decrease in cell proliferation caused by PE in MCF-7 (ER + ) cell line. Cell cycle analysis revealed that PE induced an increase in G 0 /G 1 phase followed by a decrease in G 2 /M phase. Also, PE induced apoptosis in MCF-7 (ER + ) cell line and suppressed BRCA 1 , BRCA 2 , PRAB, and Er α gene expression. Finally, we also demonstrate that no effect was observed with MDA-MB-435 cells (ER - ) after PE treatment. Taken together, the present study suggests that pitaya may have a protective effect against breast cancer.

  6. Characterization of prolactin-releasing peptide: Binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor

    Czech Academy of Sciences Publication Activity Database

    Maixnerová, Jana; Špolcová, Andrea; Pýchová, Miroslava; Blechová, Miroslava; Elbert, Tomáš; Řezáčová, M.; Železná, Blanka; Maletínská, Lenka

    2011-01-01

    Roč. 32, č. 4 (2011), s. 811-817 ISSN 0196-9781 R&D Projects: GA ČR GAP303/10/1368 Institutional research plan: CEZ:AV0Z40550506 Keywords : PrRP * pituitary cell lines * food intake Subject RIV: CC - Organic Chemistry Impact factor: 2.434, year: 2011

  7. Changes in MYCN expression in human neuroblastoma cell lines following cisplatin treatment may not be related to MYCN copy numbers

    Czech Academy of Sciences Publication Activity Database

    Procházka, Pavel; Hraběta, J.; Vícha, A.; Cipro, S.; Stejskalová, E.; Musil, Z.; Vodička, Pavel; Eckschlager, T.

    2013-01-01

    Roč. 29, č. 6 (2013), s. 2415-2421 ISSN 1021-335X Grant - others:GA ČR(CZ) GAP301/10/0356 Institutional support: RVO:68378041 Keywords : high-risk neuroblastoma cell line * multiplex ligation-dependent probe amplification * fluorescent in situ hybridization Subject RIV: CE - Biochemistry Impact factor: 2.191, year: 2013

  8. Quercetin inhibits expression of inflammatory cytokines through attenuation of NF-kappaB and p38 MAPK in HMC-1 human mast cell line.

    Science.gov (United States)

    Min, Y-D; Choi, C-H; Bark, H; Son, H-Y; Park, H-H; Lee, S; Park, J-W; Park, E-K; Shin, H-I; Kim, S-H

    2007-05-01

    Mast cell-mediated allergic inflammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce production of pro-inflammatory cytokines with immune regulatory properties. We investigated the effect of quercetin on the expression of pro-inflammatory cytokines in human mast cell line, HMC-1. HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (PMACI). Quercetin decreased the gene expression and production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8 in PMACI-stimulated HMC-1 cells. Quercetin attenuated PMACI-induced activation of NF-kappaB and p38 mitogen-activated protein kinase. Our study provides evidence that quercetin may suitable for the treatment of mast cell-derived allergic inflammatory diseases.

  9. Drug resistance in colorectal cancer cell lines is partially associated with aneuploidy status in light of profiling gene expression

    DEFF Research Database (Denmark)

    Guo, Jiao; Xu, Shaohang; Huang, Xuanlin

    2016-01-01

    A priority in solving the problem of drug resistance is to understand the molecular mechanism of how a drug induces the resistance response within cells. Because many cancer cells exhibit chromosome aneuploidy, we explored whether changes of aneuploidy status result in drug resistance. Two typical...... colorectal cancer cells, HCT116 and LoVo, were cultured with the chemotherapeutic drugs irinotecan (SN38) or oxaliplatin (QxPt), and the non- and drug-resistant cell lines were selected. Whole exome sequencing (WES) was employed to evaluate the aneuploidy status of these cells, and RNAseq and LC-MS/MS were...... the aneuploidy status in cancer cells, which was partially associated with the acquired drug resistance....

  10. Establishment of cell lines with rat spermatogonial stem cell characteristics

    NARCIS (Netherlands)

    van Pelt, Ans M. M.; Roepers-Gajadien, Hermien L.; Gademan, Iris S.; Creemers, Laura B.; de Rooij, Dirk G.; van Dissel-Emiliani, Federica M. F.

    2002-01-01

    Spermatogonial cell lines were established by transfecting a mixed population of purified rat A(s) (stem cells), A(pr) and A(al) spermatogonia with SV40 large T antigen. Two cell lines were characterized and found to express Hsp90alpha and oct-4, specific markers for germ cells and A spermatogonia,

  11. The cytotoxic effect of memantine and its effect on cytoskeletal proteins expression in metastatic breast cancer cell line

    Directory of Open Access Journals (Sweden)

    Sima Seifabadi

    2017-01-01

    Full Text Available Objective(s:Breast cancer is an important leading cause of death from cancer. Stathmin and tau proteins are regulators of cell motility, and their overexpression is associated with the progression and bad prognosis of breast cancer. Memantine, an N-methyl-D-aspartate (NMDA receptor antagonist, is the potential inhibitor of tau protein in neurons. This study determines the effect of memantine on breast cancer cell migration and proliferation, tau and stathmin gene expression in cancer cells and its synergistic effect with paclitaxel.   Materials and Methods: The cell proliferation was evaluated by MTT assay and for this purpose, MCF-7 breast cancer cells were treated with various concentration of memantine (2, 20 and 100 μg/ml. Tau and stathmin mRNA expression was evaluated through quantitative real time RT-PCR method. The migration of cancer cells treated with memantine for 24 hr was compared to non-treated cells using an in vitro transmembrane migration assay. Results: Incubation of breast cancer cells with memantine resulted in a dose dependent reduction in cell survival (P=0.0001. Paclitaxel (100 nM showed synergistic effect with memantine (P=0.0001. Memantine significantly decreased tau and stathmin mRNA expression (by RT-PCR, so that 100 µmol/l of memantine decreased tau and stathmin expression by 46% (P=0.0341 and 33% (P=0.043, respectively. Migration of cells was also decreased by memantine (P=0.0001. Conclusion: The presented data shows that memantine reduced mRNA levels of tau and stathmin proteins and also reduced cellular migration.

  12. Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

    Directory of Open Access Journals (Sweden)

    Ponti Donatella

    2011-03-01

    Full Text Available Abstract Background Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1 predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics. Materials and methods Cytotoxicity assays were performed on cells derived from fresh tumor explants of 18 human cases of malignant glioma. In addition to EGR-1, tumor cultures were investigated for genetic alterations and the expression of cancer regulating factors, related to the p53 pathway. Results We found that sensitivity to cisplatin correlates significantly with levels of EGR-1 expression in tumors with wild-type p53/INK4a/p16 status. Conclusion Increased knowledge of the mechanisms regulating EGR-1 expression in wild-type p53/INK4a/p16 cases of glioma may help in the design of new chemotherapeutic strategies for these tumors.

  13. The effects of a growth-inhibiting tripeptide, acetylGlu-Ser-GlyNH2 (Ac-ESG), on gene expression and cell cycle progression of two lymphoma cell lines.

    Science.gov (United States)

    Liu, Ying; Reichelt, Wenche H; Luna, Luisa; Elgjo, Kjell; Reichelt, Karl L

    2003-01-01

    We recently isolated and characterized a tripeptide, acetylGlu-Ser-GlyNH2 (Ac-ESG), which inhibits proliferation of lymphoid cells. In this paper we describe the effects of Ac-ESG on growth-related gene expression and cell cycle progression in two lymphoma cell lines, Ramos and Molt, representing B and T lymphocytes, respectively. RNA was extracted from Molt and Ramos cells with or without the tripeptide treatment. Gene expression was examined by semi-quantitative RT-PCR and Northern blot hybridization, and cell cycle progression was detected by flow cytometry. In the Molt cells, p53 gene expression was increased following treatment while c-myc was decreased after treatment shorter than 24 hours; N-ras expression was significantly reduced at picomolar concentration for 24-hour treatment; cyclinD1 and cdk4 expression did not show any change; DNA flow cytometry demonstrated that Molt cells were arrested or delayed predominantly in G2-M. Untreated Ramos cells had higher gene expression levels of c-myc, N-Ras and cdk4 than Molt cells, but lower p53 expression. These cells were not sensitive to Ac-ESG. The tripeptide Ac-ESG alters the expression of several growth-related genes in the Molt cell line and brings about an arrest or delay of cells in G2-M.

  14. Characterization of T cell receptor assembly and expression in a Ti gamma delta-positive cell line

    DEFF Research Database (Denmark)

    Kuhlmann, J; Caspar-Bauguil, S; Geisler, C

    1993-01-01

    - variants of the T cell Lyon were induced and found to produce all of the Ti/CD3 components, with the exception of Ti-delta. Biochemical analysis indicated that: (1) Ti-gamma/CD3 gamma, delta, epsilon complexes were formed in the endoplasmic reticulum in the absence of Ti-delta; (2) the CD3-zeta chain did...

  15. Elevated FOXC2 Expression Promotes Invasion of HCC Cell Lines and is Associated with Poor Prognosis in Hepatocellular Carcinoma.

    Science.gov (United States)

    Yang, Fang; Lv, Lizhi; Zhang, Kun; Cai, Qiucheng; Liu, Jianyong; Jiang, Yi

    2017-01-01

    Increasing evidence has indicated that Forkhead box protein C2 (FOXC2) plays an important role in carcinogenesis. However, the expression and the role of FOXC2 in hepatocellular carcinoma (HCC) have not been extensively studied. FOXC2 expression was analyzed by quantitative real-time polymerase chain reaction, Western blot analysis and immunohistochemistry in HCC tissue and cells. The relationship between FOXC2 expression and patient clinical significance and survival were assessed by Pearson's correlation and Kaplan-Meier analysis, respectively. Cell proliferation assays, colony formation assays, flow cytometric analysis and Transwell assays were employed to measure the effects of FOXC2 on HCC cells in vitro. The expression of FOXC2 was increased in HCC tissue, and high FOXC2 expression was associated with worse patient survival. Knockdown of FOXC2 inhibited HCC cell growth, migration, and invasion in vitro, as well as tumor growth. Furthermore, we found that activation of AKT-mediated MMP-2 and MMP-9 was involved in FOXC2 promoting an aggressive phenotype. Taken together, these findings demonstrate that FOXC2 is upregulated in HCC tissue and is associated with tumor size, vascular invasion and advanced TNM stage. Further investigation suggested that FOXC2 may play a vital role in promoting proliferation and invasion in HCC and serves as a novel therapeutic target in HCC. © 2017 The Author(s). Published by S. Karger AG, Basel.

  16. miR-101 suppresses HBV replication and expression by targeting FOXO1 in hepatoma carcinoma cell lines.

    Science.gov (United States)

    Wang, Yanjing; Tian, Hui

    2017-05-20

    microRNAs (miRNAs) have been identified to participate in the progression of cancers and in the infection of viruses. miR-101 expression has been found to be suppressed by HBV, however, the regulatory relationship between miR-101 and HBV replication remains elusive. In this report, miR-101 was significantly downregulated in HepG2.2.15 cells with HBV expression. miR-101 overexpression dramatically suppressed HBV replication and expression. Oppositely, overexpression of FOXO1 significantly promoted HBV replication and expression. Moreover, luciferase reporter analysis, qRT-PCR analysis and western blot assay confirmed that FOXO1 was a functional target of miR-101. Furthermore, restored FOXO1 expression abolished the inhibitory effect of miR-101 overexpression on HBV replication and expression in HepG2.2.15 cells. Our data suggested that miR-101 suppressed HBV replication and expression partially by targeting FOXO1, providing new insights into the molecular mechanisms of miR-101 in HBV-host interactions and a promising therapeutic target for HBV replication. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. [SIRT1 Influences the Sensitivity of A549 Non-small Cell Lung Cancer Cell Line to 
Cisplatin via Modulating the Noxa Expression].

    Science.gov (United States)

    Cao, Bin; He, Xiaofeng; Wang, Wengong; Shi, Minke

    2016-02-01

    The resistance of non-small cell lung cancer cells to cisplant is a common clinical phenomenon which could induce a poor therapeutic effect and should be difficult problem to be solved. SIRT1 and Noxa expression are associated with the chemotherapy for tumors. The present study focused on how SIRT1 expression influence the senstivity of non-small cell lung cancer cells and dissected the potential mechanism involved with Noxa. The difference of SIRT1 and Noxa expression between A549 cells and A549/DDP cells was detected by real-time quantitative PCR (qRT-PCR) and Western blot. SIRT1 targeted siRNA was uesed to inhibit the SIRT1 expression in A549/DDP, after transfection, Cell Titer Blue assay, flow cytometry were performed to analyze the cell viability, cell cycle and cell apoptosis in order to reveal the effect of inhibition of SIRT1 on sensitivity of A549/DDP cells to cisplant. Moreover, the expression changes of Noxa in A549/DDP cells after siRNA treatment were detected by qRT-PCR and Western blot. There was a significant difference in senstivity to cisplant between A549 and A549/DDP cells. Compared with A549 cells, the A549/DDP cells showed a higher SIRT1 expression and lower Noxa expression. After transfected with SIRT1 targeted siRNA, the cell viability decreased accompanied with a increasing apoptosis rate, meanwhile, higher percent of G2/M phase was detected after the 4 μg/mL cisplant treatment. Further more, inhibition of SIRT1 could induce the Noxa expression in A549/DDP cells. Higher SIRT1 expression may induce resistance to cisplant in A549 cells. SIRT1 inhibition may improve the sensitivity of A549/DDP cells to cisplantin though modulating the Noxa expression.
.

  18. Generation of a human immunodeficiency virus type 1 chronically infected monkey B cell line expressing low levels of endogenous TRIM5alpha.

    Science.gov (United States)

    Ridolfi, Barbara; Catone, Stefania; Sgarbanti, Marco; Sernicola, Leonardo; Battistini, Angela; Parolin, Cristina; Titti, Fausto; Borsetti, Alessandra

    2009-12-01

    Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5alpha has been shown to block human immunodeficiency virus type 1 (HIV-1) infection in several types of Old World monkey cells. Here we report a novel HIV-1 chronically infected monkey B cell line, F6/HIV-1, characterized by very low levels of TRIM5alpha expression that allows HIV-1 to overcome the restriction. Virus produced by F6/HIV-1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV-1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV-1 is the first monkey cell line chronically infected by HIV-1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV-1 pathogenesis.

  19. Higher Levels of c-Met Expression and Phosphorylation Identify Cell Lines With Increased Sensitivity to AMG-458, a Novel Selective c-Met Inhibitor With Radiosensitizing Effects

    International Nuclear Information System (INIS)

    Li Bo; Torossian, Artour; Sun, Yunguang; Du, Ruihong; Dicker, Adam P.; Lu Bo

    2012-01-01

    Purpose: c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. Methods and Materials: 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. Results: AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. Conclusions: AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor.

  20. Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

    Science.gov (United States)

    Hellweg, Christine E.; Baumstark-Khan, Christa; Horneck, Gerda

    2004-06-01

    Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

  1. Inflammatory genes are upregulated in expanded ataxin-3-expressing cell lines and spinocerebellar ataxia type 3 brains

    NARCIS (Netherlands)

    Evert, BO; Vogt, IR; Kindermann, C; Ozimek, L; de Vos, RAI; Brunt, ERP; Schmitt, [No Value; Klockgether, T; Wullner, U

    2001-01-01

    Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3. To study putative alterations of gene expression induced by expanded ataxin-3, we performed PCR-based cDNA subtractive hybridization in a cell culture

  2. Differences in Env and Gag protein expression patterns and epitope availability in feline immunodeficiency virus infected PBMC compared to infected and transfected feline model cell lines.

    Science.gov (United States)

    Roukaerts, Inge D M; Grant, Chris K; Theuns, Sebastiaan; Christiaens, Isaura; Acar, Delphine D; Van Bockstael, Sebastiaan; Desmarets, Lowiese M B; Nauwynck, Hans J

    2017-01-02

    Env and Gag are key components of the FIV virion that are targeted to the plasma membrane for virion assembly. They are both important stimulators and targets of anti-FIV immunity. To investigate and compare the expression pattern and antigenic changes of Gag and Env in various research models, infected PBMC (the natural FIV host cells) and GFox, and transfected CrFK were stained over time with various Env and Gag specific MAbs. In FIV infected GFox and PBMC, Env showed changes in epitope availability for antibody binding during processing and trafficking, which was not seen in transfected CrFK. Interestingly, epitopes exposed on intracellular Env and Env present on the plasma membrane of CrFK and GFox seem to be hidden on plasma membrane expressed Env of FIV infected PBMC. A kinetic follow up of Gag and Env expression showed a polarization of both Gag and Env expression to specific sites at the plasma membrane of PBMC, but not in other cell lines. In conclusion, mature trimeric cell surface expressed Env might be antigenically distinct from intracellular monomeric Env in PBMC and might possibly be unrecognizable by feline humoral immunity. In addition, Env expression is restricted to a small area on the plasma membrane and co-localizes with a large moiety of Gag, which may represent a preferred FIV budding site, or initiation of virological synapses with direct cell-to-cell virus transmission. Copyright © 2016. Published by Elsevier B.V.

  3. Effect of the standardized Cimicifuga foetida extract on Hsp 27 expression in the MCF-7 cell line

    OpenAIRE

    Maritza C Soler; Jessica L Molina; Hugo A Díaz; Vivian C Pinto; Yasenka L Barrios; Kan He; Marc Roller; Caroline R Weinstein-Oppenheimer

    2011-01-01

    Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose...

  4. Up regulation of K A I 1 gene expression and apoptosis effect of imatinib mesylate in gastric adenocarcinoma (AGS cell line

    Directory of Open Access Journals (Sweden)

    eyed Ataollah Sadat Shandiz

    2016-02-01

    Full Text Available Objective: To evaluate the effect of imatinib mesylate on KAI1 gene expression and apoptosis properties in human gastric carcinoma AGS cell line. Methods: Cell viability was assessed by MTT assay and quantitative real time PCR method was applied for investigation of Bax, Bcl-2, and KAI1 gene expression in AGS cells. The quantity of KAI1, Bax, and Bcl-2 compared to GAPDH gene expressions were examined using the formula 2-∆∆Ct. Furthermore, cell apoptosis/necrosis was carried out by annexin V/PI staining and quantified with flow cytometry after treatment with imatinib. Results: Imatinib mesylate was showed to have a dose-dependent toxicity effect against AGS cells. KAI1/GAPDH gene expression ratios were 1.07 ± 0.02 (P > 0.05, 1.68 ± 0.19 (P > 0.05, 3.60 ± 0.55 (P < 0.05, 6.54 ± 0.27 (P < 0.001 for 20, 50, 80 and 100 μmol/L of imatinib concentrations. The mRNA levels of Bax detected by real-time PCR after treatment with imatinib mesylate were significantly increased. Also, the number of apoptotic cells was increased from 3.72% (statistically significant; P < 0.05 in untreated AGS cells to 21.72%, 83.04% and 85.80%, respectively, following treatment with 20, 40, and 60 μmol/L imatinib mesylate. Conclusions: The results suggest that imatinib mesylate can induce apoptosis pathway in a dose-dependent mode and might modulate metastasis by up regulating KAI1 gene expression in human gastric carcinoma AGS cell line.

  5. Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line

    DEFF Research Database (Denmark)

    Nielsen, C U; Amstrup, J; Steffansen, B

    2001-01-01

    The human intestinal cell line Caco-2 was used as a model system to study the effects of epidermal growth factor (EGF) on peptide transport. EGF decreased apical-to-basolateral fluxes of [(14)C]glycylsarcosine ([(14)C]Gly-Sar) up to 50.2 +/- 3.6% (n = 6) of control values. Kinetic analysis......(max) decreased from 2.61 +/- 0.4 to 1.06 +/- 0.1 nmol x cm(-2) x min(-1) (n = 3, P T1 mRNA (using glucose-6-phosphate dehydrogenase mRNA as control......) in cells treated with EGF. Western blotting indicated a decrease in hPepT1 protein in cell lysates. We conclude that EGF treatment decreases Gly-Sar transport in Caco-2 cells by decreasing the number of peptide transporter molecules in the apical membrane....

  6. Pitaya Extracts Induce Growth Inhibition and Proapoptotic Effects on Human Cell Lines of Breast Cancer via Downregulation of Estrogen Receptor Gene Expression

    OpenAIRE

    Guimar?es, Deborah de Almeida Bauer; De Castro, Danielle dos Santos Bonfim; de Oliveira, Felipe Leite; Nogueira, Eduardo Matos; da Silva, Marco Ant?nio Mota; Teodoro, Anderson Junger

    2017-01-01

    Breast cancer is one of the most prevalent cancers in the world and is also the leading cause of cancer death in women. The use of bioactive compounds of functional foods contributes to reduce the risk of chronic diseases, such as cancer and vascular disorders. In this study, we evaluated the antioxidant potential and the influence of pitaya extract (PE) on cell viability, colony formation, cell cycle, apoptosis, and expression of BRCA1, BRCA2, PRAB, and Erα in breast cancer cell lines (MCF-7...

  7. Piceatannol promotes apoptosis via up-regulation of microRNA-129 expression in colorectal cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Haogang [Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081 (China); Jia, Ruichun [Department of Blood Transfusion, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081 (China); Wang, Chunjing; Hu, Tianming [Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081 (China); Wang, Fujing, E-mail: wangfujing-hyd@163.com [Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081 (China)

    2014-09-26

    Highlights: • Piceatannol induces apoptosis in cultured CRC cells. • Piceatannol promotes expression of miR-129. • miR-129 mediates proapoptotic effects of piceatannol. - Abstract: Piceatannol, a naturally occurring analog of resveratrol, has been confirmed as an antitumor agent by inhibiting proliferation, migration, and metastasis in diverse cancer. However, the effect and mechanisms of piceatannol on colorectal cancer (CRC) have not been well understood. This study aimed to test whether piceatannol could inhibit growth of CRC cells and reveal its underlying molecular mechanism. MTT assay was used to detect the cell viability in HCT116 and HT29 cells. Flow cytometry analysis was employed to measure apoptosis of CRC cells. Bcl-2, Bax and caspase-3 levels were analyzed by Western blot and miR-129 levels were determined by real-time RT-PCR. Our study showed that piceatannol inhibited HCT116 and HT29 cells growth in a concentration- and time-dependent manner. Piceatannol induced apoptosis by promoting expression of miR-129, and then inhibiting expression of Bcl-2, an known target for miR-129. Moreover, knock down of miR-129 could reverse the reduction of cell viability induced by piceatannol in HCT116 and HT29 cells. Taken together, our study unraveled the ability of piceatannol to suppress colorectal cancer growth and elucidated the participation of miR-129 in the anti-cancer action of piceatannol. Our findings suggest that piceatannol can be considered to be a promising anticancer agent for CRC.

  8. Human Vav1 expression in hematopoietic and cancer cell lines is regulated by c-Myb and by CpG methylation.

    Directory of Open Access Journals (Sweden)

    Lena Ilan

    Full Text Available Vav1 is a signal transducer protein that functions as a guanine nucleotide exchange factor for the Rho/Rac GTPases in the hematopoietic system where it is exclusively expressed. Recently, Vav1 was shown to be involved in several human malignancies including neuroblastoma, lung cancer, and pancreatic ductal adenocarcinoma (PDA. Although some factors that affect vav1 expression are known, neither the physiological nor pathological regulation of vav1 expression is completely understood. We demonstrate herein that mutations in putative transcription factor binding sites at the vav1 promoter affect its transcription in cells of different histological origin. Among these sites is a consensus site for c-Myb, a hematopoietic-specific transcription factor that is also found in Vav1-expressing lung cancer cell lines. Depletion of c-Myb using siRNA led to a dramatic reduction in vav1 expression in these cells. Consistent with this, co-transfection of c-Myb activated transcription of a vav1 promoter-luciferase reporter gene construct in lung cancer cells devoid of Vav1 expression. Together, these results indicate that c-Myb is involved in vav1 expression in lung cancer cells. We also explored the methylation status of the vav1 promoter. Bisulfite sequencing revealed that the vav1 promoter was completely unmethylated in human lymphocytes, but methylated to various degrees in tissues that do not normally express vav1. The vav1 promoter does not contain CpG islands in proximity to the transcription start site; however, we demonstrated that methylation of a CpG dinucleotide at a consensus Sp1 binding site in the vav1 promoter interferes with protein binding in vitro. Our data identify two regulatory mechanisms for vav1 expression: binding of c-Myb and CpG methylation of 5' regulatory sequences. Mutation of other putative transcription factor binding sites suggests that additional factors regulate vav1 expression as well.

  9. Organic anion transporter 2 transcript variant 1 shows broad ligand selectivity when expressed in multiple cell lines.

    Science.gov (United States)

    Hotchkiss, Adam G; Berrigan, Liam; Pelis, Ryan M

    2015-01-01

    Organic anion transporter 2 (OAT2) is likely important for renal and hepatic drug elimination. Three variants of the OAT2 peptide sequence have been described - OAT2 transcript variant 1 (OAT2-tv1), OAT2 transcript variant 2 (OAT2-tv2), and OAT2 transcript variant 3 (OAT2-tv3). Early studies helping to define the ligand selectivity of OAT2 failed to identify the variant used, and the studies used several heterologous expression systems. In preliminary studies using OAT2-tv1, we failed to observe transport of several previously identified substrates, leading us to speculate that ligand selectivity of OAT2 differs with variant and/or heterologous expression system. The purpose was to further investigate the ligand selectivity of the OAT2 variants expressed in multiple cell types. We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney. OAT2-tv1 and OAT2-tv2 were individually expressed in human embryonic kidney (HEK), Madin-Darby canine kidney (MDCK), or Chinese hamster ovary (CHO) cells. mRNA for OAT2-tv1 and OAT2-tv2 was demonstrated in each cell type transfected with the respective construct, indicating their expression. OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not. OAT2-tv1 transported penciclovir in all three cell types, but failed to transport para-aminohippurate, succinate, glutarate, estrone-3-sulfate, paclitaxel or dehydroepiandrosterone sulfate - previously identified substrates of OAT2-tv2. Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir. Penciclovir transport by OAT2-tv1 was sensitive to large (e.g., cyclosporine A) and small (e.g., allopurinol) organic compounds, as well as organic anions, cations and neutral compounds, highlighting the multiselectivity of OAT2-tv1. The potencies with which indomethacin, furosemide, cyclosporine A and cimetidine inhibited OAT2-tv1

  10. Ultraviolet C Irradiation Induces Different Expression of Cyclooxygenase 2 in NIH 3T3 Cells and A431 Cells: The Roles of COX-2 Are Different in Various Cell Lines

    Directory of Open Access Journals (Sweden)

    Ming-Hsiu Wu

    2012-04-01

    Full Text Available Ultraviolet C (UVC is a DNA damage inducer, and 20 J/m2 of UVC irradiation caused cell growth inhibition and induced cell death after exposure for 24–36 h. The growth of NIH 3T3 cells was significantly suppressed at 24 h after UVC irradiation whereas the proliferation of A431 cells was inhibited until 36 h after UVC irradiation. UVC irradiation increased COX-2 expression and such up-regulation reached a maximum during 3–6 h in NIH 3T3 cells. In contrast, UVC-induced COX-2 reached a maximum after 24–36 h in A431 cells. Measuring prostaglandin E2 (PGE2 level showed a biphasic profile that PGE2 release was rapidly elevated in 1–12 h after UVC irradiation and increased again at 24 h in both cell lines. Treatment with the selective COX-2 inhibitor, SC-791, during maximum expression of COX-2 induction, attenuated the UVC induced-growth inhibition in NIH 3T3 cells. In contrast, SC-791 treatment after UVC irradiation enhanced death of A431 cells. These data showed that the patterns of UVC-induced PGE2 secretion from NIH 3T3 cells and A431 cells were similar despite the differential profile in UVC-induced COX-2 up-regulation. Besides, COX-2 might play different roles in cellular response to UVC irradiation in various cell lines.

  11. ATR/TEM8 is highly expressed in epithelial cells lining Bacillus anthracis' three sites of entry: implications for the pathogenesis of anthrax infection.

    Science.gov (United States)

    Bonuccelli, Gloria; Sotgia, Federica; Frank, Philippe G; Williams, Terence M; de Almeida, Cecilia J; Tanowitz, Herbert B; Scherer, Philipp E; Hotchkiss, Kylie A; Terman, Bruce I; Rollman, Brent; Alileche, Abdelkrim; Brojatsch, Jürgen; Lisanti, Michael P

    2005-06-01

    Anthrax is a disease caused by infection with spores from the bacteria Bacillus anthracis. These spores enter the body, where they germinate into bacteria and secrete a tripartite toxin that causes local edema and, in systemic infections, death. Recent studies identified the cellular receptor for anthrax toxin (ATR), a type I membrane protein. ATR is one of the splice variants of the tumor endothelial marker 8 (TEM8) gene. ATR and TEM8 are identical throughout their extracellular and transmembrane sequence, and both proteins function as receptors for the toxin. ATR/TEM8 function and expression have been associated with development of the vascular system and with tumor angiogenesis. TEM8 is selectively upregulated in endothelial cells during blood vessel formation and tumorigenesis. However, selective expression of TEM8 in endothelial cells contradicts the presumably ubiquitous expression of the receptor. To resolve this controversial issue, we evaluated the distribution of ATR/TEM8 in a variety of tissues. For this purpose, we generated and characterized a novel anti-ATR/TEM8 polyclonal antibody. Here, we show that this novel antibody recognizes all three ATR/TEM8 isoforms, which are widely and differentially expressed in various tissue types. We found that ATR/TEM8 expression is not only associated with tumor endothelial cells, as previously described. Indeed, ATR/TEM8 is highly and selectively expressed in the epithelial cells lining those organs that constitute the anthrax toxin's sites of entry, i.e., the lung, the skin, and the intestine. In fact, we show that ATR/TEM8 is highly expressed in the respiratory epithelium of the bronchi of the lung and is particularly abundant in the ciliated epithelial cells coating the bronchi. Furthermore, immunostaining of skin biopsies revealed that ATR/TEM8 is highly expressed in the keratinocytes of the epidermis. Finally, we show that the epithelial cells lining the small intestine strongly express ATR/TEM8 isoforms. This

  12. Modulation of cell cycle and gene expression in pancreatic tumor cell lines by methionine deprivation (methionine stress): implications to the therapy of pancreatic adenocarcinoma.

    Science.gov (United States)

    Kokkinakis, Demetrius M; Liu, Xiaoyan; Neuner, Russell D

    2005-09-01

    The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

  13. Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 cells, infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants.

    Science.gov (United States)

    Cheng, Xin-Hua; Hillman, Christopher C; Zhang, Chuan-Xi; Cheng, Xiao-Wen

    2013-01-01

    During cell infection, the fp25k gene of baculoviruses frequently mutates, producing the few polyhedra (FP) per cell phenotype with reduced polyhedrin (polh) expression levels compared with wild-type baculoviruses. Here we report that the fp25k gene of the model baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), contains two hypermutable seven-adenine (A7) mononucleotide repeats (MNRs) that were mutated to A8 MNRs and a TTAA site that had host DNA insertions, producing fp25k mutants during Sf21 cell infection. The FP phenotype in Sf9 and Hi5 cells was more pronounced than in Sf21 cells. AcMNPV fp25k mutants produced similar levels of polyhedra or enhanced GFP, which were both under the control of the AcMNPV polh promoter for expression, in Sf21 cells but lower levels in Sf9 and Hi5 cells compared with AcMNPV with an intact fp25k gene. This correlated with the polh mRNA levels detected in each cell line. The majority of Sf21 cells infected with fp25 mutants showed high polh promoter-mediated GFP expression levels. Two cell lines subcloned from Sf21 cells that were infected with fp25k mutants showed different GFP expression levels. Furthermore, a small proportion of Hi5 cells infected with fp25k mutants showed higher production of polyhedra and GFP expression than the rest, and the latter was not correlated with increased m.o.i. Therefore, these data suggest that AcMNPV polh promoter-mediated gene expression activities differ in the three cell lines and are influenced by different cells within the cell line.

  14. 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.)

  15. Differential patterns of human cytomegalovirus gene expression in various T-cell lines carrying human T-cell leukemia-lymphoma virus type I: role of Tax-activated cellular transcription factors.

    Science.gov (United States)

    Beck, Zoltán; Bácsi, Attila; Liu, Xiangdong; Ebbesen, Peter; Andirkó, István; Csoma, Eszter; Kónya, József; Nagy, Etelka; Tóth, Ferenc D

    2003-09-01

    Replication of human cytomegalovirus (HCMV) was investigated in various T-cell lines expressing the tax gene product of human T-cell leukemia-lymphoma virus type I (HTLV-I). Differential patterns of HCMV replication were found in HTLV-I-carrying cell lines. HCMV gene expression was restricted to the immediate-early genes in MT-2 and MT-4 cells, whereas full replication cycle of the virus was observed in C8166-45 cells. Productive HCMV infection induced a cytopathic effect resulting in the lysis of infected cells. The results of electrophoretic mobility shift assay (EMSA) showed high levels of NF-kappaB-, CREB/ATF-1-, and SRF-specific DNA binding activity in all Tax-positive cell lines. In contrast, SP1 activity could be detected only in C8166-45 cells. Using an inducible system (Jurkat cell line JPX-9), a dramatic increase in NF-kappaB, CREB/ATF-1, SRF, and SP1 binding activity, as well as productive HCMV infection, were observed upon Tax expression. Overexpression of SP1 in MT-2 and MT-4 cells converted HCMV infection from an abortive to a productive one. These data suggest that the stimulatory effect of Tax protein on HCMV in T cells is accomplished through at least five host-related transcription factor pathways. The results of this study provide possible mechanisms whereby HCMV infections might imply suppression of adult T-cell leukemia. Copyright 2003 Wiley-Liss, Inc.

  16. [Selection and construction of cell line stably expressing survivin gene in lower level through eukaryotic plasmid vector of shRNA].

    Science.gov (United States)

    Wang, Wen-Xia; Sun, Shan-Zhen; Song, Ying

    2008-06-01

    To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly. Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test. Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01). The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).

  17. PAMPs and DAMPs stimulate the expression of pro-inflammatory cytokines in vitro in a fibroblast cell-line from rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Ingerslev, Hans-Christian; Ossum, C.G.; Nielsen, Michael Engelbrecht

    that this expression to a large extend is mediated by fibroblasts in the musculature. To investigate this, a fibroblast cell-line (RTHDF1) from the rainbow trout was stimulated with either LPS from E. coli, cell debris or supernatant from sonicated fibroblasts. Whereas LPS stimulation resulted in a significant up...... in this evolutionary lineage of the bony fishes. The expression of TLR-3 and -9 receptors were significantly up-regulated following physical damage of muscle tissue as well as in stimulated fibroblasts, where LPS induced both TLR-3 and -9, supernatant from sonicated cells only TLR-9 while debris caused no induction....... The present study reinforce the idea that fibroblasts are able to react to PAMPs and DAMPs and that non-immune cell-types play an important role in the inflammatory reaction per se. From an evolutionary perspective the facilitation of an inflammatory response through recognition of PAMPs and DAMPs by non...

  18. Epigenetic mechanisms involved in differential MDR1 mRNA expression between gastric and colon cancer cell lines and rationales for clinical chemotherapy

    Directory of Open Access Journals (Sweden)

    Kim Kyung-Jong

    2008-08-01

    Full Text Available Abstract Background The membrane transporters such as P-glycoprotein (Pgp, the MDR1 gene product, are one of causes of treatment failure in cancer patients. In this study, the epigenetic mechanisms involved in differential MDR1 mRNA expression were compared between 10 gastric and 9 colon cancer cell lines. Methods The MDR1 mRNA levels were determined using PCR and real-time PCR assays after reverse transcription. Cytotoxicity was performed using the MTT assay. Methylation status was explored by quantification PCR-based methylation and bisulfite DNA sequencing analyses. Results The MDR1 mRNA levels obtained by 35 cycles of RT-PCR in gastric cancer cells were just comparable to those obtained by 22 cycles of RT-PCR in colon cancer cells. Real-time RT-PCR analysis revealed that MDR1 mRNA was not detected in the 10 gastric cancer cell lines but variable MDR1 mRNA levels in 7 of 9 colon cancer cell lines except the SNU-C5 and HT-29 cells. MTT assay showed that Pgp inhibitors such as cyclosporine A, verapamil and PSC833 sensitized Colo320HSR (colon, highest MDR1 expression but not SNU-668 (gastric, highest and SNU-C5 (gastric, no expression to paclitaxel. Quantification PCR-based methylation analysis revealed that 90% of gastric cancer cells, and 33% of colon cancer cells were methylated, which were completely matched with the results obtained by bisulfite DNA sequencing analysis. 5-aza-2'-deoxcytidine (5AC, a DNA methyltransferase inhibitor increased the MDR1 mRNA levels in 60% of gastric cells, and in 11% of colon cancer cells. Trichostatin A (TSA, histone deacetylase inhibitor increased the MDR1 mRNA levels in 70% of gastric cancer cells and 55% of colon cancer cells. The combined treatment of 5AC with TSA increased the MDR1 mRNA levels additively in 20% of gastric cancer cells, but synergistically in 40% of gastric and 11% of colon cancer cells. Conclusion These results indicate that the MDR1 mRNA levels in gastric cancer cells are significantly

  19. Expression of surface markers on the human monocytic leukaemia cell line, THP-1, as indicators for the sensitizing potential of chemicals.

    Science.gov (United States)

    An, Susun; Kim, Seoyoung; Huh, Yong; Lee, Tae Ryong; Kim, Han-Kon; Park, Kui-Lea; Eun, Hee Chul

    2009-04-01

    Evaluation of skin sensitization potential is an important part of the safety assessment of cosmetic ingredients and topical drugs. Recently, evaluation of changes in surface marker expression induced in dendritic cells (DC) or DC surrogate cell lines following exposure to chemicals represents one approach for in vitro test methods. The study aimed to test the change of expression patterns of surface markers on THP-1 cells by chemicals as a predictive in vitro method for contact sensitization. We investigated the expression of CD54, CD86, CD83, CD80, and CD40 after a 1-day exposure to sensitizers (1-chloro-2,4-dinitrobenzene; 2,4-dinitrofluorobenzene; benzocaine; 5-chloro-2-methyl-4-isothiazolin-3-one; hexyl cinnamic aldehyde; eugenol; nickel sulfate hexahydrate; potassium dichromate; cobalt sulfate; 2-mercaptobenzothiazole; and ammonium tetrachloroplatinate) and non-sensitizers (sodium lauryl sulfate, benzalkonium chloride, lactic acid, salicylic acid, isopropanol, and dimethyl sulphoxide). The test concentrations were 0.1x, 0.5x, and 1x of the 50% inhibitory concentration, and the relative fluorescence intensity was used as an expression indicator. By evaluating the expression patterns of CD54, CD86, and CD40, we could classify the chemicals as sensitizers or non-sensitizers, but CD80 and CD83 showed non-specific patterns of expression. These data suggest that the THP-1 cells are good model for screening contact sensitizers and CD40 could be a useful marker complementary to CD54 and CD86.

  20. Esculetin Downregulates the Expression of AML1-ETO and C-Kit in Kasumi-1 Cell Line by Decreasing Half-Life of mRNA

    Directory of Open Access Journals (Sweden)

    Sharad Sawney

    2015-01-01

    Full Text Available One of the most frequent genetic aberrations in acute myeloid leukemia (AML is chromosomal translocation between AML1/RUNX1 on chromosome 21 and ETO gene on chromosome 8 resulting in the expression of chimeric oncogene AML1-ETO. Although patients with t(8;21 translocation have good prognosis, 5-year survival is observed only in 50% of the cases. AML1-ETO translocation is usually accompanied by overexpression of mutant C-Kit, a tyrosine kinase, which contributes to uncontrolled proliferation of premature blood cells leading to relapse and poor prognosis. We illustrate the potential use of esculetin on leukemic cell line, Kasumi-1, bearing t(8;21 translocation and mutated C-Kit gene. Esculetin decreases the expression of AML1-ETO at both protein and transcript level within 24 hours of treatment. Half-life of AML1-ETO mRNA was reduced from 7 hours to 1.5 hours. Similarly half-life of C-Kit mRNA was reduced to 2 hours from 5 hours in esculetin treated cells. Esculetin also perturbed the expression of ectopically expressed AML1-ETO in U937 cells. The decreased expression of AML1-ETO chimeric gene was associated with increased expression of LAT1 and RUNX3 genes, targets of AML1. We envisage that discovery of a drug candidate which could target both these mutated genes would be a considerable breakthrough for future application.

  1. Effect of Sodium Arsenite on the Expression of Antioxidant Genes (SOD2andCAT) in MCF-7 and Jurkat Cell Lines.

    Science.gov (United States)

    Fallahzadeh-Abarghooei, Leila; Samadaei-Ghadikolaie, Maryam; Saadat, Iraj; Saadat, Mostafa

    2017-02-01

    Sodium arsenite (NaAsO2) has potent cytotoxic activity in human cancer cells. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. The purpose of the present study was to evaluate the alteration of mRNA levels of catalase ( CAT ) and superoxide dismutase 2 ( SOD2 ) in MCF-7 and Jurkat cells after exposure to NaAsO 2 . Methylthiazol tetrazolium (MTT) viability assay was performed to evaluate cytotoxicity of NaAsO 2 in MCF-7 and Jurkat cells. For evaluating the expression levels of the CAT and SOD2 , we used two concentrations of NaAsO 2 (5 and 15 μM), lower than the concentrations at which 50% of cell viability were lost. The cells were treated with co-treatment of NaAsO 2 (15 μM) and N-acetyl-cysteine (NAC; 5 μM) in the media for 24 h. The control cells were maintained in sodium arsenite free growth medium. The experiments were done triplicate. Using quantitative real-time PCR, the expression levels of CAT and SOD2 were quantified. One sample student's t test was performed for comparisons of mRNA levels between treatment groups and their corresponding untreated control cells. CAT mRNA level decreased significantly in both cell lines following exposure to NaAsO 2 ( P Jurkat cells and increased in MCF-7 cells after treatment with NaAsO 2 ( P <0.05). After cells exposure to NaAsO 2 , CAT mRNA level decreased in both examined cell lines but the alterations of SOD2 mRNA level is cell specific. The NAC modulated the NaAsO 2 associated alterations of CAT and SOD2 mRNA levels, therefore, the NaAsO 2 might act through inducing reactive oxygen species.

  2. CLO : The cell line ontology

    NARCIS (Netherlands)

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

    2014-01-01

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

  3. Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

    Directory of Open Access Journals (Sweden)

    Masayuki Takeyama

    2015-01-01

    Full Text Available BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A and in vitro angiogen-esis in retinal pigment epithelium (RPE. RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF. We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

  4. Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line

    International Nuclear Information System (INIS)

    Jeang, K.T.; Cho, M.S.; Hayward, G.S.

    1984-01-01

    A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The authors isolated a clonal Ltk/sup +/ cell line which expressed the /sup 35/methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH/sub 2/p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH/sub 2/l198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors

  5. Oestrogen-mediated regulation of somatostatin receptor expression in human breast cancer cell lines assessed with {sup 99m}Tc-depreotide

    Energy Technology Data Exchange (ETDEWEB)

    Van Den Bossche, B.; D' haeninck, E.; De Vos, F.; Dierckx, R.A.; Wiele, C. van de [Division of Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, 9000B, Ghent (Belgium); Belle, S. van [Department of Medical Oncology, Ghent University Hospital, Ghent (Belgium); Bracke, M. [Laboratory of Experimental Cancerology, Ghent University Hospital, Ghent (Belgium)

    2004-07-01

    Investigating three somatostatin receptor (SSTR)-positive (+) human breast cancer cell lines, Xu et al. found a time- and dose-dependent up- or down-regulation of SSTR2 mRNA expression by 17{beta}-oestradiol (E{sub 2}) or the anti-oestrogen tamoxifen, respectively, in the two oestrogen receptor-positive (ER+) cell lines but not in the oestrogen receptor-negative (ER-) cell line. This study aimed to confirm the findings of Xu et al. at the protein level by means of western blotting and saturation binding studies using {sup 99m}Tc-depreotide (NeoSpect). The ER+/SSTR+ ZR75-1 and T47D and SSTR+/ER- MDA MB231 breast cancer cell lines were exposed to 1 nM E{sub 2} or a combination of 1 nM E{sub 2} plus 100 nM tamoxifen or ICI 182 780 (Faslodex) for 48 h. Exposed and non-exposed controls were incubated with increasing concentrations of {sup 99m}Tc-depreotide (0.5 nM-15 nM) in the absence and the presence of 20 {mu}M of octreotide. Scatchard-Rosenthal plots were derived using commercially available software. SSTR subtypes responsible for E{sub 2}-induced changes in {sup 99m}Tc-depreotide binding were identified by means of western blotting. Mean K{sub d} values for {sup 99m}Tc-depreotide were 13 nM, 7 nM and 4 nM for T47D, ZR75-1 and MDA MB231 cells, respectively. After stimulation with E{sub 2}, the ER+ cell line T47D demonstrated a mean increase of 81% (P<0.05) in {sup 99m}Tc-depreotide binding. Adding the partial agonist tamoxifen and full antagonist ICI 182 780 to E{sub 2} blocked the induced increase in T47D cells, either reducing SSTR expression or restoring it to control levels. ZR75-1 cells stimulated with E{sub 2} showed a mean decrease in {sup 99m}Tc-depreotide binding of 36% as compared to control cells; this difference, however, proved to be not statistically significant. Similarly, B{sub max} values did not change in ZR75-1 cells exposed to E{sub 2} in combination with an ER antagonist as compared to control cells. Finally, no influence of E{sub 2} on {sup 99m

  6. Comparing the Effect of Silybin and Silybin Advanced™ on Viability and HER2 Expression on the Human Breast Cancer SKBR3 Cell Line by no Serum Starvation.

    Science.gov (United States)

    Mahmoodi, Narges; Motamed, Nasrin; Paylakhi, Seyed Hassan; O Mahmoodi, Nosrat

    2015-01-01

    The polyphenol silybin has anti-oxidant and anti-cancer properties. The poor bioavailability of some polyphenols (flavonoids, and terpenoids) can be improved by binding them to phosphatidylcholine (phytosome technology). Many studies have focused on the most common phytosome, silybin-phosphatidylcholine, particularly for its hepatoprotective effects. However, in recent years, studies have also been conducted to determine its anti-cancer effect. Considering that the serum starvation should not be used for studies that are not focused on cell cycle arrest, we studied the effect of silybin-phosphatidylcholine from Silybin Advanced™ in 1:2 ratio (one part silybin bound to two parts phosphatidylcholine) on HER2 gene expression on the SKBR3 breast cancer cell line which were cultured in complete medium (not serum deprivation). The results were compared with our previous study of silybin on HER2 expression on SKBR3 cells. An MTT test was used to determine concentrations for cell treatment, and the gene expression was defined by real-time RT-PCR. Outcomes showed significant concentration- and time-dependent cell growth inhibitory effects of silybin, and silybin-phosphatidylcholine and HER2 down regulation on SKBR3 cells. Silybin-phosphatidylcholine concentrations had a much larger inhibitory and HER2 down regulate effect on cell growth than the same silybin concentrations on SKBR3 cells.

  7. BHD Tumor Cell Line and Renal Cell Carcinoma Line | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    Scientists at the National Cancer Institute  have developed a novel renal cell carcinoma (RCC) cell line designated UOK257, which was derived from the surgical kidney tissue of a patient with hereditary Birt-Hogg-Dube''''(BHD) syndrome and companion cell line UOK257-2 in which FLCN expression has been restored by lentivirus infection. The NCI Urologic Oncology Branch seeks parties interested in licensing or collaborative research to co-develop, evaluate, or commercialize kidney cancer tumor cell lines.

  8. DIF-1 inhibits the Wnt/β-catenin signaling pathway by inhibiting TCF7L2 expression in colon cancer cell lines.

    Science.gov (United States)

    Jingushi, Kentaro; Takahashi-Yanaga, Fumi; Yoshihara, Tatsuya; Shiraishi, Fumie; Watanabe, Yutaka; Hirata, Masato; Morimoto, Sachio; Sasaguri, Toshiyuki

    2012-01-01

    We previously reported that differentiation-inducing factor-1 (DIF-1), a morphogen in Dictyostelium discoideum, inhibits the proliferation of human cancer cell lines by inducing β-catenin degradation and suppressing the Wnt/β-catenin signaling pathway. To determine whether β-catenin degradation is essential for the effect of DIF-1, we examined the effect of DIF-1 on human colon cancer cell lines (HCT-116, SW-620 and DLD-1), in which the Wnt/β-catenin signaling pathway is constitutively active. DIF-1 strongly inhibited cell proliferation and arrested the cell cycle in the G(0)/G(1) phase via the suppression of cyclin D1 expression at mRNA and protein levels without reducing β-catenin protein. TCF-dependent transcriptional activity and cyclin D1 promoter activity were revealed to be inhibited via suppression of transcription factor 7-like 2 (TCF7L2) expression. Luciferase reporter assays and EMSAs using the TCF7L2 promoter fragments indicated that the binding site for the transcription factor early growth response-1 (Egr-1), which is located in the -609 to -601 bp region relative to the start codon in the TCF7L2 promoter, was involved in DIF-1 activity. Moreover, RNAi-mediated depletion of endogenous TCF7L2 resulted in reduced cyclin D1 promoter activity and protein expression, and the overexpression of TCF7L2 overrode the inhibition of the TCF-dependent transcriptional activity and cyclin D1 promoter activity induced by DIF-1. Therefore, DIF-1 seemed to inhibit the Wnt/β-catenin signaling pathway by suppressing TCF7L2 expression via reduced Egr-1-dependent transcriptional activity in these colon cancer cell lines. Our results provide a novel insight into the mechanisms by which DIF-1 inhibits the Wnt/β-catenin signaling pathway. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. The studies of DNA double-strand break (DSB) rejoining and mRNA expression of repair gene XRCCs in malignant transformed cell lines of human bronchial epithelial cells generated by α-particles

    International Nuclear Information System (INIS)

    Sun Jingfen; Sui Jianli; Geng Yu; Zhou Pingkun; Wu Dechang

    2002-01-01

    Objective: To investigate the efficiency of γ-ray-induced DNA DSB rejoining and the mRNA expression of DNA repair genes in malignantly transformed cell lines of human bronchial epithelial cells generated by exposure to a-particles. Methods: Pulsed field gel electrophoresis (PFGE) was used to detect DNA. DSBs mRNA expression was analyzed by RT-PCR. Results: The residual DNA DSB damage level after 4hrs repair following 0-150 Gy of γ-irradiation in the malignantly transformed cell lines BERP35T-1 and BERP35T-4 was significantly higher than that in their parental BEP2D cells. The analysis of mRNA level revealed a 2.5-to 6.5-fold down-regulated expression of the DNA repair genes XRCC-2, XRCC-3 and Ku80 (XRCC-5) in BERP35T-1 and BERP35T-4