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Sample records for cell line k562

  1. Anti-Proliferative and Apoptotic Effects of Beta-Ionone in Human Leukemia Cell Line K562

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    Zohreh Faezizadeh

    2016-06-01

    Full Text Available Background Beta-ionone is an aroma compound found in the Rosaceae family. Some evidence supported that beta-ionone has a great potential for cancer prevention. To date, the anti-proliferative and apoptotic effects of beta-ionone in human leukemia cell line K562 were not studied. Objectives Hence, we investigated whether beta-ionone could inhibit cell growth and induce apoptosis in the K562 cells. Materials and Methods In this experimental study, human leukemia cell line K562 was cultured and anti-proliferation effect of beta-ionone with different doses (25 - 400 µm at different times (24 - 96 hours on treated cells was evaluated by the MTT assay. To determine apoptosis rate, the Hoechst 33342 staining and flow cytometry was performed. Results The MTT assay showed that beta-ionone inhibited proliferation of K562 cells in a dose-dependent manner significantly (P = 0.0008. Moreover, the increased apoptotic rate was found after incubation of K562 cells with 200 µm beta-ionone. The Hoechst staining and flow cytometry analysis indicated that beta-ionone could increase apoptosis of K562 cells in a dose-dependent manner. Conclusions The results demonstrated that beta-ionone has anti-proliferative and apoptotic effects on K562 cells, and in the future may be used in the treatment of some leukemia sub-types.

  2. The effect of β-ionone on telomerase activity in the human leukemia cell line K562

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    Zohreh Faezizadeh

    2015-06-01

    Full Text Available Background: Telomerase is highly activated in most human cancer cells, therefore, its inhibition has been proposed as a novel and promising strategy for cancer therapy. Many plant-derived anticancer agents act through inhibition of telomerase activity and induction of apoptosis. β-ionone, a carotenoid compound isolated from Roseaceae, has been reported to possess anticancer properties. The present study was undertaken to examine the mechanism of β-ionone-induced apoptosis in human leukemia cell line K562 with special emphasis on its role in telomerase inhibition. Method: In this study the anti-proliferation effect of β-ionone on K562 cells was evaluated by MTT assay. Apoptosis rate was detected by Hoechst staining and flow cytometry analysis. Telomerase activity was measured by (TRAP ELISA assay. Results: Exposure of K562 cells to β-ionone caused a dose-dependent decrease in proliferation. Flow cytometry analysis and Hoechst staining showed that percentage of apoptotic cells markedly increased with an increase in β-ionone concentration. Compared to control cells, treatment of K562 cells with β-ionone resulted in a significant decrease of telomerase activity. Moreover, a positive correlation was detected between telomerase inhibition and apoptosis induction in the treated K562 cells. Conclusion: Based on these results, β-ionone is an appropriate candidate for inhibiting telomerase activity in K562 cells. Therefore, it may be utilized as a novel drug against some leukemia cell lines.

  3. [Effects of Aptamer-siRNA Nucleic Acid Compound on Growth and Apoptosis in Myeloid Leukemia Cell Line K562].

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    Ping, Juan; Shen, Zhi-Hui; Wang, Bao-Quan; Zhao, Na; Li, Rui; Li, Mian; Pang, Xiao-Bin; Chen, Chuan-Bo

    2015-04-01

    To explore the effects of aptamer-siRNA nucleic acid compound on growth and apoptosis in myeloid leukemia cell line K562. the changes of cellular morphology and structure were observed by using fluorescence microscope, laser confocal microscope, JEM-4000EX transmission electron microscopy; MTT assay were performed to evaluate the sensibility of K562 cells to aptamer-siRNA compound, the apoptosis was detected by DNA gel electro-phoresis. The remarkably changes of morphology and structure of K562 cells treated with 200 µmol/L aptamer-siRNA were observed under fluorescence microscopy and electromicroscopy. As compared with control, the aptamer-siRNA compound showed more inhibitory effect on K562 cells and there was significant difference (Pcompound for K562 cells was 150 µmol/L. According to agarose gel electrophoresis observation, when the aptamer-siRNA compound showed effect on K562 cells, the typical DNA lader could be observed. The aptamer-siRNA compound can significantly induce K562 cell apoptosis, and provide reference for gene therapy of patients with chronic myelocytic lenkemia.

  4. [Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

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    Wang, Lin; Fei, Chang; Huang, Zheng-Lan; Li, Hui; Liu, Zhang-Lin; Feng, Wen-Li

    2015-08-01

    To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot. The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

  5. Vorinostat enhances chemosensitivity to arsenic trioxide in K562 cell line

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    Nainong Li

    2015-05-01

    Full Text Available Objective. This study aimed to investigate the chemosensitive augmentation effect and mechanism of HDAC inhibitor Vorinostat (SAHA in combination with arsenic trioxide (ATO on proliferation and apoptosis of K562 cells.Methods. The CCK-8 assay was used to compare proliferation of the cells. Annexin-V and PI staining by flow cytometry and acridine orange/ethidium bromide stains were used to detect and quantify apoptosis. Western blot was used to detect expression of p21, Akt, pAkt, p210, Acetyl-Histone H3, and Acetyl-Histone H4 proteins.Results. SAHA and ATO inhibited proliferation of K562 cells in an additive and time- and dose-dependent manner. SAHA in combination with ATO showed significant apoptosis of K562 cells in comparison to the single drugs alone (p < 0.01. Both SAHA and ATO alone and in combination showed lower levels of p210 expression. SAHA and SAHA and ATO combined treatment showed increased levels of Acetyl-Histone H3 and Acetyl-Histone H4 protein expression. SAHA alone showed increased expression of p21, while ATO alone and in combination with SAHA showed no significant change. SAHA and ATO combined therapy showed lower levels of Akt and pAkt protein expression than SAHA or ATO alone.Conclusion. SAHA and ATO combined treatment inhibited proliferation, induced apoptosis, and showed a chemosensitive augmentation effect on K562 cells. The mechanism might be associated with increasing histone acetylation levels as well as regulating the Akt signaling pathway.

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

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

    2005-10-01

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

  7. Circumvention of acquired resistance to doxorubicin in K562 human leukemia cells by oxatomide.

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    Ishikawa, M; Fujita, R; Furusawa, S; Takayanagi, M; Sasaki, K; Satoh, S

    2001-10-01

    We studied the effect of oxatomide, an antiallergic drug, on the resistance of K562 cells to doxorubicin. Oxatomide synergistically potentiated the cytotoxicity of doxorubicin in doxorubicin-resistant K562 cells (K562/DXR) at a concentration of 1-10 microM, but had hardly any synergistic effect on the parental cell line (K562) at the same concentration. Oxatomide inhibit P-glycoprotein pump-efflux activity and the binding of [3H]-azidopine to the cell-surface protein P-glycoprotein, in a dose-related manner. These results indicate that oxatomide reverses the multidrug-resistance phenotype through direct interaction with P-glycoprotein.

  8. Putative tyrosine kinases expressed in K-562 human leukemia cells

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    Partanen, J.; Maekelae, T.P.; Lehvaeslaiho, H.; Alitalo, K.; Alitalo, R.

    1990-01-01

    Tyrosine phosphorylation is important in the transmission of growth and differentiation signals; known tyrosine kinases include several oncoproteins and growth factor receptors. Interestingly, some differentiated cell types, such as erythrocytes and platelets contain high amounts of phosphotyrosine. The authors analyzed tyrosine kinases expressed in the K-562 chronic myelogenous leukemia cell line, which has a bipotential erythroid and megakaryoblastoid differentiation capacity. Analysis of 359 polymerase chain reaction-amplified cDNA clones led to the identification of 14 different tyrosine kinase-related sequences (JTK1-14). Two of the clones (JTK2 and JTK4) represent unusual members of the fibroblast growth factor receptor gene family, and the clones JTK5, JTK11, and JTK14 may also belong to the family of receptor tyrosine kinases but lack a close relationship to any known tyrosine kinase. Each of these different genes has its own characteristic expression pattern in K-562 cells and several other human tumor cell lines. In addition, the JTK11 and JTK14 mRNAs are induced during the megakaryoblastoid differentiation of K-562 cells. These tyrosine kinases may have a role in the differentiation of megakaryoblasts or in the physiology of platelets

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

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    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

  10. Involvement of CD147 on multidrug resistance through the regulation of P-glycoprotein expression in K562/ADR leukemic cell line

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    Aoranit Somno

    2016-01-01

    Full Text Available The relationship between P-gp and CD147 in the regulation of MDR in leukemic cells has not been reported. This study aimed to investigate the correlation between CD147 and P-gp in the regulation of drug resistance in the K562/ADR leukemic cell line. The results showed that drug-resistant K562/ADR cells expressed significantly higher P-gp and CD147 levels than drug-free K562/ADR cells. To determine the regulatory effect of CD147 on P-gp expression, anti-CD147 antibody MEM-M6/6 significantly decreased P-gp and CD147 mRNA and protein levels. This is the first report to show that CD147 mediates MDR in leukemia through the regulation of P-gp expression.

  11. Chaetominine reduces MRP1-mediated drug resistance via inhibiting PI3K/Akt/Nrf2 signaling pathway in K562/Adr human leukemia cells

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    Yao, Jingyun; Wei, Xing [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai (China); Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai (China); Lu, Yanhua, E-mail: luyanhua@ecust.edu.cn [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai (China); Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai (China)

    2016-05-13

    Drug resistance limits leukemia treatment and chaetominine, a cytotoxic alkaloid that promotes apoptosis in a K562 human leukemia cell line via the mitochondrial pathway was studied with respect to chemoresistance in a K562/Adr human resistant leukemia cell line. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin (ADR) did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RT-PCR and western-blot confirmed that chaetominine suppressed highly expressed MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line. - Highlights: • Chaetominine enhanced chemosensitivity of ADR against K562/Adr cells. • Chaetominine increased intracellular ADR levels via inhibiting MRP1. • Chaetominine induced apoptosis of K562/Adr cells through upregulation of ROS and modulation of Bax/Bcl-2. • Inhibition of MRP1 and Nrf2 by chaetominine treatment was correlative with blockade of PI3K/Akt signaling.

  12. Radiation-induced apoptosis in differentially modulated by PTK inhibitora in K562 cells

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    Lee, Hyung Sik; Moon, Chang Woo; Hur, Won Joo; Jeong, Su Jin; Jeong Min Ho; Lee, Jeong Hyeon; Lim, Young Jin; Park, Heon Joo

    2000-01-01

    The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induced apoptosis in Ph-positive K562 leukemia cell line was investigated. K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X-ray irradiation and drug treatment, cultures were initiated at 2x10 6 cells/ml. The cells were irradiated with 10Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37 .deg. for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells through the cell cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bcl-2, bcl-X-L and bax protein levels. Treatment with 10 Gy X-irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electrophoresis and TUNEL assay. Genistein failed to enhance the ability of X-irradiation to induce DNA fragmentation. Enhancement of apoptosis by HMA was not attributable to downregulation of the bcl-2 or bcl-X-L anti-apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage of cells in G2/M phase decreased to 30-40% at 48 h. On the other hand, cells exposed to 10 Gy X-irradiation alone or maintained with genistein did not show marked cell cycle redistribution. We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X-irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p210 bcr/abl failed to enhance the radiation induced apoptosis in K562 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is

  13. Inhibitory effect of PTD-OD-HA fusion protein on Bcr-Abl in K562 cells

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    Miao GAO

    2012-10-01

    Full Text Available Objective To study the transduction dynamics, location of PTD-OD-HA fusion protein and its interaction with Bcr-Abl oncoprotein in K562 cell lines, and explore the influence of PTD-OD-HA fusion protein on oligomerization and tyrosine kinase activity of Bcr-Abl. Methods PTD-OD-HA fusion protein was labeled with FITC and co-cultured with K562 cells. The transduction efficiency of labeled PTD-OD-HA at different doses and time intervals was observed under fluorescence microscope. The location of labeled PTD-OD-HA fusion protein in K562 cells was detected by confocal microscopy. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was confirmed by coimmunoprecipitation. The phosphorylation of Bcr-Abl oncoprotein was detected by Western blotting. Results PTD-OD-HA fusion protein labeled with FITC was transduced into K562 cells in a dose- and time-dependent manner. PTD-OD-HA fusion protein was located in the cytoplasm of K562 cells and was consistent with the location of Bcr-Abl oncoprotein. The interaction of PTD-OD-HA fusion protein with Bcr-Abl oncoprotein was proved in K562 cells. This interaction could interrupt the homologous oligomerization of Bcr-Abl oncoprotein and reduce the phosphorylation of Bcr-Abl oncoprotein. Conclusion PTD-OD-HA fusion protein could be transduced into K562 cells efficiently, inhibit the oligomerization and reduce the phosphorylation of Bcr-Abl oncoprotein.

  14. PENGARUH EKSTRAK JAMU TERHADAP AKTIVITAS SEL NATURAL KILLER DALAM MELISIS ALUR SEL LEUKIMIA (K-562 SECARA IN VITRO [The Effects of Commercial “Jamu” Extracts on Natural Killer Cell Activity in Lysing Leukemic Cell Line (K-562 in vitro

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    Elisa Veronica D.C. 2

    2002-04-01

    Full Text Available Natural killer (NK cell consitutes white blood cells which specifically functions in lysing tumor and virus invected cells. In this research, a commercial “Jamu” was tested to observe its effect on NK cells activity against leukemic cell lines (K562 in vitro. Jamu was extracted with hot water, diluted and added into cell cultures consisted of a mixture of human peripheric limphocyte cells, as the source of the effector NK cells, and K562 cell line i.e., the target cells which were cell line derived from human leukemia and had been labelled with H3-thymidine. The mixture of the cells were made by culturing the two cells at the ratio of 50:1 and 100 : 1, respectively. The results showed that lysing activity of NK cells in the presence of “Jamu” water extract measured as lysing percentage and lysing index increased only slightly, which were not statiscally significant. It should be considered that the test used in this research represents only a part of the lysing mechanism by NK cells against the target cells. An in vivo test for a period of time will be recessary to elucidate ffurther this NK cell activity.

  15. Inhibitiory properties of cytoplasmic extract of Lactobacilli isolated from common carp intestine on human chronic myelocytic leukemia K562 cell line: an in vitro study

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    Kabiri F

    2011-03-01

    Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 st1":*{behavior:url(#ieooui } /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Lactobacillus species are genetically diverse groups of Lactic Acid Bacteria (LAB that have been introduced as probiotics, because of some characteristics such as their anti-tumor properties, helping the intestinal flora balance, production of antibiotics, stimulation of host immune response, etc. The aim of this study was to investigate the effects of cytoplasmic extraction and cell wall of Lactobacillus species isolated from the intestine of common carp on human chronic myelocytic leukemia or K562 cancer cell lines."n"nMethods: The intestinal contents of 115 common carp captured from the natural resources of West Azerbaijan province in Iran were examined for LAB. After isolation, the identification of Lactobacilli was done according to traditional and molecular bacteriological tests. Subsequently, a suspension of each bacterium was prepared and the protein content of the cytoplasm was extracted. Cell wall disintegration was done by cell lysis buffer and sonication. The effects of cytoplasmic extraction and cell wall on K562 cell line proliferation were investigated by MTT assays."n"nResults: The cytoplasmic extraction of the isolated Lactobacilli had significant (p<0.05 anti

  16. The Effects of Royal Jelly on In-Vitro Cytotoxicity of K562 Cells and Peripheral Blood Mononuclear Cells

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    SE Hosseini

    2014-02-01

    Full Text Available Abstract Background & aim: Royal jelly, secreted by worker bees, has different biological activities on cells and tissues. The aim of this study was to evaluate the effects of royal jelly on peripheral blood mononuclear cells and on the tumor category of K562 cell line. Methods: In the present experimental study, three subjects were selected separately with three repetitions. K562 (104 cells and PBMC (105 cells with different concentrations of royal jelly (5, 10, 25, 50 and 100 mg/ml were cultured under standard conditions for 48 and 72 h separately. The fatality rate on PBMC cells and K562 cancer cells was evaluated by using MTT (Tetrazolium Dye-Reduction Assay. The number of viable cells in PBMC that were exposed for 48 hours with Royal Jelly was evaluated by trypan blue staining. Data were analyzed by ANOVA. Results: The royal jelly had no cytotoxicity effect on PBMC cells but at concentration of 50 and 100 mg/mL the cytotoxicity effect were observed on k562 cells whereas, at 10 and 25 mg/ml the number of PBMC viable cells increased. Conclusion: Due to the lack of lethality of royal jelly on PBMC cells and PBMC cell viability and an increase in the fatality rate of cancer cells in the future, royal jelly can be used as a potential candidate for treatment of leukemia. Keywords: Royal jelly, K562, peripheral blood mononuclear cell

  17. Expression of human gamma-globin genes in human erythroleukemia (K562) cells.

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    Donovan-Peluso, M; Acuto, S; Swanson, M; Dobkin, C; Bank, A

    1987-12-15

    K562 cells express embryonic (epsilon) and fetal (gamma) globins and hemoglobins but not adult (beta) globin. To define the cis acting regulatory elements involved in the discrimination between gamma and beta genes, we have constructed chimeric genes composed of portions of gamma and beta and evaluated their expression in stable K562 transfectants. A gamma beta fusion gene containing gamma 5' sequences to the EcoRI site in exon 3 and beta sequences 3' is expressed at 10-40% that of the endogenous gamma level. In 50% of the lines, this fusion gene appropriately increases its expression in response to hemin, an inducer of endogenous globin gene expression in K562 cells. In contrast, a beta gamma fusion gene, containing beta sequences 5' to the EcoRI site in exon 3 and gamma sequences 3', is neither expressed nor correctly initiated. A beta gene containing gamma-intervening sequence (IVS) 2 accumulates an mRNA transcript when analyzed with a 3' beta probe. However, no correctly initiated beta mRNA is observed. A gamma gene with beta-IVS 2 is only inducible in one of six expressing clones. All the results are consistent with the presence of stage-specific trans acting factors in K562 cells that stimulate expression of gamma genes and suggest a significant role for gamma-IVS 2 in gamma gene expression.

  18. Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation

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    Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Xie, Ji-Sheng [Youjiang Medical College for Nationalities, Guangxi 533000 (China); Meng, Xin; Guan, Yifu [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Wang, Hua-Qin, E-mail: wanghq_doctor@hotmail.com [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)

    2010-01-01

    Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2{alpha} inhibitor, or overexpression of dominant negative mutants of PERK or eIF2{alpha}, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2{alpha} branch of UPR in RES-induced inhibition of cell proliferation.

  19. Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation

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    Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang; Xie, Ji-Sheng; Meng, Xin; Guan, Yifu; Wang, Hua-Qin

    2010-01-01

    Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2α (eIF2α), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2α inhibitor, or overexpression of dominant negative mutants of PERK or eIF2α, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2α branch of UPR in RES-induced inhibition of cell proliferation.

  20. Multidrug resistance in tumour cells: characterisation of the multidrug resistant cell line K562-Lucena 1

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    VIVIAN M. RUMJANEK

    2001-03-01

    Full Text Available Multidrug resistance to chemotherapy is a major obstacle in the treatment of cancer patients. The best characterised mechanism responsible for multidrug resistance involves the expression of the MDR-1 gene product, P-glycoprotein. However, the resistance process is multifactorial. Studies of multidrug resistance mechanisms have relied on the analysis of cancer cell lines that have been selected and present cross-reactivity to a broad range of anticancer agents. This work characterises a multidrug resistant cell line, originally selected for resistance to the Vinca alkaloid vincristine and derived from the human erythroleukaemia cell K562. This cell line, named Lucena 1, overexpresses P-glycoprotein and have its resistance reversed by the chemosensitisers verapamil, trifluoperazine and cyclosporins A, D and G. Furthermore, we demonstrated that methylene blue was capable of partially reversing the resistance in this cell line. On the contrary, the use of 5-fluorouracil increased the resistance of Lucena 1. In addition to chemotherapics, Lucena 1 cells were resistant to ultraviolet A radiation and hydrogen peroxide and failed to mobilise intracellular calcium when thapsigargin was used. Changes in the cytoskeleton of this cell line were also observed.A resistência a múltiplos fármacos é o principal obstáculo no tratamento de pacientes com câncer. O mecanismo responsável pela resistência múltipla mais bem caracterizado envolve a expressão do produto do gene MDR-1, a glicoproteína P. Entretanto, o processo de resistência tem fatores múltiplos. Estudos de mecanismos de resistência m��ltipla a fármacos têm dependido da análise de linhagens celulares tumorais que foram selecionadas e apresentam reatividade cruzada a uma ampla faixa de agentes anti-tumorais. Este trabalho caracteriza uma linhagem celular com múltipla resistência a fármacos, selecionada originalmente pela resistência ao alcalóide de Vinca vincristina e derivado

  1. Effects of P-Glycoprotein and Its Inhibitors on Apoptosis in K562 Cells

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    Yaqiong Zu

    2014-08-01

    Full Text Available P-glycoprotein (P-gp is a major factor in multidrug resistance (MDR which is a serious obstacle in chemotherapy. P-gp has also been implicated in causing apoptosis of tumor cells, which was shown to be another important mechanism of MDR recently. To study the influence of P-gp in tumor cell apoptosis, K562/A cells (P-gp+ and K562/S cells (P-gp− were subjected to doxorubicin (Dox, serum withdrawal, or independent co-incubation with multiple P-gp inhibitors, including valspodar (PSC833, verapamil (Ver and H108 to induce apoptosis. Apoptosis was simultaneously detected by apoptotic rate, cell cycle by flow cytometry and cysteine aspartic acid-specific protease 3 (caspase 3 activity by immunoassay. Cytotoxicity and apoptosis induced by PSC833 were evaluated through an MTT method and apoptosis rate, and cell cycle combined with caspase 3 activity, respectively. The results show that K562/A cells are more resistant to apoptosis and cell cycle arrest than K562/S cells after treatment with Dox or serum deprivation. The apoptosis of K562/A cells increased after co-incubation with each of the inhibitors of P-gp. P-gp inhibitors also enhanced cell cycle arrest in K562/A cell. PSC833 most strikingly decreased viability and led to apoptosis and S phase arrest of cell cycle in K562/A cells. Our study demonstrates that P-gp inhibits the apoptosis of tumor cells in addition to participating in the efflux of intracellular chemotherapy drugs. The results of the caspase 3 activity assay also suggest that the role of P-gp in apoptosis avoidance is caspase-related.

  2. Musashi2 modulates K562 leukemic cell proliferation and apoptosis involving the MAPK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Huijuan; Tan, Shi; Wang, Juan; Chen, Shana; Quan, Jing; Xian, Jingrong; Zhang, Shuai shuai; He, Jingang; Zhang, Ling, E-mail: lingzhang@cqmu.edu.cn

    2014-01-01

    The RNA-binding protein Musashi2 (Msi2) has been identified as a master regulator within a variety of stem cell populations via the regulation of translational gene expression. A recent study has suggested that Msi2 is strongly expressed in leukemic cells of acute myeloid leukemia patients, and elevated Msi2 is associated with poor prognosis. However, the potential role of Msi2 in leukemogenesis is still not well understood. Here, we investigated the effect of Msi2 knockdown on the biological properties of leukemic cells. High expression of Msi2 was found in K562 and KG-1a leukemic cell lines, and low expression was observed in the U937 cell line. We transduced K562 cells with two independent adenoviral shRNA vectors targeting Msi2 and confirmed knockdown of Msi2 at the mRNA and protein levels. Msi2 silencing inhibited cell growth and caused cell cycle arrest by increasing the expression of p21 and decreasing the expression of cyclin D1 and cdk2. In addition, knockdown of Msi2 promoted cellular apoptosis via the upregulation of Bax and downregulation of Bcl-2 expression. Furthermore, Msi2 knockdown resulted in the inactivation of the ERK/MAPK and p38/MAPK pathways, but no remarkable change in p-AKT was observed. These data provide evidence that Msi2 plays an important role in leukemogenesis involving the MAPK signaling pathway, which indicates that Msi2 may be a novel target for leukemia treatment. - Highlights: • Knockdown of Msi2 inhibited K562 cell growth and arrested cell cycle progression. • Knockdown of Msi2 induced K562 cell apoptosis via the regulation of Bax and Bcl-2. • The MAPK pathway was involved in the process of Msi2-mediated leukemogenesis. • Our data indicate that Msi2 is a potential new target for leukemia treatment.

  3. Analysis of the Effects of δ-Tocopherol on RAW264.7 and K562 Cells Based on 1H NMR Metabonomics.

    Science.gov (United States)

    Lu, Yang; Li, Hui; Geng, Yue

    2018-01-31

    δ-Tocopherol (δ-TOH) is a form of vitamin E with higher bioactivity. In this study, we studied the bioactivity of δ-TOH using the IC 50 of δ-TOH on RAW264.7 (80 μM) and K562 (110 μM) cells. We compared the differential metabolites from the cell lines with and without δ-TOH treatment by 1 H NMR metabonomics analysis. It was found that δ-TOH affected the protein biosynthesis, betaine metabolism, and urea cycle in various ways in both cell lines. Metabolic levels of the cell lines were changed after treatment with δ-TOH as differential metabolites were produced. The betaine level in RAW264.7 cells was reduced significantly, while the l-lactic acid level in K562 cells was significantly enhanced. The metabolic changes might contribute to the switch of the respiration pattern from aerobic respiration to anaerobic respiration in K562 cells. These results are helpful in further understanding the subtoxicity of δ-TOH.

  4. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells.

    Science.gov (United States)

    Suriguga; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. © 2013.

  5. The best time of cytotoxicity for extracted cell wall from Lactobacillus casei and paracasei in K562 cell line

    Directory of Open Access Journals (Sweden)

    Riki M

    2013-02-01

    Full Text Available Background: The aim of this study was to evaluate the effect of extracted cell walls from Lactobacillus casei and Lactobacillus paracasei as probiotic bacteria (isolated from common carp intestine on K562 and the role of cell concentration on the results of MTT [3-(4,5-Dimethylthiazol-2-yl2,5- Diphenyl tetrazolium Bromide] test.Methods: For this purpose, bacteria were cultured in specific medium (MRS broth at anaerobic condition for 24-48 hour. After incubation period culture medium was centri-fuged, then the cells were washed twice with PBS buffer to remove additional medium. Finally, collected bacterial cell disrupted by Sonication and cell walls were separated from other components by centrifugation. After that, different concentrations of cell walls (500, 1000, 2000 and 4000 µg/ml were prepared in RPMI medium for each bacteria, separately. Then anticancer properties of the cell walls were determined in vitro at 12, 24, 48 and 72 h, also the effect of K562 concentration was assayed with MTT technique.Results: The results showed extracted cell wall from both probiotic statistically (P=0.098 have anti turmeric properties in K562 and their properties will arise in relation with concentration. As well as, we found that the number of cell had not any affect on the result of MTT assay.Conclusion: We conclude that the cytotoxicity property of extracted cell wall is related in the type of bacteria, but this anticancer property would warrant further study on the clinical application of extracted cell wall.

  6. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun, E-mail: yizc@buaa.edu.cn

    2013-12-15

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation.

  7. The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

    International Nuclear Information System (INIS)

    Suriguga,; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

    2013-01-01

    Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. - Highlights: • Catechol enhanced hemin-induced hemoglobin accumulation. • COMT-catalyzed methylation acted as detoxication of catechol. • COMT involved in catechol-enhanced erythroid differentiation

  8. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

    Directory of Open Access Journals (Sweden)

    Giuseppe Fiume

    2016-11-01

    Full Text Available The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03% of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7% and 698 downregulated (54.3% RNAs. In K562 cells, 1959 (3.1% of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7% and 906 downregulated (46.3%. Only 137 transcripts (0.22% were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  9. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

    Science.gov (United States)

    Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

    2016-11-07

    The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  10. Retinoic acid receptor gamma impacts cellular adhesion, Alpha5Beta1 integrin expression and proliferation in K562 cells.

    Science.gov (United States)

    Kelley, Melissa D; Phomakay, Raynin; Lee, Madison; Niedzwiedz, Victoria; Mayo, Rachel

    2017-01-01

    The interplay between cellular adhesion and proliferation is complex; however, integrins, particularly the α5β1 subset, play a pivotal role in orchestrating critical cellular signals that culminate in cellular adhesion and growth. Retinoids modify the expression of a variety of adhesive/proliferative signaling proteins including α5β1 integrins; however, the role of specific retinoic acid receptors involved in these processes has not been elucidated. In this study, the effect of all-trans-retinoic acid receptor (RAR) agonists on K562 cellular adhesion, proliferation, and α5β1 integrin cell surface expression was investigated. RARγ agonist exposure increased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin and FN-120 in a time- and concentration dependent manner, while RARα or RARβ agonist treatment had no effect on cellular adhesion. Due to the novel RARγ- dependent cellular adhesion response exhibited by K562 cells, we examined α5 and β1 integrin subunit expression when K562 cells were exposed to retinoid agonists or vehicle for 24, 48, 72 or 96 hours. Our data demonstrates no differences in K562 cell surface expression of the α5 integrin subunit when cells were exposed to RARα, RARβ, or RARγ agonists for all time points tested. In contrast, RARγ agonist exposure resulted in an increase in cell surface β1 integrin subunit expression within 48 hours that was sustained at 72 and 96 hours. Finally, we demonstrate that while exposure to RARα or RARβ agonists have no effect on K562 cellular proliferation, the RARγ agonist significantly dampens K562 cellular proliferation levels in a time- and concentration- dependent manner. Our study is the first to report that treatment with a RARγ specific agonist augments cellular adhesion to α5β1 integrin substrates, increases cell surface levels of the β1 integrin subunit, and dampens cellular proliferation in a time and concentration dependent manner in a human

  11. The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

    International Nuclear Information System (INIS)

    Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming; Wang, Yan; Wang, Jie; Li, Yang; Suriguga,; Zhang, Guang-Yao; Yi, Zong-Chun

    2012-01-01

    Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.

  12. The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming; Wang, Yan; Wang, Jie; Li, Yang; Suriguga,; Zhang, Guang-Yao; Yi, Zong-Chun, E-mail: yizc@buaa.edu.cn

    2012-11-15

    Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.

  13. 5-(2-Carboxyethenyl) isatin derivative induces G2/M cell cycle arrest and apoptosis in human leukemia K562 cells

    International Nuclear Information System (INIS)

    Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin; Yang, Yao; Song, Bin-Bin; Guo, Qian-Nan; Fan, Zhen-Chuan; Zhang, Yong-Min; Teng, Yu-Ou; Yu, Peng

    2014-01-01

    Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G 2 /M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC 50 ) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G 2 /M phase and accumulated subsequently in the sub-G 1 phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G 2 /M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation

  14. Comparison of different methods for erythroid differentiation in the K562 cell line.

    Science.gov (United States)

    Shariati, Laleh; Modaress, Mehran; Khanahmad, Hossein; Hejazi, Zahra; Tabatabaiefar, Mohammad Amin; Salehi, Mansoor; Modarressi, Mohammad Hossein

    2016-08-01

    To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis. Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001). Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

  15. Regulation of hTERT by BCR-ABL at multiple levels in K562 cells

    International Nuclear Information System (INIS)

    Chai, Juin Hsien; Zhang, Yong; Tan, Wei Han; Chng, Wee Joo; Li, Baojie; Wang, Xueying

    2011-01-01

    The cytogenetic characteristic of Chronic Myeloid Leukemia (CML) is the formation of the Philadelphia chromosome gene product, BCR-ABL. Given that BCR-ABL is the specific target of Gleevec in CML treatment, we investigated the regulation of the catalytic component of telomerase, hTERT, by BCR-ABL at multiple levels in K562 cells. Molecular techniques such as over expression, knockdown, real-time PCR, immunoprecipitation, western blotting, reporter assay, confocal microscopy, telomerase assays and microarray were used to suggest that hTERT expression and activity is modulated by BCR-ABL at multiple levels. Our results suggest that BCR-ABL plays an important role in regulating hTERT in K562 (BCR-ABL positive human leukemia) cells. When Gleevec inhibited the tyrosine kinase activity of BCR-ABL, phosphorylation of hTERT was downregulated, therefore suggesting a positive correlation between BCR-ABL and hTERT. Gleevec treatment inhibited hTERT at mRNA level and significantly reduced telomerase activity (TA) in K562 cells, but not in HL60 or Jurkat cells (BCR-ABL negative cells). We also demonstrated that the transcription factor STAT5a plays a critical role in hTERT gene regulation in K562 cells. Knockdown of STAT5a, but not STAT5b, resulted in a marked downregulation of hTERT mRNA level, TA and hTERT protein level in K562 cells. Furthermore, translocation of hTERT from nucleoli to nucleoplasm was observed in K562 cells induced by Gleevec. Our data reveal that BCR-ABL can regulate TA at multiple levels, including transcription, post-translational level, and proper localization. Thus, suppression of cell growth and induction of apoptosis by Gleevec treatment may be partially due to TA inhibition. Additionally, we have identified STAT5a as critical mediator of the hTERT gene expression in BCR-ABL positive CML cells, suggesting that targeting STAT5a may be a promising therapeutic strategy for BCR-ABL positive CML patients

  16. H-ferritin-regulated microRNAs modulate gene expression in K562 cells.

    Directory of Open Access Journals (Sweden)

    Flavia Biamonte

    Full Text Available In a previous study, we showed that the silencing of the heavy subunit (FHC offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC comparing it with K562 transduced with scrambled RNA (K562shRNA. Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, "Cell Death and Survival, Hematological System Development and Function, Hematopoiesis", is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.

  17. 5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin; Yang, Yao; Song, Bin-Bin; Guo, Qian-Nan [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Fan, Zhen-Chuan [Key Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Ministry of Education, Tianjin 300457 (China); Obesita and Algaegen LLC, College Station, TX 77845 (United States); Zhang, Yong-Min [Université Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire UMR CNRS 8232, 4 Place Jussieu, 75005 Paris (France); Teng, Yu-Ou, E-mail: tyo201485@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Yu, Peng, E-mail: yupeng@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China)

    2014-08-08

    Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.

  18. Modulation of P-glycoprotein-mediated multidrug resistance in K562 leukemic cells by indole-3-carbinol

    International Nuclear Information System (INIS)

    Arora, Annu; Seth, Kavita; Kalra, Neetu; Shukla, Yogeshwer

    2005-01-01

    Resistance to chemotherapeutic drugs is one of the major problems in the treatment of cancer. P-glycoprotein (P-gp) encoded by the mdr gene is a highly conserved protein, acts as a multidrug transporter, and has a major role in multiple drug resistance (MDR). Targeting of P-gp by naturally occurring compounds is an effective strategy to overcome MDR. Indole-3-carbinol (I3C), a glucosinolates present in cruciferous vegetables, is a promising chemopreventive agent as it is reported to possess antimutagenic, antitumorigenic, and antiestrogenic properties in experimental studies. In the present investigation, the potential of I3C to modulate P-gp expression was evaluated in vinblastine (VBL)-resistant K562 human leukemic cells. The resistant K562 cells (K562/R10) were found to be cross-resistant to vincristine (VCR), doxorubicin (DXR), and other antineoplastic agents. I3C at a nontoxic dose (10 x 10 -3 M) enhanced the cytotoxic effects of VBL time dependently in VBL-resistant human leukemia (K562/R10) cells but had no effect on parent-sensitive cells (K562/S). The Western blot analysis of K 562/R 10 cells showed that I3C downregulates the induced levels of P-gp in resistant cells near to normal levels. The quantitation of immunocytochemically stained K562/R10 cells showed 24%, 48%, and 80% decrease in the levels of P-gp by I3C for 24, 48, and 72 h of incubation. The above features thus indicate that I3C could be used as a novel modulator of P-gp-mediated multidrug resistance in vitro and may be effective as a dietary adjuvant in the treatment of MDR cancers

  19. Delayed K562 cell apoptosis promoted by cleaved LyGDI after 60Co γ-rays irradiation

    International Nuclear Information System (INIS)

    Sun Huali; Duan Weiming; Shao Yanyan; Xiao Hainan; Zhou Xinwen

    2010-01-01

    Objective: To elucidate the function and regulatory mechanism of LyGDI involved delayed cell death in the human K562 cells and HL-60 cells induced by 60 Co γ-rays. Methods: Erythrosine B cells staining was used to count the apoptosis rate. PI staining and flow cytometry were applied to check the cell cycle. The expression of LYGDI and Rac1 was resolved by Western blot by using monoclonal antibody of LyGDI and Rac1. The distribution of Rac1 protein in cells was observed with immunofluorescence by using the confocal microscope. Results: The K562 cells showed G 2 /M phase arrest and the percent age was 71.3%. The apoptosis rate was very low at early post-irradiation stage in the K562 cells. The apoptosis rate was 14% in the K562 cells at 24 h post-irradiation with 8 Gy of γ-rays, and delayed cell apoptosis was present. LyGDI was cleaved in the K562 cells irradiated by 4 Gy 60 Co γ-rays after 24 hours post-irradiation. The expression of Rac1 protein was not altered at all, but the distribution was changed in the irradiated cells while the Rac1 protein moved to cell membrane and a little in cell nucleus. The Rac1 was activated with the losing the binding affinity with the LyGDI. Conclusion: LyGDI could promote the delayed cell apoptosis, which is through the activation of the Rac1. (authors)

  20. Regulation of HtrA2 on WT1 gene expression under imatinib stimulation and its effects on the cell biology of K562 cells.

    Science.gov (United States)

    Zhang, Lixia; Li, Yan; Li, Xiaoyan; Zhang, Qing; Qiu, Shaowei; Zhang, Qi; Wang, Min; Xing, Haiyan; Rao, Qing; Tian, Zheng; Tang, Kejing; Wang, Jianxiang; Mi, Yingchang

    2017-09-01

    exhibited no notable effect on the inhibition of the ERK1/2 pathway. HtrA2 and its regulatory effect on WT1 may affect the sensitivity of BCR/ABL(+) cell lines to target therapy drugs through different mechanisms. Regulation of WT1 by HtrA2 occurs in K562 cells, and the regulation may affect the apoptosis of K562 cells under the stress caused by chemotherapeutic treatment. The p38 MAPK signaling pathway, which serves an important role in cell apoptosis, is a downstream pathway of this regulation.

  1. Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells

    International Nuclear Information System (INIS)

    Shi, Lin; Song, Quansheng; Zhang, Yingmei; Lou, Yaxin; Wang, Yanfang; Tian, Linjie; Zheng, Yi; Ma, Dalong; Ke, Xiaoyan; Wang, Ying

    2010-01-01

    Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.

  2. Apoptosis induction in MV4-11 and K562 human leukemic cells by Pereskia sacharosa (Cactaceae) leaf crude extract.

    Science.gov (United States)

    Asmaa, Mat Jusoh Siti; Al-Jamal, Hamid Ali Nagi; Ang, Cheng Yong; Asan, Jamaruddin Mat; Seeni, Azman; Johan, Muhammad Farid

    2014-01-01

    Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia. Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting. P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells. These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.

  3. Effect of JTV1 gene on the proliferation and apoptosis of K562 cells and its mechanism

    Directory of Open Access Journals (Sweden)

    Yan WU

    2011-05-01

    Full Text Available Objective To investigate the effect of tumor-suppressing gene JTV1 on proliferation and apoptosis of leukemic K562 cells,and the changes in apoptosis factors Bcl-2,C-myc and Bax genes.Methods The recombinate vector pcDNA3.1-JTV1,and the empty vector pcDNA3.1 were transfected into K562 cells as control.The cell proliferation of K562 cells was evaluated by colony formation assay;the cell cycle and apoptosis rate were assessed by flow cytometry(FCM;the mRNA levels of apoptosis related genes Bax,Bcl-2 and C-myc were determined by RT-PCR;the protein levels of Bax,Bcl-2 and C-myc were assayed by Western Blotting.Results The colony formation assay showed that the proliferation of K562 cells decreased when the expression of JTV1 gene was up-regulated.FCM assay showed that the G phase cells in pcDNA3.1-JTV1 positive transfection group increased compared with that of the control group and the pcDNA3.1 empty vector transfected group,and the differences were statistically significant(P < 0.05.Compared with the control group and the empty vector group,the mRNA transcription level and the protein translation level of Bax gene increased significantly,and the mRNA transcription level and the protein translation level of Bcl-2 and C-myc gene were reduced significantly(P < 0.05.Conclusions The expressions of Bcl-2 and C-myc gene are inhibited when the gene JTV1 is up-regulated,leading to an increase in Bax gene expression,inhibition of K562 cell proliferation,and promotion of tumor cells apoptosis.Over expression of JTV1 gene can inhibit the proliferation of K562 cells and promote cell apoptosis by inhibiting Bcl-2 and C-myc expression and up-regulating that of Bax.

  4. A nanocomplex of Cu(II) with theophylline drug; synthesis, characterization, and anticancer activity against K562 cell line

    Science.gov (United States)

    Sahlabadi, Maryam; Daryanavard, Marzieh; Hadadzadeh, Hassan; Amirghofran, Zahra

    2018-03-01

    A new mononuclear of copper (II), [Cu(theophylline)2(H2O)3]·2H2O, has been synthesized by reaction of theophylline (1,3-dimethyl-7H-purine-2,6-dione) with copper (II) nitrate in water. Further, its nanocomplex has been prepared through the three different methods including sonication, grinding, and a combination thereof, sonication-grinding. The prepared nanocomplex was characterized using different techniques including FT-IR, UV-Vis, X-ray diffraction (XRD) analysis, and field-emission scanning electron microscopy (FE-SEM). Moreover, the anticancer activity of the precursor complex, nanocomplex, free theophylline ligand, and the starting copper salt (Cu(NO3)2·3H2O) was investigated against the K562 cell line. The results show that the nanocomplex is an effective nano metal-based anticancer agent with IC50 = 11.7 μM.

  5. Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA.

    Science.gov (United States)

    Ojima, Yoshihiro; Duncan, Mark Thompson; Nurhayati, Retno Wahyu; Taya, Masahito; Miller, William Martin

    2013-08-15

    The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

    Science.gov (United States)

    Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

    2016-08-01

    Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Macrophage inflammatory protein-3α influences growth of K562 leukemia cells in co-culture with anticancer drug-pretreated HS-5 stromal cells

    International Nuclear Information System (INIS)

    Lee, Y.C.; Chiou, T.-J.; Tzeng, W.-F.; Chu, S.T.

    2008-01-01

    Stromal cell monolayers have been an important means of studying the regulation of hematopoiesis, because they produce cytokines. Cytosine arabinoside, vincristine, daunorubicin, and doxorubicin are common drugs for hematological cancer therapy, and they may have some effects on bone marrow stroma during chemotherapy. The aim of this study was to elucidate interactions between the bone marrow stromal microenvironment and leukemic cells after drug treatment. We tested the hypothesis that human HS-5 stromal cells, pretreated with anticancer drugs, affected the growth of leukemic K562 cells by changing the cytokines in the culture microenvironment. Thereafter, proliferation of K562 cells increased nearly 2.5-fold compared the co-cultivation with drugs-pretreated HS-5 stromal cells and drugs-untreated HS-5 stromal cells. The results indicated that co-cultivation with HS-5 stromal cells pretreated with drugs caused significant K562 cell proliferation. Cytokines in the microenvironment were detected via the RayBio Human Cytokine Antibody Array Membrane. The levels of the cytokines CKβ, IL-12, IL-13, IGFBP-2, MCP-1, MCP-3, MCP-4, MDC, MIP-1β and MIP-1δ were decreased, with a particularly marked decrease in MIP-3α. In co-culture medium, there was a 20-fold decrease in MIP-3α in daunorubicin-pretreated HS-5 cells and at least a 3-fold decrease in Ara-C-pretreated cells. This indicated a significant effect of anticancer drugs on the stromal cell line. Using phosphorylated Erk and pRb proteins as cell proliferation markers, we found that phosphorylation of these markers in K562 cells was inhibited during co-cultivation with drug-pretreated stromal cells in MIP-3α-supplemented medium and restored by MIP-3α antibody supplement. In conclusion, anticancer drug pretreatment suppresses the negative control exerted by HS-5 cells on leukemic cell proliferation, via modulation of cytokines in the microenvironment, especially at the level of MIP-3α

  8. CLEC4M慢病毒载体的构建及其在K562细胞中的表达

    Directory of Open Access Journals (Sweden)

    WANG Yuanyuan

    2014-06-01

    Full Text Available ObjectiveTo construct the lentiviral vector encoding CLEC4M and prepare K-562 cells with stable overexpression of CLEC4M. MethodsThe gene sequence of normal CLEC4M was cloned by RT-PCR and then inserted into GV166 vector to construct GV166-CLEC4M lentiviral expression vector, and then lentiviral packaging was performed by transfection of 293T cells. The obtained lentiviral liquid was used to infect human leukemia cell line K-562. Real-time PCR and Western blot were used to detect the overexpression of CLEC4M in K-562 cells. ResultsSequencing showed that the recombinant lentiviral expression plasmid GV166-CLEC4M was successfully constructed. Lentiviruses could efficiently infect K-562 cells, according to real-time PCR. CLEC4M was successfully over-expressed in K-562 cells at mRNA and protein levels. ConclusionThe construction of lentiviral vector encoding CLEC4M lays a foundation for further study of CLEC4M gene involved in HCV entry into host cells.

  9. PaDef defensin from avocado (Persea americana var. drymifolia) is cytotoxic to K562 chronic myeloid leukemia cells through extrinsic apoptosis.

    Science.gov (United States)

    Flores-Alvarez, Luis José; Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Salgado-Garciglia, Rafael; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

    2018-06-01

    Plant defensins, a group of antimicrobial peptides, show selective cytotoxicity toward cancer cells. However, their mechanisms of action remain poorly understood. Here, we evaluated the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on K562 chronic myeloid leukemia cells and analyzed the pathway involved in the induction of cell death. The defensin PaDef was not cytotoxic against human PBMCs; however, it was cytotoxic for K562 cell line (IC 50  = 97.3 μg/ml) activating apoptosis at 12 h. PaDef did not affect the mitochondrial membrane potential (ΔΨm), neither the transmembranal potential or the release of intracellular calcium. Also, PaDef induced gene expression of caspase 8 (∼2 fold), TNF-α (∼4 fold) and TNFR1 (∼10 fold). In addition, the activation of caspase 8 was detected at 24 h, whereas caspase 9 activity was not modified, suggesting that the extrinsic apoptosis pathway could be activated. In conclusion, PaDef induces apoptosis on K562 cells, which is related to the activation of caspase 8 and involves the participation of TNF-α, which is a novel property for a plant defensin. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Anticancer activity of Pupalia lappacea on chronic myeloid leukemia K562 cells.

    Science.gov (United States)

    Ravi, Alvala; Alvala, Mallika; Sama, Venkatesh; Kalle, Arunasree M; Irlapati, Vamshi K; Reddy, B Madhava

    2012-12-05

    Cancer is one of the most prominent human diseases which has enthused scientific and commercial interest in the discovery of newer anticancer agents from natural sources. Here we demonstrated the anticancer activity of ethanolic extract of aerial parts of Pupalia lappacea (L) Juss (Amaranthaceae) (EAPL) on Chronic Myeloid Leukemia K562 cells. Antiproliferative activity of EAPL was determined by MTT assay using carvacrol as a positive control. Induction of apoptosis was studied by annexin V, mitochondrial membrane potential, caspase activation and cell cycle analysis using flow cytometer and modulation in protein levels of p53, PCNA, Bax and Bcl2 ratio, cytochrome c and cleavage of PARP were studied by Western blot analysis. The standardization of the extract was performed through reverse phase-HPLC using Rutin as biomarker. The results showed dose dependent decrease in growth of K562 cells with an IC50 of 40 ± 0.01 μg/ml by EAPL. Induction of apoptosis by EAPL was dose dependent with the activation of p53, inhibition of PCNA, decrease in Bcl2/Bax ratio, decrease in the mitochondrial membrane potential resulting in release of cytochrome c, activation of multicaspase and cleavage of PARP. Further HPLC standardization of EAPL showed presence 0.024% of Rutin. Present study significantly demonstrates anticancer activity of EAPL on Chronic Myeloid Leukemia (K562) cells which can lead to potential therapeutic agent in treating cancer. Rutin, a known anti cancer compound is being reported and quantified for the first time from EAPL.

  11. Complete genome of Phenylobacterium zucineum – a novel facultative intracellular bacterium isolated from human erythroleukemia cell line K562

    Directory of Open Access Journals (Sweden)

    Sun Jie

    2008-08-01

    Full Text Available Abstract Background Phenylobacterium zucineum is a recently identified facultative intracellular species isolated from the human leukemia cell line K562. Unlike the known intracellular pathogens, P. zucineum maintains a stable association with its host cell without affecting the growth and morphology of the latter. Results Here, we report the whole genome sequence of the type strain HLK1T. The genome consists of a circular chromosome (3,996,255 bp and a circular plasmid (382,976 bp. It encodes 3,861 putative proteins, 42 tRNAs, and a 16S-23S-5S rRNA operon. Comparative genomic analysis revealed that it is phylogenetically closest to Caulobacter crescentus, a model species for cell cycle research. Notably, P. zucineum has a gene that is strikingly similar, both structurally and functionally, to the cell cycle master regulator CtrA of C. crescentus, and most of the genes directly regulated by CtrA in the latter have orthologs in the former. Conclusion This work presents the first complete bacterial genome in the genus Phenylobacterium. Comparative genomic analysis indicated that the CtrA regulon is well conserved between C. crescentus and P. zucineum.

  12. 9-cis-retinoic Acid and troglitazone impacts cellular adhesion, proliferation, and integrin expression in K562 cells.

    Science.gov (United States)

    Hanson, Amanda M; Gambill, Jessica; Phomakay, Venusa; Staten, C Tyler; Kelley, Melissa D

    2014-01-01

    Retinoids are established pleiotropic regulators of both adaptive and innate immune responses. Recently, troglitazone, a PPAR gamma agonist, has been demonstrated to have anti-inflammatory effects. Separately, retinoids and troglitazone are implicated in immune related processes; however, their combinatory role in cellular adhesion and proliferation has not been well established. In this study, the effect of 9-cis-retinoic acid (9-cis-RA) and troglitazone on K562 cellular adhesion and proliferation was investigated. Troglitazone exposure decreased K562 cellular adhesion to RGD containing extracellular matrix proteins fibronectin, FN-120, and vitronectin in a concentration and time-dependent manner. In the presence of troglitazone, 9-cis-retinoic acid restores cellular adhesion to levels comparable to vehicle treatment alone on fibronectin, FN-120, and vitronectin substrates within 72 hours. Due to the prominent role of integrins in attachment to extracellular matrix proteins, we evaluated the level of integrin α5 subunit expression. Troglitazone treatment results in decrease in α5 subunit expression on the cell surface. In the presence of both agonists, cell surface α5 subunit expression was restored to levels comparable to vehicle treatment alone. Additionally, troglitazone and 9-cis-RA mediated cell adhesion was decreased in the presence of a function blocking integrin alpha 5 inhibitor. Further, through retinoid metabolic profiling and HPLC analysis, our study demonstrates that troglitazone augments retinoid availability in K562 cells. Finally, we demonstrate that troglitazone and 9-cis-retinoic acid synergistically dampen cellular proliferation in K562 cells. Our study is the first to report that the combination of troglitazone and 9-cis-retinoic acid restores cellular adhesion, alters retinoid availability, impacts integrin expression, and dampens cellular proliferation in K562 cells.

  13. Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice.

    Science.gov (United States)

    Verrax, Julien; Stockis, Julie; Tison, Aurélie; Taper, Henryk S; Calderon, Pedro Buc

    2006-09-14

    The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy.

  14. Use of zinc-finger nucleases to knock out the WAS gene in K562 cells: a human cellular model for Wiskott-Aldrich syndrome

    Directory of Open Access Journals (Sweden)

    Miguel G. Toscano

    2013-03-01

    Mutations in the WAS gene cause Wiskott-Aldrich syndrome (WAS, which is characterized by eczema, immunodeficiency and microthrombocytopenia. Although the role of WASP in lymphocytes and myeloid cells is well characterized, its role on megakaryocyte (MK development is poorly understood. In order to develop a human cellular model that mimics the megakaryocytic-derived defects observed in WAS patients we used K562 cells, a well-known model for study of megakaryocytic development. We knocked out the WAS gene in K562 cells using a zinc-finger nuclease (ZFN pair targeting the WAS intron 1 and a homologous donor DNA that disrupted WASP expression. Knockout of WASP on K562 cells (K562WASKO cells resulted in several megakaryocytic-related defects such as morphological alterations, lower expression of CD41ɑ, lower increments in F-actin polymerization upon stimulation, reduced CD43 expression and increased phosphatidylserine exposure. All these defects have been previously described either in WAS-knockout mice or in WAS patients, validating K562WASKO as a cell model for WAS. However, K562WASPKO cells showed also increased basal F-actin and adhesion, increased expression of CD61 and reduced expression of TGFβ and Factor VIII, defects that have never been described before for WAS-deficient cells. Interestingly, these phenotypic alterations correlate with different roles for WASP in megakaryocytic differentiation. All phenotypic alterations observed in K562WASKO cells were alleviated upon expression of WAS following lentiviral transduction, confirming the role of WASP in these phenotypes. In summary, in this work we have validated a human cellular model, K562WASPKO, that mimics the megakaryocytic-related defects found in WAS-knockout mice and have found evidences for a role of WASP as regulator of megakaryocytic differentiation. We propose the use of K562WASPKO cells as a tool to study the molecular mechanisms involved in the megakaryocytic-related defects observed in WAS

  15. Unravelling pathways downstream Sox6 induction in K562 erythroid cells by proteomic analysis

    KAUST Repository

    Barbarani, Gloria; Ronchi, Antonella; Ruoppolo, Margherita; Santorelli, Lucia; Steinfelder, Robert; Elangovan, Sudharshan; Fugazza, Cristina; Caterino, Marianna

    2017-01-01

    are accompanied with a reduced survival of Sox6-/- red blood cells, resulting in a compensated anemia. Sox6-overexpression in K562cells and in human primary ex vivo erythroid cultures enhances erythroid differentiation and leads to hemoglobinization, the hallmark

  16. The proteomic study of sodium butyrate antiproliferative/cytodifferentiation effects on K562 cells

    Czech Academy of Sciences Publication Activity Database

    Grebeňová, D.; Kuželová, K.; Pluskalová, M.; Pešlová, G.; Halada, Petr; Hrkal, Z.

    2006-01-01

    Roč. 37, - (2006), s. 210-217 ISSN 1079-9796 R&D Projects: GA MZd NL7681 Institutional research plan: CEZ:AV0Z50200510 Keywords : k562 * cell cycle * hemoglobin synthesis Subject RIV: EE - Microbiology, Virology Impact factor: 2.678, year: 2006

  17. Aqueous extract of Crataegus azarolus protects against DNA damage in human lymphoblast Cell K562 and enhances antioxidant activity.

    Science.gov (United States)

    Mustapha, Nadia; Bouhlel, Inès; Chaabane, Fadwa; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

    2014-02-01

    The present study was carried out to characterize the cellular antioxidant effect of the aqueous extract of Crataegus azarolus and its antigenotoxic potential using human myelogenous cells, K562. The antioxidant capacity of this extract was evaluated by determining its cellular antioxidant activity (CAA) in K562 cells. Also, preceding antigenotoxicity assessment, its eventual genotoxicity property was investigated by evaluating its capacity to induce the DNA degradation of treated cell nuclei. As no genotoxicity was detected at different exposure times, its ability to protect cell DNA against H2O2 oxidative effect was investigated, using the "comet assay." It appears that 800 μg/mL of extract inhibited the genotoxicity induced by H2O2 with a rate of 41.30 %, after 4 h of incubation. In addition, this extract revealed a significant cellular antioxidant capacity against the reactive oxygen species in K562 cells.

  18. Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

    Science.gov (United States)

    van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

    2015-01-01

    Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

  19. Induced apoptosis by mild hyperthermia occurs via telomerase inhibition on the three human myeloid leukemia cell lines: TF-1, K562, and HL-60.

    Science.gov (United States)

    Deezagi, Abdolkhaleg; Manteghi, Sanaz; Khosravani, Pardis; Vaseli-Hagh, Neda; Soheili, Zahra-Soheila

    2009-09-01

    The purpose of this research was to understand the effect of hyperthermia on the telomerase activity in human leukemic cell lines (HL-60, K562, and TF-1). The cells were treated by hyperthermia at the range of 41-44 degrees C for 120 min and incubated for 96 h. Then telomerase activity, cell proliferation, and apoptosis were assessed. The results indicated that hyperthermia significantly induced apoptosis on the cells. The cells exhibited pre-apoptotic pattern at 41 and 42 degrees C at 60-120 min and apoptotic pattern at 43 and 44 degrees C over 30 min after hyperthermia. Telomerase activity (that was assayed immediately after hyperthermia) was stable at 41-42 degrees C for 60 min but decreased to 35-40% at 120 min. However, at severe hyperthermia (43-44 degrees C) telomerase activity was decreased in a time- and dose-dependent manner. Following hyperthermia (41-44 degrees C up to 120 min), the cells were incubated for 96 h. In these conditions, the telomerase activity was decreased by about 60-80% in comparison with that untreated control cells.

  20. Apoptosis of leukemia K562 and Molt-4 cells induced by emamectin benzoate involving mitochondrial membrane potential loss and intracellular Ca2+ modulation.

    Science.gov (United States)

    Yun, Xinming; Rao, Wenbing; Xiao, Ciying; Huang, Qingchun

    2017-06-01

    Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca 2+ concentration ([Ca 2+ ] i ) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10μM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48h exposure to 10μM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca 2+ ] i level was peaked and persisted within 70s in K562 cells whilst 50s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

    Science.gov (United States)

    Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

    1997-03-01

    We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

  2. c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min-Sun; Kim, Sun-Young; Arunachalam, Sankarganesh [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Hwang, Pyoung-Han [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Yi, Ho-Keun [Department of Biochemistry, School of Dentistry, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Nam, Sang-Yun [Department of Alternative Therapy, School of Alternative Medicine and Health Science, Jeonju University, Jeonju 561-712 (Korea, Republic of); Lee, Dae-Yeol, E-mail: leedy@chonbuk.ac.kr [Department of Pediatrics, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of); Research Institute of Clinical Medicine, School of Medicine, Chonbuk National University, Jeonju 561-712 (Korea, Republic of)

    2009-07-17

    c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.

  3. c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

    International Nuclear Information System (INIS)

    Kim, Min-Sun; Kim, Sun-Young; Arunachalam, Sankarganesh; Hwang, Pyoung-Han; Yi, Ho-Keun; Nam, Sang-Yun; Lee, Dae-Yeol

    2009-01-01

    c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, and IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.

  4. Caveolin-1 contributes to realgar nanoparticle therapy in human chronic myelogenous leukemia K562 cells

    Directory of Open Access Journals (Sweden)

    Shi D

    2016-11-01

    Full Text Available Dan Shi,1,* Yan Liu,1,* Ronggang Xi,1 Wei Zou,2 Lijun Wu,3 Zhiran Zhang,1 Zhongyang Liu,1 Chao Qu,1 Baoli Xu,1 Xiaobo Wang1 1Department of Pharmacy, The 210th Hospital of People’s Liberation Army, 2College of Life Science, Liaoning Normal University, Dalian, Liaoning, 3Department of Pharmaceutics, College of Pharmacy, Harbin Medical University, Harbin, Heilongjiang, People’s Republic of China *These authors contributed equally to this work Abstract: Chronic myelogenous leukemia (CML is characterized by the t(9;22 (q34;q11-associated Bcr-Abl fusion gene, which is an essential element of clinical diagnosis. As a traditional Chinese medicine, realgar has been widely used for the treatment of various diseases for >1,500 years. Inspired by nano-drug, realgar nanoparticles (NPs have been prepared with an average particle size of <100 nm in a previous work. Compared with coarse realgar, the realgar NPs have higher bioavailability. As a principal constituent protein of caveolae, caveolin-1 (Cav-1 participates in regulating various cellular physiological and pathological processes including tumorigenesis and tumor development. In previous studies, it was found that realgar NPs can inhibit several types of tumor cell proliferation. However, the therapeutic effect of realgar NPs on CML has not been fully elucidated. In the present paper, it was demonstrated that realgar NPs can inhibit the proliferation of K562 cells and degrade Bcr-Abl fusion protein effectively. Both apoptosis and autophagy were activated in a dose-dependent manner in realgar NPs treated cells, and the induction of autophagy was associated with class I phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway. Morphological analysis indicated that realgar NPs induced differentiation effectively in CML cells. Furthermore, it was identified that Cav-1 might play a crucial role in realgar NP therapy. In order to study the effects of Cav-1 on K562 cells during

  5. Antileukemic Effect of Tualang Honey on Acute and Chronic Leukemia Cell Lines

    Directory of Open Access Journals (Sweden)

    Nik Muhd Khuzaimi Nik Man

    2015-01-01

    Full Text Available Complementary medicine using natural product as antitumor is on the rise. Much research has been performed on Tualang Honey and it was shown to have therapeutic potential in wound healing, and antimicrobial activity and be antiproliferative against several cancer models such as human osteosarcoma (HOS, human breast (MCF-7 and MDA-MB-231, and cervical (HeLa cancer cell lines. To date, there was limited study on antileukemic properties of Tualang (Koompassia excelsa Honey. The aim of this study was to evaluate the antileukemic effect of Tualang Honey on acute and chronic leukemia cell lines. Leukemia cell lines (K562 and MV4-11 and human mononuclear cell isolated from peripheral blood were grown in RPM1 1640 culture medium. The cells were incubated with increasing concentrations of Tualang Honey. After incubation, the evaluation of viability and apoptosis was performed. The morphological changes of leukemia cells were the presence of cytoplasmic blebs followed by apoptotic bodies and round shape of cells. IC50 against K562 and MV4-11 was determined. Tualang Honey gave 53.9% and 50.6% apoptosis activity on K562 and MV4-11, respectively, while on human mononuclear cell it was 37.4%. Tualang Honey has the apoptosis-inducing ability for acute and chronic myeloid leukemia (K562 and MV4-11 cell lines.

  6. Dual Functions of the C5a Receptor as a Connector for the K562 Erythroblast-Like Cell-THP-1 Macrophage-Like Cell Island and as a Sensor for the Differentiation of the K562 Erythroblast-Like Cell during Haemin-Induced Erythropoiesis

    Directory of Open Access Journals (Sweden)

    Hiroshi Nishiura

    2012-01-01

    Full Text Available The transcriptional nuclear factor binding to the Y box of human leukocyte antigen genes (NF-Y for the C5a receptor (C5aR gene is active in erythroblasts. However, the roles of the C5aR in erythropoiesis are unclear. We have previously demonstrated that apoptotic cell-derived ribosomal protein S19 (RP S19 oligomers exhibit extraribosomal functions in promoting monocyte chemotaxis and proapoptosis via the C5aR without receptor internalisation. In contrast to the extraribosomal functions of the RP S19, a proapoptotic signal in pro-EBs, which is caused by mutations in the RP S19 gene, is associated with the inherited erythroblastopenia, Diamond-Blackfan anaemia. In this study, we detected C5aR expression and RP S19 oligomer generation in human erythroleukemia K562 cells during haemin-induced erythropoiesis. Under monocell culture conditions, the differentiation into K562 erythrocyte-like cells was enhanced following the overexpression of Wild-type RP S19. Conversely, the differentiation was repressed following the overexpression of mutant RP S19. An RP S19 oligomer inhibitor and a C5aR inhibitor blocked the association of the K562 basophilic EB-like cells and the THP-1 macrophage-like cells under coculture conditions. When bound to RP S19 oligomers, the C5aR may exhibit dual functions as a connector for the EB-macrophage island and as a sensor for EB differentiation in the bone marrow.

  7. 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

  8. Unravelling pathways downstream Sox6 induction in K562 erythroid cells by proteomic analysis

    KAUST Repository

    Barbarani, Gloria

    2017-10-20

    The Sox6 transcription factor is crucial for terminal maturation of definitive red blood cells. Sox6-null mouse fetuses present misshapen and nucleated erythrocytes, due to impaired actin assembly and cytoskeleton stability. These defects are accompanied with a reduced survival of Sox6-/- red blood cells, resulting in a compensated anemia. Sox6-overexpression in K562cells and in human primary ex vivo erythroid cultures enhances erythroid differentiation and leads to hemoglobinization, the hallmark of erythroid maturation. To obtain an overview on processes downstream to Sox6 expression, we performed a differential proteomic analysis on human erythroid K562cells overexpressing Sox6. Sox6-overexpression induces dysregulation of 64 proteins, involved in cytoskeleton remodeling and in protein synthesis, folding and trafficking, key processes for erythroid maturation. Moreover, 43 out of 64 genes encoding for differentially expressed proteins contain within their proximal regulatory regions sites that are bound by SOX6 according to ENCODE ChIP-seq datasets and are possible direct SOX6 targets. SAR1B, one of the most induced proteins upon Sox6 overexpression, shares a conserved regulatory module, composed by a double SOX6 binding site and a GATA1 consensus, with the adjacent SEC24 A gene. Since both genes encode for COPII components, this element could concur to the coordinated expression of these proteins during erythropoiesis.

  9. Co-ordinate expression of activin A and its type I receptor mRNAs during phorbol ester-induced differentiation of human K562 erythroleukemia cells.

    Science.gov (United States)

    Hildén, K; Tuuri, T; Erämaa, M; Ritvos, O

    1999-07-20

    Activins were originally isolated based on their ability to stimulate follicle-stimulating hormone secretion but later they have been shown to regulate a number of different cellular functions such as nerve cell survival, mesoderm induction during early embryogenesis as well as hematopoiesis. We studied the regulation of activin A, a homodimer of betaA-subunits, mRNA and protein in K562 erythroleukemia cells, which are known to be induced toward the erythroid lineage in response to activin or TGF-beta or toward the megakaryocytic lineage by the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Here we show by Northern blot analysis as well as by Western and ligand blotting that TPA strongly promotes activin betaA-subunit mRNA and activin A protein expression in K562 cells in time- and concentration dependent manner. In contrast, neither activin A nor TGF-beta induced betaA-subunit mRNA expression during erythroid differentiation in K562 cells. Interestingly, whereas activin type II receptors are not regulated during K562 cell differentiation (Hilden et al. (1994) Blood 83, 2163-2170), we now show that the activin type I and IB receptor mRNAs are clearly induced by TPA but not by activin or TGF-beta. We also show that the inducing effect of TPA on expression of activin betaA-subunit mRNA is potentiated by the protein kinase A activator 8-bromo-cAMP. We conclude that activin A and its type I receptors appear to be co-ordinately up-regulated during megakaryocytic differentiation of K562 cells.

  10. Bcr-Abl-independent mechanism of resistance to imatinib in K562 cells: Induction of cyclooxygenase-2 (COX-2) by histone deacetylases (HDACs).

    Science.gov (United States)

    Kalle, Arunasree M; Sachchidanand, Sachchidanand; Pallu, Reddanna

    2010-09-01

    Our previous studies have shown that overexpression of MDR1 and cyclooygenase-2 (COX-2) resulted in resistance development to imatinib in chronic myelogenous leukemia (CML) K562 (IR-K562) cells. In the present study, the regulatory mechanism of MDR1 induction by COX-2 was investigated. A gradual overexpression of MDR1 and COX-2 during the process of development was observed. Furthermore, down regulation of MDR1 upon COX-2 knockdown by siRNA showed a decrease in the PKC levels and activation of PKC by addition of PGE(2) to K562 cells, suggesting a role for PKC in the COX-2 mediated induction of MDR1. The present study demonstrates COX-2 induction by HDACs and MDR1 induction by COX-2 via PGE(2)-cAMP-PKC-mediated pathway. Copyright 2010 Elsevier Ltd. All rights reserved.

  11. Evaluation of Synergetic Anticancer Activity of Berberine and Curcumin on Different Models of A549, Hep-G2, MCF-7, Jurkat, and K562 Cell Lines

    Directory of Open Access Journals (Sweden)

    Acharya Balakrishna

    2015-01-01

    Full Text Available Ayurvedic system of medicine is using Berberis aristata and Curcuma longa herbs to treat different diseases including cancer. The study was performed to evaluate the synergetic anticancer activity of Berberine and Curcumin by estimating the inhibition of the cell proliferation by cytotoxicity assay using MTT method on specified human cell lines (A549, Hep-G2, MCF-7, Jurkat, and K562. All the cells were harvested from the culture and seeded in the 96-well assay plates at seeding density of 2.0 × 104 cells/well and were incubated for 24 hours. Test items Berberine with Curcumin (1 : 1, Curcumin 95% pure, and Berberine 95% pure were exposed at the concentrations of 1.25, 0.001, and 0.5 mg/mL, respectively, and incubated for a period of 48 hours followed by dispensing MTT solution (5 mg/mL. The cells were incubated at 37 ± 1°C for 4 hours followed by addition of DMSO for dissolving the formazan crystals and absorbance was read at 570 nm. Separate wells were prepared for positive control, controls (only medium with cells, and blank (only medium. The results had proven the synergetic anticancer activity of Berberine with Curcumin inducing cell death greater percentage of >77% when compared to pure curcumin with <54% and pure Berberine with <45% on average on all cell line models.

  12. In Vitro Characterization and Evaluation of the Cytotoxicity Effects of Nisin and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines.

    Science.gov (United States)

    Goudarzi, Fariba; Asadi, Asadollah; Afsharpour, Maryam; Jamadi, Robab Hassanvand

    2018-05-01

    The aim of this study was an in vitro evaluation and comparison of the cytotoxic effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical properties, including formation, size, and shape, were studied using hemolysis, fluorescein isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading efficiency and release kinetics were examined to assess the potential impact of this formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays. Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange (AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter nanoparticles was about 85-90%. The hemolysis test represented their non-cytotoxicities and the FITC images indicated their entrance inside the cells. An increase in the percentage of apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.

  13. 33 CFR 183.562 - Metallic fuel lines.

    Science.gov (United States)

    2010-07-01

    ...) BOATING SAFETY BOATS AND ASSOCIATED EQUIPMENT Fuel Systems Manufacturer Requirements § 183.562 Metallic fuel lines. (a) Each metallic fuel line that is mounted to the boat structure must be connected to the engine by a flexible fuel line. (b) Each metallic fuel line must be attached to the boat's structure...

  14. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    International Nuclear Information System (INIS)

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui; Cheng, Tian-Lu; Lin, Shinne-Ren; Chang, Long-Sen

    2015-01-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression

  15. Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates.

    Science.gov (United States)

    Shinkai, Yasuhiro; Kashihara, Shinichi; Minematsu, Go; Fujii, Hirofumi; Naemura, Madoka; Kotake, Yojiro; Morita, Yasutaka; Ohnuki, Koichiro; Fokina, Alesya A; Stetsenko, Dmitry A; Filichev, Vyacheslav V; Fujii, Masayuki

    2017-06-01

    Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideβ7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideβ7 is a promising candidate for in vivo use and therapeutic applications.

  16. Small Molecule TH-39 Potentially Targets Hec1/Nek2 Interaction and Exhibits Antitumor Efficacy in K562 Cells via G0/G1 Cell Cycle Arrest and Apoptosis Induction.

    Science.gov (United States)

    Zhu, Yongxia; Wei, Wei; Ye, Tinghong; Liu, Zhihao; Liu, Li; Luo, Yong; Zhang, Lidan; Gao, Chao; Wang, Ningyu; Yu, Luoting

    2016-01-01

    Cancer is still a major public health issue worldwide, and new therapeutics with anti-tumor activity are still urgently needed. The anti-tumor activity of TH-39, which shows potent anti-proliferative activity against K562 cells with an IC50 of 0.78 µM, was investigated using immunoblot, co-immunoprecipitation, the MTT assay, and flow cytometry. Mechanistically, TH-39 may disrupt the interaction between Hec1 and Nek2 in K562 cells. Moreover, TH-39 inhibited cell proliferation in a concentration- and time-dependent manner by influencing the morphology of K562 cells and inducing G0/G1 phase arrest. G0/G1 phase arrest was associated with down-regulation of CDK2-cyclin E complex and CDK4/6-cyclin D complex activities. Furthermore, TH-39 also induced cell apoptosis, which was associated with activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax. TH-39 could also decrease mitochondrial membrane potential (Δψm) and increase reactive oxygen species (ROS) accumulation in K562 cells. The results indicated that TH-39 might induce apoptosis via the ROS-mitochondrial apoptotic pathway. This study highlights the potential therapeutic efficacy of the anti-cancer compound TH-39 in treatment-resistant chronic myeloid leukemia. © 2016 The Author(s) Published by S. Karger AG, Basel.

  17. Nuclear topography of beta-like globin gene cluster in IL-3-stimulated human leukemic K-562 cells

    Czech Academy of Sciences Publication Activity Database

    Galiová-Šustáčková, Gabriela; Bártová, Eva; Kozubek, Stanislav

    2004-01-01

    Roč. 33, č. 1 (2004), s. 4-14 ISSN 1079-9796 R&D Projects: GA ČR GA301/01/0186; GA AV ČR KSK5052113; GA AV ČR IAA5004306; GA ČR GA202/04/0907; GA MŠk ME 565 Institutional research plan: CEZ:AV0Z5004920 Keywords : beta-like globin gene cluster * K-562 cells * nuclear topography Subject RIV: BO - Biophysics Impact factor: 2.549, year: 2004

  18. 6′-Hydroxy Justicidin B Triggers a Critical Imbalance in Ca2+ Homeostasis and Mitochondrion-Dependent Cell Death in Human Leukemia K562 Cells

    Directory of Open Access Journals (Sweden)

    Jiaoyang Luo

    2018-06-01

    Full Text Available Justicia procumbens (J. procumbens is a traditional Chinese herbal medicine which was used for the treatment of fever, pain, and cancer. A compound 6′-hydroxy justicidin B (HJB isolated from J. procumbens exhibits promising biological properties. However, the mechanism of action and the in vivo behavior of HJB remain to be elucidated. In this study, we investigated the mechanism of action of HJB on human leukemia K562 cells and its pharmacokinetic properties in rats. The results demonstrated that HJB significantly inhibited the proliferation of K562 cells and promoted apoptosis. Besides, HJB resulted in decreased mitochondrial membrane potential deltaPSIm, increased the level of the calcium homeostasis regulator protein TRPC6 and cytosolic calcium. The activity of caspase-8, caspase-9 and the expression of p53 were significantly increased after treatment with HJB. Additionally, HJB has rapid absorption rate and relative long elimination t1/2, indicating a longer residence time in vivo. The results indicate that HJB inhibited the proliferation of K562 cells and induced apoptosis by affecting the function of mitochondria and calcium homeostasis to activate the p53 signaling pathway. The pharmacokinetic study of HJB suggested it is absorbed well and has moderate metabolism in vivo. These results present HJB as a potential novel alternative to standard human leukemia therapies.

  19. MiR-27a Promotes Hemin-Induced Erythroid Differentiation of K562 Cells by Targeting CDC25B

    Directory of Open Access Journals (Sweden)

    Dongsheng Wang

    2018-03-01

    Full Text Available Background/Aims: MicroRNAs (miRNAs play a crucial role in erythropoiesis. MiR-23a∼27a∼24-2 clusters have been proven to take part in erythropoiesis via some proteins. CDC25B (cell division control Cdc2 phosphostase B is also the target of mir-27a; whether it regulates erythropoiesis and its mechanism are unknown. Methods: To evaluate the potential role of miR-27a during erythroid differentiation, we performed miR-27a gain- and loss-of-function experiments on hemin-induced K562 cells. We detected miR-27a expression after hemin stimulation at different time points. At the same time, the γ-globin gene also was measured via real-time PCR. According to the results of the chips, we screened the target protein of miR-27a through a dual-luciferase reporter assay and identified it via Western blot analyses. To evaluate the function of CDC25B, benzidine staining and flow cytometry were employed to detect the cell differentiation and cell cycle. Results: We found that miR-27a promotes hemin-induced erythroid differentiation of human K562 cells by targeting cell division cycle 25 B (CDC25B. Overexpression of miR-27a promotes the differentiation of hemin-induced K562 cells, as demonstrated by γ-globin overexpression. The inhibition of miR-27a expression suppresses erythroid differentiation, thus leading to a reduction in the γ-globin gene. CDC25B was identified as a new target of miR-27a during erythroid differentiation. Overexpression of miR-27a led to decreased CDC25B expression after hemin treatment, and CDC25B was up-regulated when miR-27a expression was inhibited. Moreover, the inhibition of CDC25B affected erythroid differentiation, as assessed by γ-globin expression. Conclusion: This study is the first report of the interaction between miR-27a and CDC25B, and it improves the understanding of miRNA functions during erythroid differentiation.

  20. Overcoming imatinib resistance using Src inhibitor CGP76030, Abl inhibitor nilotinib and Abl/Lyn inhibitor INNO-406 in newly established K562 variants with BCR-ABL gene amplification.

    Science.gov (United States)

    Morinaga, Koji; Yamauchi, Takahiro; Kimura, Shinya; Maekawa, Taira; Ueda, Takanori

    2008-06-01

    Because imatinib (IM) resistance in chronic myeloid leukemia is primarily caused by the re-establishment of Abl kinase, new inhibitors may be efficacious. We evaluated 3 new agents against 2 new K562 variants, IM-R1 and IM-R2 cells, which were developed having 7- and 27-fold greater IM resistance, respectively, than the parental K562 cells. Both variants possessed BCR-ABL gene amplification along with elevated levels of its transcript and protein. Greater BCR-ABL gene amplification was observed in IM-R2 cells than in IM-R1 cells, which was consistent with the higher mRNA and protein levels of Bcr-Abl, and ultimately correlated with the greater IM resistance in IM-R2 cells. No mutation in the Abl kinase domain was detected in either variant. Despite the absence of Lyn overexpression, the Src kinase inhibitor CGP76030 showed positive cooperability with IM in inhibiting cell growth of not only K562 cells but also these 2 variants. This might be because of the augmented inhibition of Erk1/2 phosphorylation. The new Abl kinase inhibitor nilotinib was 10-fold more potent than IM in inhibiting the growth of K562 cells. Nilotinib inhibited the growth of IM-R1 and IM-R2 cells as potently as K562 cells. The combination of nilotinib with CGP76030 showed little additivity, because the potency of nilotinib masked the efficacy of CGP76030. The new dual Abl/Lyn inhibitor INNO-406 (formerly NS-187) was slightly more potent than nilotinib in inhibiting the growth of all 3 cell lines. Because BCR-ABL gene amplification occurs in blast crisis, these inhibitors might overcome IM resistance in such patients' leukemia. (c) 2008 Wiley-Liss, Inc.

  1. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl+ K562 and Jak2(V617F)+ HEL Leukemia Cells

    International Nuclear Information System (INIS)

    Weber, Axel; Borghouts, Corina; Brendel, Christian; Moriggl, Richard; Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd

    2015-01-01

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl + K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells, Stat5

  2. Comparative Study of Different Nano-Formulations of Curcumin for Reversal of Doxorubicin Resistance in K562R Cells.

    Science.gov (United States)

    Dash, Tapan K; Konkimalla, V Badireenath

    2017-02-01

    Curcumin is very well established as a chemo-therapeutic, chemo-preventive and chemo-sensitizing agent in diverse disease conditions. As the isolated pure form has poor solubility and pharmacokinetic problems, therefore it is encapsulated in to several nano-formulations to improve its bioavailability. Here in the current study, we aim to compare different nano-formulations of curcumin for their chemo-sensitizing activity in doxorubicin (DOX) resistant K562 cells. Four different curcumin formulations were prepared namely DMSO assisted curcumin nano-dispersion (CurD, 260 nm), liposomal curcumin (CurL, 165 nm), MPEG-PCL micellar curcumin (CurM, 18 nm) and cyclodextrin encapsulated curcumin (CurN, 37 nm). The formulations were subjected to particle characterizations (size, zeta potential, release studies), followed by biological assays such as cellular uptake, P-gp inhibitory activity and reversal of DOX resistance by co-treatment with DOX. Curcumin uptake in K562N and K562R cells was mildly reduced when treated with CurL and CurM, while for CurD and CurN the uptake remained equivalent. However, CurL retained P-gp inhibitory activity of curcumin and with a considerable chemo-sensitizing effect but CurM showed no P-gp inhibitory activity. CurN retained above biological activities, but requires a secondary carrier under in vivo conditions. From the results, CurM was found to be most suitable for solubilization of curcumin where as CurL can be considered as most suitable nano-formulation for reversal of DOX resistance.

  3. Effects of light irradiation upon photodynamic therapy based on 5-aminolevulinic acid–gold nanoparticle conjugates in K562 cells via singlet oxygen generation

    Directory of Open Access Journals (Sweden)

    Xu H

    2012-09-01

    Full Text Available Hao Xu, Chen Liu, Jiansheng Mei, Cuiping Yao, Sijia Wang, Jing Wang, Zheng Li, Zhenxi ZhangKey Laboratory of Biomedical Information Engineering of Education Ministry, Institute of Biomedical Analytical Technology and Instrumentation, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, Shannxi, People’s Republic of ChinaPurpose: As a precursor of the potent photosensitizer protoporphyrin IX (PpIX, 5-aminolevulinic acid (5-ALA, was conjugated onto cationic gold nanoparticles (GNPs to improve the efficacy of photodynamic therapy (PDT.Methods: Cationic GNPs reduced by branched polyethyleneimine and 5-ALA were conjugated onto the cationic GNPs by creating an electrostatic interaction at physiological pH. The efficacy of ALA-GNP conjugates in PDT was investigated under irradiation with a mercury lamp (central wavelength of 395 nm and three types of light-emitting diode arrays (central wavelengths of 399, 502, and 621 nm, respectively. The impacts of GNPs on PDT were then analyzed by measuring the intracellular PpIX levels in K562 cells and the singlet oxygen yield of PpIX under irradiation.Results: The 2 mM ALA-GNP conjugates showed greater cytotoxicity against K562 cells than ALA alone. Light-emitting diode (505 nm irradiation of the conjugates caused a level of K562 cell destruction similar to that with irradiation by a mercury lamp, although it had no adverse effects on drug-free control cells. These results may be attributed to the singlet oxygen yield of PpIX, which can be enhanced by GNPs.Conclusion: Under irradiation with a suitable light source, ALA-GNP conjugates can effectively destroy K562 cells. The technique offers a new strategy of PDT.Keywords: nonradiative energy transfer, photodamage, protoporphyrin IX, selective destruction, singlet oxygen sensor green reagent, surface plasmon resonance

  4. Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

    Science.gov (United States)

    Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

    2010-06-15

    CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation. 2010 Elsevier Ltd. All rights reserved.

  5. Stat5 Exerts Distinct, Vital Functions in the Cytoplasm and Nucleus of Bcr-Abl{sup +} K562 and Jak2(V617F){sup +} HEL Leukemia Cells

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Axel [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany); Borghouts, Corina [Ganymed Pharmaceuticals AG, Mainz 55131 (Germany); Brendel, Christian [Boston Children’s Hospital, Division of Hematology/Oncology, Boston, MA 02115 (United States); Moriggl, Richard [Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna 1090 (Austria); Delis, Natalia; Brill, Boris; Vafaizadeh, Vida; Groner, Bernd, E-mail: Groner@em.uni-frankfurt.de [Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt am Main 60596 (Germany)

    2015-03-19

    Signal transducers and activators of transcription (Stats) play central roles in the conversion of extracellular signals, e.g., cytokines, hormones and growth factors, into tissue and cell type specific gene expression patterns. In normal cells, their signaling potential is strictly limited in extent and duration. The persistent activation of Stat3 or Stat5 is found in many human tumor cells and contributes to their growth and survival. Stat5 activation plays a pivotal role in nearly all hematological malignancies and occurs downstream of oncogenic kinases, e.g., Bcr-Abl in chronic myeloid leukemias (CML) and Jak2(V617F) in other myeloproliferative diseases (MPD). We defined the mechanisms through which Stat5 affects growth and survival of K562 cells, representative of Bcr-Abl positive CML, and HEL cells, representative for Jak2(V617F) positive acute erythroid leukemia. In our experiments we suppressed the protein expression levels of Stat5a and Stat5b through shRNA mediated downregulation and demonstrated the dependence of cell survival on the presence of Stat5. Alternatively, we interfered with the functional capacities of the Stat5 protein through the interaction with a Stat5 specific peptide ligand. This ligand is a Stat5 specific peptide aptamer construct which comprises a 12mer peptide integrated into a modified thioredoxin scaffold, S5-DBD-PA. The peptide sequence specifically recognizes the DNA binding domain (DBD) of Stat5. Complex formation of S5-DBD-PA with Stat5 causes a strong reduction of P-Stat5 in the nuclear fraction of Bcr-Abl-transformed K562 cells and a suppression of Stat5 target genes. Distinct Stat5 mediated survival mechanisms were detected in K562 and Jak2(V617F)-transformed HEL cells. Stat5 is activated in the nuclear and cytosolic compartments of K562 cells and the S5-DBD-PA inhibitor most likely affects the viability of Bcr-Abl{sup +} K562 cells through the inhibition of canonical Stat5 induced target gene transcription. In HEL cells

  6. Phenethyl isothiocyanate inhibits growth of human chronic myeloid leukemia K562 cells via reactive oxygen species generation and caspases.

    Science.gov (United States)

    Wang, Yating; Wei, Sixi; Wang, Jishi; Fang, Qin; Chai, Qixiang

    2014-07-01

    Phenethyl isothiocyanate (PEITC), a potential cancer chemopreventive constituent of cruciferous vegetables, including watercress, has been reported to inhibit cancer cell growth by arresting the cell cycle and inducing apoptosis in various human cancer cell models. However, the role of PEITC in the inhibition of human chronic myeloid leukemia (CML) K562 cell growth and its underlying mechanisms have yet to be elucidated. In the present study, PEITC was found to induce cell death through the induction of reactive oxygen species (ROS) stress and oxidative damage. Heme oxygenase‑1 (HO‑1), which participates in the development of numerous tumors and the sensitivity of these tumors to chemotherapeutic drugs, plays a protective role by modulating oxidative injury. Therefore, the present study assessed the inhibitory effect of PEITC on K562 cells and whether HO‑1 facilitated cell apoptosis and ROS generation. PEITC was found to suppress cell growth and cause apoptosis by promoting Fas and Fas ligand expression, increasing ROS generation and by the successive release of cytochrome c as well as the activation of caspase‑9 and caspase‑3. PEITC was also combined with the HO‑1 inhibitor zinc protoporphyrin IX and the inducer hemin to assess whether HO‑1 determines cell survival and ROS generation. The results of the present study suggest that PEITC may be a potential anti‑tumor compound for CML therapy, and that HO‑1 has a critical function in PEITC‑induced apoptosis and ROS generation.

  7. 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.

  8. Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

    Science.gov (United States)

    Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

    2016-05-01

    Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

  9. Natural and semi-synthetic clerodanes of Croton cajucara and their cytotoxic effects against ehrlich carcinoma and human K562 leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Maciel, Maria Aparecida M. [Universidade Federal do Rio Grande do Norte, Natal, RN (Brazil). Dept. de Quimica; Martins, Jenilce R.; Pinto, Angelo C.; Kaiser, Carlos R. [Universidade Federal, Rio de Janeiro, RJ (Brazil). Inst. de Quimica; Esteves-Souza, Andressa; Echevarria, Aurea [Universidade Federal Rural do Rio de Janeiro, Seropedica, RJ (Brazil). Dept. de Quimica]. E-mail: echevarr@ufrrj.br

    2007-03-15

    The clerodane-type diterpene, trans-dehydrocrotonin (1) the major component of Croton cajucara has shown striking correlation with its therapeutic use in traditional folk medicine. Phytochemical investigations led to the isolation of the metabolites 1, cajucarinolide (6), isocajucarinolide (7), trans-crotonin (2), trans-cajucarin B (3), cis-cajucarin B (4), trans-cajucarin A (5), N-methyltyrosine, vanillic acid and 4-hydroxy-benzoic acid. 6 and 7 were synthesized in good yield by regiospecific oxidation of 1 using singlet-oxygen. All clerodanes were studied for their cytotoxic effects against human K562 leukemia and Ehrlich carcinoma cells. Ehrlich carcinoma assays with IC{sub 50} = 166 {mu}M (1), 164 {mu}M (2), 65 {mu}M (6) and 10 {mu}M (7) related to cell growth inhibitory effects were dose dependent. Furthermore, moderate cytotoxic activity against K562 leukemia cells was observed with IC{sub 50} = 38 {mu}M (3), 33 {mu}M (5), 36 {mu}M (6) and 43 {mu}M (7). The semi-synthetic 2, 6 and 7 showed similar results when compared to the corresponding natural clerodanes. (author)

  10. Natural and semi-synthetic clerodanes of Croton cajucara and their cytotoxic effects against ehrlich carcinoma and human K562 leukemia cells

    International Nuclear Information System (INIS)

    Maciel, Maria Aparecida M.; Martins, Jenilce R.; Pinto, Angelo C.; Kaiser, Carlos R.; Esteves-Souza, Andressa; Echevarria, Aurea

    2007-01-01

    The clerodane-type diterpene, trans-dehydrocrotonin (1) the major component of Croton cajucara has shown striking correlation with its therapeutic use in traditional folk medicine. Phytochemical investigations led to the isolation of the metabolites 1, cajucarinolide (6), isocajucarinolide (7), trans-crotonin (2), trans-cajucarin B (3), cis-cajucarin B (4), trans-cajucarin A (5), N-methyltyrosine, vanillic acid and 4-hydroxy-benzoic acid. 6 and 7 were synthesized in good yield by regiospecific oxidation of 1 using singlet-oxygen. All clerodanes were studied for their cytotoxic effects against human K562 leukemia and Ehrlich carcinoma cells. Ehrlich carcinoma assays with IC 50 = 166 μM (1), 164 μM (2), 65 μM (6) and 10 μM (7) related to cell growth inhibitory effects were dose dependent. Furthermore, moderate cytotoxic activity against K562 leukemia cells was observed with IC 50 = 38 μM (3), 33 μM (5), 36 μM (6) and 43 μM (7). The semi-synthetic 2, 6 and 7 showed similar results when compared to the corresponding natural clerodanes. (author)

  11. SENYAWA BIOAKTIF RIMPANG JAHE (Zingiber officinale Roscue MENINGKATKAN RESPON SITOLITIK SEL NK TERHADAP SEL KANKER DARAH K-562 IN VITRO [Ginger Root Bioactive Compounds Increased Cytolitic Response of Natural Killer (NK Cells Against Leucemic Cell Line K-562 In Vitro

    Directory of Open Access Journals (Sweden)

    Fransiska Rungkat Zakaria 2

    2006-08-01

    Full Text Available Natural killer (NK cell, a kind of lymphocyte cells, plays an important role in attacking infectious, immature, and cancer cell. Its function could be modulated by food bioactive compounds. This experiment was conducted to investigate the effects of ginger root bioactive compounds such as oleoresin, gingerol, and shogaol on cytolitic response of NK cell in vitro. Lymphocyte cells were isolated by centrifugation on ficoll-hypaque density (1,77 ?0,001 g/ml method. Leukemic cells line K-562 as target cells(TC labelled by [3H]-timidin, together with lymphocyte as effector cell (EC were cultured in two ratio levels of EC : TC equal to 1:50 and 1:100, and two culture conditions, for 4 hours, respectively. Paraquate dichloride (1,1-dimethyl-4,4-bipyridilium dichloride 3 mM was used to induce stress oxidative circumstance. Cytolytic capacity of NK cells was determined by percentage of TC lysed by NK cells, in normal and oxidative stress conditions. Statistical analysis showed that the effects of ginger bioactive compounds on cytolytic response of NK cell depended on the culture conditions, as shown by cultures in the presence of oleoresin, and gingerol, but not shogaol. In the lymphocyte culture without stress oxidative, oleoresin, gingerol and shogaol compounds increased significantly cytolytic response of NK cells cultured at a ratio of TC : EC equal to 1:50, with the highest increament of 65 % at oleoresin concentration of 50 ?g/ml. However, in culture at a ratio of TC : EC equals to 1:100, only oleoresin at a concentration of 50 ?g/ml increased significantly cytolytic response of NK cells with the highest increament of 8 %. Shogaol did not affect significantly NK cells cytolytic response. Under stress oxidative conditions, shogaol increased significantly cytolytic response of NK cells cultured at a ratio of TC:EC equal to 1:50, but the highest increament of 56 % , was by oleoresin at concentration of 50 ?g/ml. Meanwhile, oleoresin and gingerol did

  12. 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)

  13. Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Seong-Hoon Yun

    2018-04-01

    Full Text Available Marine triterpene glycosides are attractive candidates for the development of anticancer agents. Holotoxin A1 is a triterpene glycoside found in the edible sea cucumber, Apostichopus (Stichopus japonicus. We previously showed that cladoloside C2, the 25(26-dihydro derivative of holotoxin A1, induced apoptosis in human leukemia cells by activating ceramide synthase 6. Thus, we hypothesized that holotoxin A1, which is structurally similar to cladoloside C2, might induce apoptosis in human leukemia cells through the same molecular mechanism. In this paper, we compared holotoxin A1 and cladoloside C2 for killing potency and mechanism of action. We found that holotoxin A1 induced apoptosis more potently than cladoloside C2. Moreover, holotoxin A1 induced apoptosis in K562 cells by activating caspase-8 and caspase-3, but not by activating caspase-9. During holotoxin A1-induced apoptosis, acid sphingomyelinase (SMase and neutral SMase were activated in both K562 cells and human primary leukemia cells. Specifically inhibiting acid SMase and neutral SMаse with chemical inhibitors or siRNAs significantly inhibited holotoxin A1–induced apoptosis. These results indicated that holotoxin A1 might induce apoptosis by activating acid SMase and neutral SMase. In conclusion, holotoxin A1 represents a potential anticancer agent for treating leukemia. Moreover, the aglycone structure of marine triterpene glycosides might affect the mechanism involved in inducing apoptosis.

  14. A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe.

    Directory of Open Access Journals (Sweden)

    Macario Martinez-Castillo

    Full Text Available Curcumin is extensively investigated as a good chemo-preventive agent in the development of many cancers and particularly in leukemia, including treatment of chronic myelogenous leukemia and it has been proposed as an adjuvant for leukemia therapies. Human chronic myeloid leukemia cells (K562, were treated with 20 μM of curcumin, and we found that a subpopulation of these cells were arrested and accumulate in the G2/M phase of the cell cycle. Characterization of this cell subpopulation showed that the arrested cells presented nuclear morphology changes resembling those described for mitotic catastrophe. Mitotic cells displayed abnormal chromatin organization, collapse of the mitotic spindle and abnormal chromosome segregation. Then, these cells died in an apoptosis dependent manner and showed diminution in the protein levels of BCL-2 and XIAP. Moreover, our results shown that a transient activation of the nuclear factor κB (NFκB occurred early in these cells, but decreased after 6 h of the treatment, explaining in part the diminution of the anti-apoptotic proteins. Additionally, P73 was translocated to the cell nuclei, because the expression of the C/EBPα, a cognate repressor of the P73 gene, was decreased, suggesting that apoptosis is trigger by elevation of P73 protein levels acting in concert with the diminution of the two anti-apoptotic molecules. In summary, curcumin treatment might produce a P73-dependent apoptotic cell death in chronic myelogenous leukemia cells (K562, which was triggered by mitotic catastrophe, due to sustained BAX and survivin expression and impairment of the anti-apoptotic proteins BCL-2 and XIAP.

  15. Time-series analysis in imatinib-resistant chronic myeloid leukemia K562-cells under different drug treatments.

    Science.gov (United States)

    Zhao, Yan-Hong; Zhang, Xue-Fang; Zhao, Yan-Qiu; Bai, Fan; Qin, Fan; Sun, Jing; Dong, Ying

    2017-08-01

    Chronic myeloid leukemia (CML) is characterized by the accumulation of active BCR-ABL protein. Imatinib is the first-line treatment of CML; however, many patients are resistant to this drug. In this study, we aimed to compare the differences in expression patterns and functions of time-series genes in imatinib-resistant CML cells under different drug treatments. GSE24946 was downloaded from the GEO database, which included 17 samples of K562-r cells with (n=12) or without drug administration (n=5). Three drug treatment groups were considered for this study: arsenic trioxide (ATO), AMN107, and ATO+AMN107. Each group had one sample at each time point (3, 12, 24, and 48 h). Time-series genes with a ratio of standard deviation/average (coefficient of variation) >0.15 were screened, and their expression patterns were revealed based on Short Time-series Expression Miner (STEM). Then, the functional enrichment analysis of time-series genes in each group was performed using DAVID, and the genes enriched in the top ten functional categories were extracted to detect their expression patterns. Different time-series genes were identified in the three groups, and most of them were enriched in the ribosome and oxidative phosphorylation pathways. Time-series genes in the three treatment groups had different expression patterns and functions. Time-series genes in the ATO group (e.g. CCNA2 and DAB2) were significantly associated with cell adhesion, those in the AMN107 group were related to cellular carbohydrate metabolic process, while those in the ATO+AMN107 group (e.g. AP2M1) were significantly related to cell proliferation and antigen processing. In imatinib-resistant CML cells, ATO could influence genes related to cell adhesion, AMN107 might affect genes involved in cellular carbohydrate metabolism, and the combination therapy might regulate genes involved in cell proliferation.

  16. Cellular uptake, nuclear localization and cytotoxicity of 125I-labelled DNA minor groove binding ligands in K562, human erythroleukaemia cells

    International Nuclear Information System (INIS)

    Karagiannis, T.C.; Lobachevsky, P.N.; Martin, R.F.

    2000-01-01

    Full text: Iodine-125 decays by orbital electron capture and internal conversion resulting in the emission of numerous Auger electrons which produce a highly localised radiochemical damage in the immediate vicinity of the site of decay. Given the requirement to deliver 125 I to the nuclear DNA, a minor groove binding bibenzimidazole, 125 I-iodoHoechst 33258 was investigated. It has been noted that this analogue may be prone to de-iodination in vitro and in vivo, given the presence of an orthoiodophenol moiety which is analogous to that in thyroxins. Therefore, an 125 I -iodoHoechst analogue without the hydroxyl group was also studied. The 125 I -iodoHoechst 33258 analogue was prepared by direct iodination of Hoechst 33258 and 125 I iodoHoechst was prepared by demetallation of a trimethylstannyl precursor. DNA binding studies indicated that both iodo-analogues bind to calf thymus DNA, K D = 89 ± 30nM, n = 0.018 bp - 1 for iodoHoechst 33258 and K D = 121 ± 31nM, n = 0.024 bp -1 for iodoHoechst. Similarly, nuclear localization following incubation with 5μM of either ligand at 37 deg C was observed in K562 cells by fluorescence microscopy. Flow cytometry was used to investigate the kinetics of drug uptake and efflux in K562 cells. The results indicated that when 10 6 cells were incubated with 5μM ligand at 37 deg C, the uptake reached a plateau at approximately 43 minutes for iodoHoechst 33258 and approximately 52 minutes for iodoHoechst. Ligand efflux results indicated two-phase kinetics. The initial phase which involves 50-60% of drug was characterised by a half-life time (t 1/2 ) of 55.4 minutes for efflux of iodoHoechst 33258 and a t 1/2 of 10.3 minutes for efflux of iodoHoechst, at 37 deg C. Furthermore, the results suggested that the DNA binding sites in a 10 6 cell/ml suspension were saturated by incubation with 3μM iodoHoechst 33258 and 5μM iodoHoechst. In the initial cytotoxicity experiments using 125 I-iodoHoechst 33258, K562 cells were incubated for 1

  17. Change of cell cycle arrest of tumor cell lines after 60Co γ-irradiation

    International Nuclear Information System (INIS)

    Tang Yi; Liu Wenli; Zhou Jianfeng; Gao Qinglei; Wu Jianhong

    2003-01-01

    Objective: To observe the cell cycle arrest changes in peripheral blood mononuclear cells (PBMNCs) of normal persons and several kinds of tumor cell lines after 60 Co γ-irradiation. Methods: PBMNCs of normal persons, HL-60, K562, SiHA and 113 tumor cell lines were irradiated with 60 Co γ-rays at the absorbed doses of 6, 10,15 Gy. Cell cycles changes were checked 6, 12, 24, 48 and 60 h after the irradiation. Results: A stasis state was observed in normal person PBMNCs, 95 percents of which were in G 1 phase, and they still remained stasis after the irradiation. Except the 113 cell line manifesting G 1 phase arrest, all other tumor cell lines showed G 2 /M phase arrest after irradiation. The radiation sensitivity of HL-60 was higher than that of SiHA cell line. Conclusion: Different cell lines have different cell cycle arrest reaction to radiation and their radiation sensitivity are also different

  18. Celecoxib sensitizes imatinib-resistant K562 cells to imatinib by inhibiting MRP1-5, ABCA2 and ABCG2 transporters via Wnt and Ras signaling pathways.

    Science.gov (United States)

    Dharmapuri, Gangappa; Doneti, Ravinder; Philip, Gundala Harold; Kalle, Arunasree M

    2015-07-01

    Imatinib mesylate, a tyrosine kinase inhibitor, is very effective in the treatment of chronic myeloid leukemia (CML). However, development of resistance to imatinib therapy is also a very common mechanism observed with long-term administration of the drug. Our previous studies have highlighted the role of cyclooxygenase-2 (COX-2) in regulating the expression of multidrug resistant protein-1 (MDR1), P-gp, in imatinib-resistant K562 cells (IR-K562) via PGE2-cAMP-PKC-NF-κB pathway and inhibition of COX-2 by celecoxib, a COX-2 specific inhibitor, inhibits this pathway and reverses the drug resistance. Studies have identified that not only MDR1 but other ATP-binding cassette transport proteins (ABC transporters) are involved in the development of imatinib resistance. Here, we tried to study the role of COX-2 in the regulation of other ABC transporters such as MRP1, MRP2, MRP3, ABCA2 and ABCG2 that have been already implicated in imatinib resistance development. The results of the study clearly indicated that overexpression of COX-2 lead to upregulation of MRP family proteins in IR-K562 cells and celecoxib down-regulated the ABC transporters through Wnt and MEK signaling pathways. The study signifies that celecoxib in combination with the imatinib can be a good alternate treatment strategy for the reversal of imatinib resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. H ferritin silencing induces protein misfolding in K562 cells: A Raman analysis

    KAUST Repository

    Zolea, Fabiana

    2015-10-09

    The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation.

  20. H ferritin silencing induces protein misfolding in K562 cells: A Raman analysis

    KAUST Repository

    Zolea, Fabiana; Biamonte, Flavia; Candeloro, Patrizio; Di Sanzo, Maddalena; Cozzi, Anna; Di Vito, Anna; Quaresima, Barbara; Lobello, Nadia; Trecroci, Francesca; Di Fabrizio, Enzo M.; Levi, Sonia; Cuda, Giovanni; Costanzo, Francesco

    2015-01-01

    The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation.

  1. Efecto de extractos de la esponja calcarea Leucetta aff. floridana sobre el ciclo de líneas celulares leucemoides Effect of extracts from the calcareous sponge Leucetta aff. floridana on the cell cycle of leukemoid cell lines

    Directory of Open Access Journals (Sweden)

    Diana Margarita Márquez Fernández

    2012-12-01

    .Introduction: Leucetta aff. floridana sponge produces compounds with differential antiproliferative activity on lung and breast cancer. Nevertheless, this activity in other tumour cell lines has not yet been tested and it remains unknown whether its antiproliferative potential is correlated with the cell progression through cell cycle or not. Objective: To evaluate the antiproliferative and anticlonogenic potential and the effect of methanolic and hexanic extracts of sponge L. aff. floridana from the Colombian Caribbean region on the cell cycle of Jurkat and K562 leukemoid cell lines. Methods: The viability and antiproliferative effect were determined using trypan blue assay at 0, 24, 48, 72 and 96 hours. Clongenicity and effect on cell cycle were assayed at 10 and 100 µg/mL Data obtained were analyzed using multifactorial ANOVA and Tukey's test. Results: The hexanic extract presented antiproliferative activity in both Jurkat and K652 cell lines; Jurkat being more sensitive than K652. These results were confirmed by clongenicity assays. The hexanic extract also showed its effect on the dose-dependent accumulation of Sub-G1 cells, although it was different in the two cell lines. The duration of the treatment with the hexanic extract was not significant for K562 cell line, but it was for Jurkat cells. Additionally, the percentage of cell accumulation in Sub-G1 was higher in K562 than in Jurkat cells. The methanolic extract showed antiproliferative effect similar to that of the hexanic extract, but more potent at the lowest concentration (10 µg/mL in K652 cell line clonegenicity. The effect on cell cycle was also similar to that of the hexanic extract, but in this case the duration of treatment was not significant in the cell accumulation in Sub-G1. Conclusions: Altogether these results show the differential potential of the extracts on the cell cycle of the evaluated leukemoid cell lines.

  2. RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Houcai; Yu, Jing; Zhang, Lixia; Xiong, Yuanyuan; Chen, Shuying; Xing, Haiyan; Tian, Zheng; Tang, Kejing; Wei, Hui; Rao, Qing; Wang, Min; Wang, Jianxiang, E-mail: wangjx@ihcams.ac.cn

    2014-04-18

    Highlights: • RPS27a expression was up-regulated in advanced-phase CML and AL patients. • RPS27a knockdown changed biological property of K562 and K562/G01 cells. • RPS27a knockdown affected Raf/MEK/ERK, P21 and BCL-2 signaling pathways. • RPS27a knockdown may be applicable for new combination therapy in CML patients. - Abstract: Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL) patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.

  3. Analysis of the role of COP9 Signalosome (CSN subunits in K562; the first link between CSN and autophagy

    Directory of Open Access Journals (Sweden)

    Bunce Christopher M

    2009-04-01

    Full Text Available Abstract Background The COP9/signalosome (CSN is a highly conserved eight subunit complex that, by deneddylating cullins in cullin-based E3 ubiquitin ligases, regulates protein degradation. Although studied in model human cell lines such as HeLa, very little is known about the role of the CSN in haemopoietic cells. Results Greater than 95% knockdown of the non-catalytic subunit CSN2 and the deneddylating subunit CSN5 of the CSN was achieved in the human myeloid progenitor cell line K562. CSN2 knockdown led to a reduction of both CSN5 protein and mRNA whilst CSN5 knockdown had little effect on CSN2. Both knockdowns inhibited CSN deneddylase function as demonstrated by accumulation of neddylated Cul1. Furthermore, both knockdowns resulted in the sequential loss of Skp2, Cdc4 and β-TrCP F-box proteins. These proteins were rescued by the proteasome inhibitor MG132, indicating the autocatalytic degradation of F-box proteins upon loss of CSN2 or CSN5. Interestingly, altered F-box protein gene expression was also observed in CSN2 and CSN5 knockdowns, suggesting a potential role of the CSN in regulating F-box protein transcription. Loss of either CSN subunit dramatically reduced cell growth but resulted in distinct patterns of cell death. CSN5 knockdown caused mitotic defects, G2/M arrest and apoptotic cell death. CSN2 knockdown resulted in non-apoptotic cell death associated with accumulation of both the autophagy marker LC3-II and autophagic vacuoles. Treatment of vector control K562 cells with the autophagy inhibitors 3-methyladenine and bafilomycin A1 recapitulated the growth kinetics, vacuolar morphology and LC3-II accumulation of CSN2 knockdown cells indicating that the cellular phenotype of CSN2 cells arises from autophagy inhibition. Finally, loss of CSN2 was associated with the formation of a CSN5 containing subcomplex. Conclusion We conclude that CSN2 is required for CSN integrity and the stability of individual CSN subunits, and postulate

  4. Heme-binding plasma membrane proteins of K562 erythroleukemia cells: Adsorption to heme-microbeads, isolation with affinity chromatography

    International Nuclear Information System (INIS)

    Majuri, R.

    1989-01-01

    Heme-microbeads attached themselves to the surface of viable K562 cells in a manner inhibitable by free hemin, indicating heme-recptor interaction. The microbeads were at first evenly distributed, but after prolonged incubation at 37 deg. C they formed a cap on one pole of the cells indicating clustering of the membrane heme receptors. Membrane proteins were labeled by culturing the cells in the presence of 35 S-methionine and were then solubilized with Triton X-114. The hydrophobic proteins contained about 20% of the total bound label. The solubilized membrane proteins were subsequently adsorbed to a heme-Sepharose affinity gel. According to SDS-electrophorsis and subsequent autoradiography, the immobilized heme captures two proteins or a protein with two polypeptides of 20 000 and 32 000 daltons. The larger of these was only wekly labeled with 35 S. The same two bands were observed if the cell surface proteins were labeled with 125 I by the lactoperoxidase method and the subsequently solubilized membrane proteins were isolated with heme-Sepharose. (author)

  5. Inhibition of Siah2 Ubiquitin Ligase by Vitamin K3 Attenuates Chronic Myeloid Leukemia Chemo-Resistance in Hypoxic Microenvironment.

    Science.gov (United States)

    Huang, Jixian; Lu, Ziyuan; Xiao, Yajuan; He, Bolin; Pan, Chengyun; Zhou, Xuan; Xu, Na; Liu, Xiaoli

    2018-02-05

    BACKGROUND A hypoxic microenvironment is associated with resistance to tyrosine kinase inhibitors (TKIs) and a poor prognosis in chronic myeloid leukemia (CML). The E3 ubiquitin ligase Siah2 plays a vital role in the regulation of hypoxia response, as well as in leukemogenesis. However, the role of Siah2 in CML resistance is unclear, and it is unknown whether vitaminK3 (a Siah2 inhibitor) can improve the chemo-sensitivity of CML cells in a hypoxic microenvironment. MATERIAL AND METHODS The expression of Siah2 was detected in CML patients (CML-CP and CML-BC), K562 cells, and K562-imatinib-resistant cells (K562-R cells). We measured the expression of PHD3, HIF-1α, and VEGF in both cell lines under normoxia and hypoxic conditions, and the degree of leukemic sensitivity to imatinib and VitaminK3 were evaluated. RESULTS Siah2 was overexpressed in CML-BC patients (n=9) as compared to CML-CP patients (n=13). Similarly, K562-imatinib-resistant cells (K562-R cells) showed a significantly higher expression of Siah2 as compared to K562 cells in a hypoxic microenvironment. Compared to normoxia, under hypoxic conditions, both cell lines had lower PHD3, higher HIF-1α, and higher VEGF expression. Additionally, Vitamin K3 (an inhibitor of Siah2) reversed these changes and promoted a higher degree of leukemic sensitivity to imatinib. CONCLUSIONS Our findings indicate that the Siah2-PHD3- HIF-1α-VEGF axis is an important hypoxic signaling pathway in a leukemic microenvironment. An inhibitor of Siah2, combined with TKIs, might be a promising therapy for relapsing and refractory CML patients.

  6. 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....

  7. CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

    Directory of Open Access Journals (Sweden)

    Hensel Nancy

    2005-04-01

    Full Text Available Abstract In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.

  8. Cytotoxic activity of vitamins K1, K2 and K3 against human oral tumor cell lines.

    Science.gov (United States)

    Okayasu, H; Ishihara, M; Satoh, K; Sakagami, H

    2001-01-01

    Vitamin K1, K2 and K3 were compared for their cytotoxic activity, radical generation and O2- scavenging activity. Among these compounds, vitamin K3 showed the highest cytotoxic activity against human oral tumor cell lines (HSC-2, HSG), human promyelocytic leukemic cell line (HL-60) and human gingival fibroblast (HGF). Vitamin K3 induced internucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 or HSG cells. The cytotoxic activity of vitamins K2 and K1 was one and two orders lower, respectively, than K3. Vitamin K2, but not vitamin K3, showed tumor-specific cytotoxic action. ESR spectroscopy showed that only vitamin K3 produced radical(s) under alkaline condition and most potently enhanced the radical intensity of sodium ascorbate and scavenged O2- (generated by hypoxanthine-xanthine oxidase reaction system); vitamin K2 was much less active whereas vitamin K1 was inactive. These data suggest that the cytotoxic activity of vitamin K3 is generated by radical-mediated oxidation mechanism and that this vitamin has two opposing actions (that is, antioxidant and prooxidant), depending on the experimental conditions.

  9. Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix.

    Science.gov (United States)

    Neri, L M; Bortul, R; Zweyer, M; Tabellini, G; Borgatti, P; Marchisio, M; Bareggi, R; Capitani, S; Martelli, A M

    1999-06-01

    The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the

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

    International Nuclear Information System (INIS)

    Li Xiaoming; Song Tianbao

    1999-01-01

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

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

    Science.gov (United States)

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

    1997-02-01

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

  12. Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells

    Czech Academy of Sciences Publication Activity Database

    Pluskalová, M.; Pešlová, G.; Grebeňová, D.; Halada, Petr; Hrkal, Z.

    2006-01-01

    Roč. 83, - (2006), s. 205-212 ISSN 1011-1344 R&D Projects: GA MZd NL7681 Institutional research plan: CEZ:AV0Z50200510 Keywords : k562 * bcr -abl * photodynamic treatment Subject RIV: EE - Microbiology, Virology Impact factor: 1.909, year: 2006

  13. Carbon monoxide induced erythroid differentiation of K562 cells mimics the central macrophage milieu in erythroblastic islands.

    Directory of Open Access Journals (Sweden)

    Shlomi Toobiak

    Full Text Available Growing evidence supports the role of erythroblastic islands (EI as microenvironmental niches within bone marrow (BM, where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule--CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage.Based on the above, the study hypothesized CO to have a role in erythroid maturation. Thus, the effect of CO gas as a potential erythroid differentiation inducer on the common model for erythroid progenitors, K562 cells, was explored. Cells were kept under oxygen lacking environment to mimic BM conditions. Nitrogen anaerobic atmosphere (N₂A served as control for CO atmosphere (COA. Under both atmospheres cells proliferation ceased: in N₂A due to cell death, while in COA as a result of erythroid differentiation. Maturation was evaluated by increased glycophorin A expression and Hb concentration. Addition of 1%CO only to N₂A, was adequate for maintaining cell viability. Yet, the average Hb concentration was low as compared to COA. This was validated to be the outcome of diversified maturation stages of the progenitor's population.In fact, the above scenario mimics the in vivo EI conditions, where at any given moment only a minute portion of the progenitors proceeds into terminal differentiation. Hence, this model might provide a basis for further molecular investigations of the EI structure/function relationship.

  14. Efficacy of the dual PI3K and mTOR inhibitor NVP-BEZ235 in combination with imatinib mesylate against chronic myelogenous leukemia cell lines

    Directory of Open Access Journals (Sweden)

    Xin P

    2017-04-01

    Full Text Available Pengliang Xin, Chuntuan Li, Yan Zheng, Qunyi Peng, Huifang Xiao, Yuanling Huang, Xiongpeng Zhu Department of Haematology, First Hospital of Quanzhou Affiliated to Fujian Medical University, Licheng, Quanzhou, Fujian Province, China Background: Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR pathway is a therapy target of cancer. We aimed to confirm the effect of dual PI3K/mTOR inhibitor NVP-BEZ235 on proliferation, apoptosis, and autophagy of chronic myelogenous leukemia (CML cells and sensitivity of tyrosine kinase inhibitor in vitro.Methods: Two human CML cell lines, K562 and KBM7R (T315I mutant strain, were used. The proliferation of CML cells was detected by MTS (Owen’s reagent assay. Cell cycle and apoptosis assay were examined by flow cytometric analysis. The phosphorylation levels and the expression levels were both evaluated by Western blot analysis. NVP-BEZ235 in combination with imatinib was also used to reveal the effect on proliferation and apoptosis.Results: NVP-BEZ235 significantly inhibited the proliferation in a time- and dose-dependent manner, and the half-maximal inhibitory concentration values of NVP-BEZ235 inhibiting the proliferation of K562 and KBM7R were 0.37±0.21 and 0.43±0.27 µmol/L, respectively, after 48 h. Cell apoptosis assay showed that NVP-BEZ235 significantly increased the late apoptotic cells. Cell cycle analysis indicated that the cells were mostly arrested in G1/G0 phase after treatment by NVP-BEZ235. In addition, results also found that, after treatment by NVP-BEZ235, phosphorylation levels of Akt kinase and S6K kinase significantly reduced, and the expression levels of cleaved caspase-3 significantly increased; meanwhile, the expression levels of caspase-3, B-cell lymphoma-2, cyclin D1, and cyclin D2 significantly decreased, and the ratio of LC3II/LC3I was significantly increased with increased LC3II expression level. Moreover, imatinib in combination with NVP-BEZ235

  15. Gene mutation in ATM/PI3K region of nasopharyngeal carcinoma cell lines

    International Nuclear Information System (INIS)

    Wang Hongmei; Wu Xinyao; Xia Yunfei

    2002-01-01

    Objective: To define the correlation between nasopharyngeal carcinoma (NPC) cell radiosensitivity and gene mutation in the ATM/PI3K coding region. Methods: The gene mutation in the ATM/PI3K region of nasopharyngeal carcinoma cell lines which vary in radiosensitivity, was monitored by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-marked ddNTP cycle sequencing technique. Results: No gene mutation was detected in the ATM/PI3K region of either CNE1 or CNE2. Conclusion: Disparity in intrinsic radiosensitivity between different NPC cell lines depends on some other factors and mechanism without being related to ATM/PI3K mutations

  16. Retroviral-mediated transfer and expression of human β-globin genes in cultured murine and human erythroid cells

    International Nuclear Information System (INIS)

    Weber-Benarous, A.; Cone, R.D.; London, I.M.; Mulligan, R.C.

    1988-01-01

    The authors cloned human β-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 β-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the β-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human β-globin gene transferred into mouse erythroleukemia cells in the same way. They have also introduced the normal human β-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal β-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, they suggest that the lack of expression of the endogenous β-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for β-lobin gene transcription. Retroviral-mediated transfer of the human β-globin gene may, however, uniquely influence expression of the gene K562 cells

  17. Induction of Mitochondrial Dependent Apoptosis in Human Leukemia K562 Cells by Meconopsis integrifolia: A Species from Traditional Tibetan Medicine

    Directory of Open Access Journals (Sweden)

    Jianping Fan

    2015-06-01

    Full Text Available Objectives: Meconopsis integrifolia (M. integrifolia is one of the most popular members in Traditional Tibetan Medicine. This study aimed to investigate the anticancer effect of M. integrifolia and to detect the underlying mechanisms of these effects. Methods: 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT assay and trypan blue assay were used to evaluate the cytotoxicity of M. integrifolia. Changes in cell nuclear morphology and reactive oxygen species (ROS level were observed by fluorescent microscopy. Apoptosis ratio, DNA damage and mitochondrial membrane potential (MMP loss were analyzed by flow cytometry. Western blotting assay was adopted to detect the proteins related to apoptosis. Immunofluorescence was used to observe the release of cytochrome C. Results: The obtained data revealed that M. integrifolia could significantly inhibit K562 cell viability, mainly by targeting apoptosis induction and cell cycle arrest in G2/M phase. Collapse in cell morphology, chromatin condensation, DNA damage and ROS accumulation were observed. Further mechanism detection revealed that mitochondrion might be a key factor in M. integrifolia-induced apoptosis. Conclusions: M. integrifolia could induce mitochondria mediated apoptosis and cell cycle arrest in G2/M phase with little damage to normal cells, suggesting that M. integrifolia might be a potential and efficient anticancer agent that deserves further investigation.

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

    Science.gov (United States)

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

    2016-08-01

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

  19. Irradiated KHYG-1 retains cytotoxicity: potential for adoptive immunotherapy with a natural killer cell line.

    Science.gov (United States)

    Suck, G; Branch, D R; Keating, A

    2006-05-01

    To evaluate gamma-irradiation on KHYG-1, a highly cytotoxic natural killer (NK) cell line and potential candidate for cancer immunotherapy. The NK cell line KHYG-1 was irradiated at 1 gray (Gy) to 50 Gy with gamma-irradiation, and evaluated for cell proliferation, cell survival, and cytotoxicity against tumor targets. We showed that a dose of at least 10 Gy was sufficient to inhibit proliferation of KHYG-1 within the first day but not its cytolytic activity. While 50 Gy had an apoptotic effect in the first hours after irradiation, the killing of K562 and HL60 targets was not different from non-irradiated cells but was reduced for the Ph + myeloid leukemia lines, EM-2 and EM-3. gamma-irradiation (at least 10 Gy) of KHYG-1 inhibits cell proliferation but does not diminish its enhanced cytolytic activity against several tumor targets. This study suggests that KHYG-1 may be a feasible immunotherapeutic agent in the treatment of cancers.

  20. Structural Characteristics of the Novel Polysaccharide FVPA1 from Winter Culinary-Medicinal Mushroom, Flammulina velutipes (Agaricomycetes), Capable of Enhancing Natural Killer Cell Activity against K562 Tumor Cells.

    Science.gov (United States)

    Jia, Wei; Feng, Jie; Zhang, Jing-Song; Lin, Chi-Chung; Wang, Wen-Han; Chen, Hong-Ge

    2017-01-01

    FVPA1, a novel polysaccharide, has been isolated from fruiting bodies of the culinary-medicinal mushroom Flammulina velutipes, a historically popular, widely cultivated and consumed functional food with an attractive taste, beneficial nutraceutical properties such as antitumor and immunomodulatory effects, and a number of essential biological activities. The average molecular weight was estimated to be ~1.8 × 104 Da based on high-performance size exclusion chromatography. Sugar analyses, methylation analyses, and 1H, 13C, and 2-dimensional nuclear magnetic resonance spectroscopy revealed the following structure of the repeating units of the FVPA1 polysaccharide Identification of this structure would conceivably lead to better understanding of the nutraceutical functions of this very important edible fungus. Bioactivity tests in vitro indicated that FVPA1 could significantly enhance natural killer cell activity against K562 tumor cells.

  1. Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Juan Liu

    2016-04-01

    Full Text Available Although dasatinib is effective in most imatinib mesylate (IMT-resistant chronic myeloid leukemia (CML patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT. Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt/ mammalian target of rapamycin (mTOR signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells.

  2. Cancer stemness and metastatic potential of the novel tumor cell line K3: an inner mutated cell of bone marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Qian, Hui; Ding, Xiaoqing; Zhang, Jiao; Mao, Fei; Sun, Zixuan; Jia, Haoyuan; Yin, Lei; Wang, Mei; Zhang, Xu; Zhang, Bin; Yan, Yongmin; Zhu, Wei; Xu, Wenrong

    2017-06-13

    Mesenchymal stem cells (MSCs) transplantation has been used for therapeutic applications in various diseases. Here we report MSCs can malignantly transform in vivo. The novel neoplasm was found on the tail of female rat after injection with male rat bone marrow-derived MSCs (rBM-MSCs) and the new tumor cell line, K3, was isolated from the neoplasm. The K3 cells expressed surface antigens and pluripotent genes similar to those of rBM-MSCs and presented tumor cell features. Moreover, the K3 cells contained side population cells (SP) like cancer stem cells (CSCs), which might contribute to K3 heterogeneity and tumorigenic capacity. To investigate the metastatic potential of K3 cells, we established the nude mouse models of liver and lung metastases and isolated the corresponding metastatic cell lines K3-F4 and K3-B6. Both K3-F4 and K3-B6 cell lines with higher metastatic potential acquired more mesenchymal and stemness-related features. Epithelial-mesenchymal transition is a potential mechanism of K3-F4 and K3-B6 formation.

  3. Identifying and validating a combined mRNA and microRNA signature in response to imatinib treatment in a chronic myeloid leukemia cell line.

    Directory of Open Access Journals (Sweden)

    Steven Bhutra

    Full Text Available Imatinib, a targeted tyrosine kinase inhibitor, is the gold standard for managing chronic myeloid leukemia (CML. Despite its wide application, imatinib resistance occurs in 20-30% of individuals with CML. Multiple potential biomarkers have been identified to predict imatinib response; however, the majority of them remain externally uncorroborated. In this study, we set out to systematically identify gene/microRNA (miRNA whose expression changes are related to imatinib response. Through a Gene Expression Omnibus search, we identified two genome-wide expression datasets that contain expression changes in response to imatinib treatment in a CML cell line (K562: one for mRNA and the other for miRNA. Significantly differentially expressed transcripts/miRNAs post imatinib treatment were identified from both datasets. Three additional filtering criteria were applied 1 miRbase/miRanda predictive algorithm; 2 opposite direction of imatinib effect for genes and miRNAs; and 3 literature support. These criteria narrowed our candidate gene-miRNA to a single pair: IL8 and miR-493-5p. Using PCR we confirmed the significant up-regulation and down-regulation of miR-493-5p and IL8 by imatinib treatment, respectively in K562 cells. In addition, IL8 expression was significantly down-regulated in K562 cells 24 hours after miR-493-5p mimic transfection (p = 0.002. Furthermore, we demonstrated significant cellular growth inhibition after IL8 inhibition through either gene silencing or by over-expression of miR-493-5p (p = 0.0005 and p = 0.001 respectively. The IL8 inhibition also further sensitized K562 cells to imatinib cytotoxicity (p < 0.0001. Our study combined expression changes in transcriptome and miRNA after imatinib exposure to identify a potential gene-miRNA pair that is a critical target in imatinib response. Experimental validation supports the relationships between IL8 and miR-493-5p and between this gene-miRNA pair and imatinib sensitivity in a CML cell

  4. Overexpression of Hiwi Inhibits the Growth and Migration of Chronic Myeloid Leukemia Cells.

    Science.gov (United States)

    Wang, Yalin; Jiang, Yan; Ma, Ning; Sang, Bailu; Hu, Xiaolin; Cong, Xiaofeng; Liu, Ziling

    2015-09-01

    Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by dysregulated growth and proliferation of hematopoietic stem/progenitor cells in bone marrow and excessive expansion of hematopoietic compartments in peripheral blood. Expression deletion of Hiwi, a human Piwi homolog, has been reported to be implicated in leukemogenesis. We here explored Hiwi's role in CML pathogenesis by determining how and whether its forced overexpression could affect CML cell growth and migration. The present results showed that lentivirus-mediated overexpression of Hiwi significantly suppressed cell proliferation and induced obvious apoptosis in K562 cells, a CML line cell line. Tumors in BALB/c nude mice generated by the K562 cells expressing Hiwi were much smaller than those formed by the control cells. Like in vitro, Hiwi upregulation induced cell apoptosis in the tumor tissues in vivo. Additionally, Hiwi elevation suppressed K562 cell migration and inhibited the activity and expression of matrix metalloproteinase-2 and -9. In summary, our study demonstrates that Hiwi overexpression inhibits CML cell growth and migration, providing insights into its role in CML pathogenesis.

  5. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most....... Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance...... property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production....

  6. The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line

    DEFF Research Database (Denmark)

    Xu, Xun; Pan, Shengkai; Liu, Xin

    2011-01-01

    Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of...

  7. Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate (DCA) on THP-1 cells: a multiparameter analysis

    NARCIS (Netherlands)

    Verhoeven, H.A.; Griensven, van L.J.L.D.

    2012-01-01

    Two human leukemia cells K562 and THP-1, the breast cancer lines MCF-7 and ZR-75-1, and the melanoma line MDA-MB-435S were compared by flowcytometry for their behaviour at increasing levels of 3BP. K562 and THP-1 responded to 3BP by membrane depolarization and increased ROS; MCF-7 and ZR-75-1 showed

  8. The Recognition of N-Glycans by the Lectin ArtinM Mediates Cell Death of a Human Myeloid Leukemia Cell Line

    Science.gov (United States)

    Carvalho, Fernanda Caroline; Soares, Sandro Gomes; Tamarozzi, Mirela Barros; Rego, Eduardo Magalhães; Roque-Barreira, Maria-Cristina

    2011-01-01

    ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment. PMID:22132163

  9. The recognition of N-glycans by the lectin ArtinM mediates cell death of a human myeloid leukemia cell line.

    Directory of Open Access Journals (Sweden)

    Fernanda Caroline Carvalho

    Full Text Available ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus (jackfruit, interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC(50 = 10 µg/mL, as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment.

  10. Acadesine kills chronic myelogenous leukemia (CML cells through PKC-dependent induction of autophagic cell death.

    Directory of Open Access Journals (Sweden)

    Guillaume Robert

    Full Text Available CML is an hematopoietic stem cell disease characterized by the t(9;22 (q34;q11 translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.

  11. Identification of pyrogallol as an antiproliferative compound present in extracts from the medicinal plant Emblica officinalis: effects on in vitro cell growth of human tumor cell lines.

    Science.gov (United States)

    Khan, Mahmud Tareq Hassan; Lampronti, Ilaria; Martello, Dino; Bianchi, Nicoletta; Jabbar, Shaila; Choudhuri, Mohammad Shahabuddin Kabir; Datta, Bidduyt Kanti; Gambari, Roberto

    2002-07-01

    In this study we compared the in vitro antiproliferative activity of extracts from medicinal plants toward human tumor cell lines, including human erythromyeloid K562, B-lymphoid Raji, T-lymphoid Jurkat, erythroleukemic HEL cell lines. Extracts from Emblica officinalis were the most active in inhibiting in vitro cell proliferation, after comparison to those from Terminalia arjuna, Aphanamixis polystachya, Oroxylum indicum, Cuscuta reflexa, Aegle marmelos, Saraca asoka, Rumex maritimus, Lagerstroemia speciosa, Red Sandalwood. Emblica officinalis extracts have been studied previously, due to their hepatoprotective, antioxidant, antifungal, antimicrobial and anti-inflammatory medicinal activities. Gas chromatography/mass spectrometry analyses allowed to identify pyrogallol as the common compound present both in unfractionated and n-butanol fraction of Emblica officinalis extracts. Antiproliferative effects of pyrogallol were therefore determined on human tumor cell lines thus identifying pyrogallol as an active component of Emblica officinalis extracts.

  12. [Cytotoxic effect of physalis peruviana in cell culture of colorectal and prostate cancer and chronic myeloid leukemia].

    Science.gov (United States)

    Quispe-Mauricio, Angel; Callacondo, David; Rojas, José; Zavala, David; Posso, Margarita; Vaisberg, Abraham

    2009-01-01

    The plants have been used as drugs for centuries. However, limited research has been done on its great potential as sources of new therapeutic agents. The purpose of this study was to evaluate Physalis peruviana cytotoxic activity on cell lines HT-29, PC-3, K-562 and VERO. The HT-29 cell lines, PC-3, K-562 and VERO, were exposed to four concentrations of P. peruviana ethanolic leave and stem extracts, also at different concentrations of cisplatin and 5-fluorouracil (5-FU), which were used as positive controls. We found rates of growth within 48 hours, then we determined the inhibitory concentration 50 (IC50) using linear regression analysis and the index of selectivity of each sample. The P. peruviana ethanolic leave and stem extracts showed cytotoxic activity. The IC50 in g/mL in leaves and stems were, 0.35 (r =-0.95 p peruviana leaves and steams ethanolic extracts were more cytotoxic than cisplatin and 5 FU, on the lines HT-29, PC-3 and K562. Furthermore the P. peruviana cytotoxic effects were less than cisplatin and 5-FU for VERO control cells lines.

  13. Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

    Science.gov (United States)

    Verrax, Julien; Vanbever, Stéphanie; Stockis, Julie; Taper, Henryk; Calderon, Pedro Buc

    2007-03-15

    Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells. (c) 2006 Wiley-Liss, Inc.

  14. RUNX3 is involved in caspase-3-dependent apoptosis induced by a combination of 5-aza-CdR and TSA in leukaemia cell lines.

    Science.gov (United States)

    Zhai, Feng-Xian; Liu, Xiang-Fu; Fan, Rui-Fang; Long, Zi-Jie; Fang, Zhi-Gang; Lu, Ying; Zheng, Yong-Jiang; Lin, Dong-Jun

    2012-03-01

    Epigenetic therapy has had a significant impact on the management of haematologic malignancies. The aim of this study was to assess whether 5-aza-CdR and TSA inhibit the growth of leukaemia cells and induce caspase-3-dependent apoptosis by upregulating RUNX3 expression. K562 and Reh cells were treated with 5-aza-CdR, TSA or both compounds. RT-PCR and Western blot analyses were used to examine the expression of RUNX3 at the mRNA and protein levels, respectively. Immunofluorescence microscopy was used to detect the cellular location of RUNX3. Additionally, after K562 cells were transfected with RUNX3, apoptosis and proliferation were studied using Annexin V staining and MTT assays. The expression of RUNX3 in leukaemia cell lines was markedly less than that in the controls. Demethylating drug 5-aza-CdR could induce RUNX3 expression, but the combination of TSA and 5-aza-CdR had a greater effect than did treatment with a single compound. The combination of 5-aza-CdR and TSA induced the translocation of RUNX3 from the cytoplasm into the nucleus. TSA enhanced apoptosis induced by 5-aza-CdR, and Annexin V and Hoechst 33258 staining showed that the combination induced apoptosis but not necrosis. Furthermore, apoptosis was dependent on the caspase-3 pathway. RUNX3 overexpression in K562 cells led to growth inhibition and apoptosis and potentiated the effects of 5-aza-CdR induction. RUNX3 plays an important role in leukaemia cellular functions, and the induction of RUNX3-mediated effects may contribute to the therapeutic value of combination TSA and 5-aza-CdR treatment.

  15. Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

    Science.gov (United States)

    Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

    2016-04-01

    First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro

  16. Characterization of Camptothecin-induced Genomic Changes in the Camptothecin-resistant T-ALL-derived Cell Line CPT-K5

    DEFF Research Database (Denmark)

    Kjeldsen, Eigil; Nielsen, Christine J F; Roy, Amit

    2018-01-01

    -K5 and its parental cell line. We identified copy number alterations affecting genes important for maintaining genome integrity and reducing CPT-induced DNA damage. We show for the first time that short tandem repeats are targets for TOP1 cleavage, that can be differentially stimulated by CPT.......Acquisition of resistance to topoisomerase I (TOP1)-targeting camptothecin (CPT) derivatives is a major clinical problem. Little is known about the underlying chromosomal and genomic mechanisms. We characterized the CPT-K5 cell line expressing mutant CPT-resistant TOP1 and its parental T......-cell derived acute lymphoblastic leukemia CPT-sensitive RPMI-8402 cell line by karyotyping and molecular genetic methods, including subtractive oligo-based array comparative genomic hybridization (soaCGH) analysis. Karyotyping revealed that CPT-K5 cells had acquired additional structural aberrations...

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

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  18. Spectrofluorometric determination of intracellular levels of reactive oxygen species in drug-sensitive and drug-resistant cancer cells using the 2',7'-dichlorofluorescein diacetate assay

    Energy Technology Data Exchange (ETDEWEB)

    Loetchutinat, Chatchanok [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand); Kothan, Suchart [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand); Dechsupa, Samarn [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand); Meesungnoen, Jintana [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand); Departement de medecine nucleaire et de radiobiologie, Faculte de medecine, Groupe en sciences des radiations, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4 (Canada); Jay-Gerin, Jean-Paul [Departement de medecine nucleaire et de radiobiologie, Faculte de medecine, Groupe en sciences des radiations, Universite de Sherbrooke, Sherbrooke, Quebec, J1H 5N4 (Canada); Mankhetkorn, Samlee [Laboratory of Physical Chemistry, Molecular and Cellular Biology, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131 (Thailand)]. E-mail: samlee@bucc4.buu.ac.th

    2005-02-01

    This article examines a non-invasive spectrofluorometric method using the 2',7'-dichlorofluorescein diacetate (DCHF-DA) assay for quantifying the intracellular reactive oxygen species (ROS{sub i}) produced in four cultured cancer cell lines: drug-sensitive (K562) and drug-resistant (K562/adr) human erythromyelogenous leukemia cell lines, and drug-sensitive (GLC4) and drug-resistant (GLC4/adr) human small cell lung carcinoma cell lines. The oxidation of the probe to the fluorescent dichlorofluorescein (DCF) was continuously monitored by following the DCF fluorescence intensity as a function of time using a standard spectrofluorometer in the presence of an extracellular DCF fluorescence quencher (Co{sup 2+}). By fitting the spectrofluorometric data to a kinetic model based on the following two reactions: (i) deacetylation of DCHF-DA to the oxidant-sensitive compound 2',7'-dichlorofluorescein (DCHF) by cellular esterase enzymes (pseudo-first-order rate constant: k{sub e}) and (ii) oxidation of DCHF by ROS{sub i} (second-order rate constant: k{sub 2}), the parameters intervening in DCF formation, k{sub e} and the product of k{sub 2} by the ROS{sub i} concentration, were quantitatively determined for the different cell lines studied. The results revealed that the intracellular esterase content or activity is similar in K562, K562/adr, and GLC4 cells, but 5-fold higher in GLC4/adr cells. The product k{sub 2}[ROS{sub i}] was found to be similar in the four cell lines considered, with a mean value of (5.3+/-0.9)x10{sup -7}cell{sup -1}s{sup -1}. Assuming that H{sub 2}O{sub 2} (in combination with peroxidases) is the primary responsible species for DCHF oxidation in intact cells, and using the rate constant value k2=790+/-62M-1s-1 established in our laboratory for the reaction of DCHF with H{sub 2}O{sub 2} in the presence of horseradish peroxidase, the mean value of the intracellular levels of ROS{sub i} in those cells was estimated to be 0.67+/-0.16n

  19. Encapsulation of Piper cabralanum (Piperaceae) nonpolar extract in poly(methyl methacrylate) by miniemulsion and evaluation of increase in the effectiveness of antileukemic activity in K562 cells.

    Science.gov (United States)

    Mendes, Anderson Nogueira; Filgueiras, Lívia Alves; Siqueira, Monica Regina Pimentel; Barbosa, Gleyce Moreno; Holandino, Carla; de Lima Moreira, Davyson; Pinto, José Carlos; Nele, Marcio

    2017-01-01

    This study aimed to synthesize and characterize nanoparticles (NPs) of poly(methyl methacrylate) (PMMA) and evaluate their ability to incorporate plant extracts with antitumor activity and low dissolution in aqueous media. The extract used was n -hexane partition of the methanol extract of Piper cabralanum (PCA-HEX). PMMA NPs were obtained using the mini-emulsion method, which was able to encapsulate almost 100% of PCA-HEX. The synthesized polymeric particles presented with a size of 200 nm and a negative charge. Cytotoxicity tests by MTT and trypan blue assays showed that NPs without PCA-HEX did not kill leukemic cells (K562 cells). NPs containing PCA-HEX were able to enhance cell death when compared to pure extract. The results showed that PMMA NPs could be useful as a drug delivery system as they can enhance the antitumor activity of the PCA-HEX extract by more than 20-fold. PMMA NPs containing plant extracts with antitumor activities may be an alternative to control the evolution of diseases such as leukemia.

  20. Downregulation of an Aim-1 Kinase Couples with Megakaryocytic Polyploidization of Human Hematopoietic Cells

    Science.gov (United States)

    Kawasaki, Akira; Matsumura, Itaru; Miyagawa, Jun-ichiro; Ezoe, Sachiko; Tanaka, Hirokazu; Terada, Yasuhiko; Tatsuka, Masaaki; Machii, Takashi; Miyazaki, Hiroshi; Furukawa, Yusuke; Kanakura, Yuzuru

    2001-01-01

    During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3–dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-rasG12V), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes. PMID:11266445

  1. Mycoplasma orale infection affects K+ and Cl- currents in the HSG salivary gland cell line.

    Science.gov (United States)

    Izutsu, K T; Fatherazi, S; Belton, C M; Oda, D; Cartwright, F D; Kenny, G E

    1996-06-01

    The relations between K+ channel and Cl- channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K+ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K+ channels and K+ channel expression as estimated by ionomycin- or hypotonically induced K+ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl- channels, but only the latter decrease was statistically significant. Also, Cl- currents could be elicited more frequently than K+ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K+ channels relatively more than Cl- channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurred in vivo.

  2. Nitric oxide-releasing nanoparticles: synthesis, characterization, and cytotoxicity to tumorigenic cells

    Energy Technology Data Exchange (ETDEWEB)

    Pelegrino, Milena T. [Universidade Federal de São Paulo, Exact and Earth Sciences Department (Brazil); Silva, Letícia C.; Watashi, Carolina M. [Universidade Federal do ABC, UFABC, Center of Natural and Human Sciences (Brazil); Haddad, Paula S. [Universidade Federal de São Paulo, Exact and Earth Sciences Department (Brazil); Rodrigues, Tiago; Seabra, Amedea B., E-mail: amedea.seabra@ufabc.edu.br [Universidade Federal do ABC, UFABC, Center of Natural and Human Sciences (Brazil)

    2017-02-15

    Nitric oxide (NO) is involved in several biological processes, including toxicity against tumor cells. The aim of this study was to synthesize, characterize, and evaluate the cytotoxicity of NO-releasing chitosan nanoparticles. A thiol-containing molecule, mercaptosuccinic acid (MSA), was encapsulated (encapsulation efficiency of 99%) in chitosan/sodium tripolyphosphate nanoparticles (CS NPs). The obtained nanoparticles showed an average hydrodynamic size of 108.40 ± 0.96 nm and polydispersity index of 0.26 ± 0.01. MSA-CS NPs were nitrosated leading to S-nitroso-MSA-CS NPs, which act as NO donor. The cytotoxicity of CS NPs, MSA-CS NPs, and S-nitroso-MSA-CS NPs were evaluated in several tumor cells, including human hepatocellular carcinoma (HepG2), mouse melanoma (B16F10), and human chronic myeloid leukemia (K562) cell lines and Lucena-1, a vincristine-resistant K562 cell line. Both CS NPs and MSA-CS NPs did not cause toxic effects in these cells, whereas S-nitroso-MSA-CS NPs caused potent cytotoxic effects in all the tested tumor cell lines. The half-maximal inhibitory concentration values of S-nitroso-MSA-CS NPs were 19.7, 10.5, 22.8, and 27.8 μg·mL{sup −1} for HepG2, B16F10, K562, and Lucena-1 cells, respectively. In contrast, S-nitroso-MSA-CS NPs exhibited lower cytotoxic to non-tumorigenic melanocytes (Melan-A) when compared with melanoma B16F10. Therefore, the results highlight the potential use of NO-releasing CS NPs in antitumor chemotherapy.

  3. Chemical composition of Schinus molle essential oil and its cytotoxic activity on tumour cell lines.

    Science.gov (United States)

    Díaz, Cecilia; Quesada, Silvia; Brenes, Oscar; Aguilar, Gilda; Cicció, José F

    2008-01-01

    The leaf essential oil hydrodistilled from Schinus molle grown in Costa Rica was characterised in terms of its chemical composition, antioxidant activity, ability to induce cytotoxicity and the mechanism of cell death involved in the process. As a result, 42 constituents, accounting for 97.2% of the total oil, were identified. The major constituents of the oil were beta-pinene and alpha-pinene. The antioxidant activity showed an IC(50) of 36.3 microg mL(-1). The essential oil was cytotoxic in several cell lines, showing that it is more effective on breast carcinoma and leukemic cell lines. The LD(50) for cytotoxicity at 48 h in K562 corresponded to 78.7 microg mL(-1), which was very similar to the LD(50) obtained when apoptosis was measured. The essential oil did not induce significant necrosis up to 200 microg mL(-1), which together with the former results indicate that apoptosis is the main mechanism of toxicity induced by S. molle essential oil in this cell line. In conclusion, the essential oil tested was weak antioxidant and induced cytotoxicity in different cell types by a mechanism related to apoptosis. It would be interesting to elucidate the role that different components of the oil play in the effect observed here, since some of them could have potential anti-tumoural effects, either alone or in combination.

  4. Ubiquitous expression of MAKORIN-2 in normal and malignant hematopoietic cells and its growth promoting activity.

    Directory of Open Access Journals (Sweden)

    King Yiu Lee

    Full Text Available Makorin-2 (MKRN2 is a highly conserved protein and yet its functions are largely unknown. We investigated the expression levels of MKRN2 and RAF1 in normal and malignant hematopoietic cells, and leukemia cell lines. We also attempted to delineate the role of MKRN2 in umbilical cord blood CD34+ stem/progenitor cells and K562 cell line by over-expression and inhibition of MKRN2 through lentivirus transduction and shRNA nucleofection, respectively. Our results provided the first evidence on the ubiquitous expression of MKRN2 in normal hematopoietic cells, embryonic stem cell lines, primary leukemia and leukemic cell lines of myeloid, lymphoid, erythroid and megakaryocytic lineages. The expression levels of MKRN2 were generally higher in primary leukemia samples compared with those in age-matched normal BM cells. In all leukemia subtypes, there was no significant correlation between expression levels of MKRN2 and RAF1. sh-MKRN2-silenced CD34+ cells had a significantly lower proliferation capacity and decreased levels of the early stem/progenitor subpopulation (CFU-GEMM compared with control cultures. Over-expression of MKRN2 in K562 cells increased cell proliferation. Our results indicated possible roles of MKRN2 in normal and malignant hematopoiesis.

  5. Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Mizutani, Naoki [Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya (Japan); College of Life and Health Sciences, Chubu University, Kasugai (Japan); Omori, Yukari [Department of Pathophysiological Laboratory Science, Nagoya University Graduate School of Medicine, Nagoya (Japan); Kawamoto, Yoshiyuki; Sobue, Sayaka; Ichihara, Masatoshi [College of Life and Health Sciences, Chubu University, Kasugai (Japan); Suzuki, Motoshi [Division of Molecular Carcinogenesis, Nagoya University Graduate School of Medicine, Nagoya (Japan); Kyogashima, Mamoru [Department of Microbiology and Molecular Biology, Nihon Pharmaceutical University, Saitama (Japan); Nakamura, Mitsuhiro [Department of Drug Information, Gifu Pharmaceutical University, Gifu (Japan); Tamiya-Koizumi, Keiko [College of Life and Health Sciences, Chubu University, Kasugai (Japan); Nozawa, Yoshinori [Tokai Gakuin University, Kakamigahara (Japan); Murate, Takashi, E-mail: murate@isc.chubu.ac.jp [College of Life and Health Sciences, Chubu University, Kasugai (Japan)

    2016-02-19

    Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.

  6. Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

    International Nuclear Information System (INIS)

    Mizutani, Naoki; Omori, Yukari; Kawamoto, Yoshiyuki; Sobue, Sayaka; Ichihara, Masatoshi; Suzuki, Motoshi; Kyogashima, Mamoru; Nakamura, Mitsuhiro; Tamiya-Koizumi, Keiko; Nozawa, Yoshinori; Murate, Takashi

    2016-01-01

    Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.

  7. Furano diterpenes from Pterodon pubescens Benth with selective in vitro anticancer activity for prostate cell line

    International Nuclear Information System (INIS)

    Spindola, Humberto M.; Carvalho, Joao E. de; Ruiz, Ana Lucia T.G.; Rodrigues, Rodney A. F.; Denny, Carina; Sousa, Ilza M. de Oliveira; Foglio, Mary Ann; Tamashiro, Jorge Y.

    2009-01-01

    Activity guided fractionation of Pterodon pubescens Benth. methylene chloride-soluble fraction afforded novel 6α-acetoxi 7β-hydroxy-vouacapan 1 and four known diterpene furans 2, 3, 4, 5. The compounds were evaluated for in vitro cytotoxic activities against human normal cells and tumour cell lines UACC-62 (melanoma), MCF-7 (breast), NCI-H460 (lung, non-small cells), OVCAR-03 (ovarian), PC-3 (prostate), HT-29 (colon), 786-0 (renal), K562 (leukemia) and NCI-ADR/RES (ovarian expressing phenotype multiple drugs resistance). Results were expressed by three concentration dependent parameters GI 50 (concentration that produces 50% growth inhibition), TGI (concentration that produces total growth inhibition or cytostatic effect) and LC 50 (concentration that produces .50% growth, a cytotoxicity parameter). Also, in vitro cytotoxicity was evaluated against 3T3 cell line (mouse embryonic fibroblasts). Antiproliferative properties of compounds 1, 4 and 5 are herein reported for the first time. These compounds showed selectivity in a concentration-dependent way against human PC-3. Compound 1 demonstrated selectivity 26 fold more potent than the positive control, doxorubicin, for PC-3 (prostrate) cell line based on GI 50 values, causing cytostatic effect (TGI value) at a concentration fifteen times less than positive control. Moreover comparison of 50% lethal concentration (LC 50 value) with positive control (doxorubicin) suggested that compound 1 was less toxic. (author)

  8. Expression profile of CREB knockdown in myeloid leukemia cells

    International Nuclear Information System (INIS)

    Pellegrini, Matteo; Cheng, Jerry C; Voutila, Jon; Judelson, Dejah; Taylor, Julie; Nelson, Stanley F; Sakamoto, Kathleen M

    2008-01-01

    The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution. To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA. By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA. We have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

  9. Metabolism modifications and apoptosis induction after Cellfood™ administration to leukemia cell lines.

    Science.gov (United States)

    Catalani, Simona; Carbonaro, Valentina; Palma, Francesco; Arshakyan, Marselina; Galati, Rossella; Nuvoli, Barbara; Battistelli, Serafina; Canestrari, Franco; Benedetti, Serena

    2013-09-09

    Cellfood™ (CF) is a nutritional supplement containing deuterium sulphate, minerals, amino acids, and enzymes, with well documented antioxidant properties. Its organic and inorganic components are extracted from the red algae Lithothamnion calcareum, whose mineral extract has shown growth-inhibitory effect both on in vitro and in vivo models. The purpose of this study was to evaluate the antiproliferative effects of CF on leukemic cells. In fact, according to its capacity to modulate O2 availability and to improve mitochondrial respiratory metabolism, we wondered if CF could affect cancer cell metabolism making cells susceptible to apoptosis. Three leukemic cell lines, Jurkat, U937, and K562, were treated with CF 5 μl/ml up to 72 hours. Cell viability, apoptosis (i.e. caspase-3 activity and DNA fragmentation), hypoxia inducible factor 1 alpha (HIF-1α) concentration, glucose transporter 1 (GLUT-1) expression, lactate dehydrogenase (LDH) activity and lactate release in the culture medium were detected and compared with untreated cells. CF significantly inhibited leukemic cell viability by promoting cell apoptosis, as revealed by caspase-3 activation and DNA laddering. In particular, CF treated cells showed lower HIF-1α levels and lower GLUT-1 expression as compared to untreated cells. At the same time, CF was able to reduce LDH activity and, consequently, the amount of lactate released in the extracellular environment. We supplied evidence for an antiproliferative effect of CF on leukemia cell lines by inducing cell death through an apoptotic mechanism and by altering cancer cell metabolism through HIF-1α and GLUT-1 regulation. Thanks to its antioxidative and proapoptotic properties, CF might be a good candidate for cancer prevention.

  10. Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism.

    Science.gov (United States)

    Bonilla-Porras, Angelica R; Jimenez-Del-Rio, Marlene; Velez-Pardo, Carlos

    2011-06-10

    Secondary therapy-related acute lymphoblastic leukemia might emerge following chemotherapy and/or radiotherapy for primary malignancies. Therefore, other alternatives should be pursued to treat leukemia. It is shown that vitamin K3- or vitamin C- induced apoptosis in leukemia cells by oxidative stress mechanism involving superoxide anion radical and hydrogen peroxide generation, activation of NF-κB, p53, c-Jun, protease caspase-3 activation and mitochondria depolarization leading to nuclei fragmentation. Cell death was more prominent when Jurkat and K562 cells are exposed to VC and VK3 in a ratio 1000:1 (10 mM: 10 μM) or 100:1 (300 μM: 3 μM), respectively. We provide for the first time in vitro evidence supporting a causative role for oxidative stress in VK3- and VC-induced apoptosis in Jurkat and K562 cells in a domino-like mechanism. Altogether these data suggest that VK3 and VC should be useful in the treatment of leukemia.

  11. Synthesis of Nanodiamond-Daunorubicin Conjugates to Overcome Multidrug Chemoresistance in Leukemia

    Science.gov (United States)

    Man, Han B.; Kim, Hansung; Kim, Ho-Joong; Robinson, Erik; Liu, Wing Kam; Chow, Edward Kai-Hua; Ho, Dean

    2013-01-01

    Nanodiamonds (NDs) are promising candidates in nanomedicine, demonstrating significant potential as gene/drug delivery platforms for cancer therapy. We have synthesized ND vectors capable of chemotherapeutic loading and delivery with applications towards chemoresistant leukemia. The loading of Daunorubicin (DNR) onto NDs was optimized by adjusting reaction parameters such as acidity and concentration. The resulting conjugate, a novel therapeutic payload for NDs, was characterized extensively for size, surface charge, and loading efficiency. A K562 human myelogenous leukemia cell line, with multidrug resistance conferred by incremental DNR exposure, was used to demonstrate the efficacy enhancement resulting from ND-based delivery. While resistant K562 cells were able to overcome treatment from DNR alone, as compared with non-resistant K562 cells, NDs were able to improve DNR delivery into resistant K562 cells. By overcoming efflux mechanisms present in this resistant leukemia line, ND-enabled therapeutics have demonstrated the potential to improve cancer treatment efficacy, especially towards resistant strains. PMID:23916889

  12. Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

    Science.gov (United States)

    Collins, Denis M; Gately, Kathy; Hughes, Clare; Edwards, Connla; Davies, Anthony; Madden, Stephen F; O'Byrne, Kenneth J; O'Donovan, Norma; Crown, John

    2017-09-01

    Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high

  13. Effects of ultraviolet irradiation on natural killer cell function in systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Nived, O.; Johansson, I.; Sturfelt, G. (University Hospital, Lund (Sweden). Dept. of Rheumatology)

    1992-06-01

    In vitro irradiation with long wavelength ultraviolet light (UV-A), in clinically relevant dosages, of a natural killer cell line containing cell preparations from 17 control subjects reduced natural killer cell cytotoxicity with the cell line K562 as target. The spontaneous function of natural killer cells from 12 patients with systematic lupus erythematosus (SLE) correlated inversely with the one hour erythrocyte sedimentation rate, but not with glucocorticoid doses. After UV-A exposure, natural killer cells from patients with SLE exert either increased or decreased cytotoxicity, and the direction of change is inversely correlated with the spontaneous natural killer cell function. (Author).

  14. Effects of ultraviolet irradiation on natural killer cell function in systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Nived, O.; Johansson, I.; Sturfelt, G.

    1992-01-01

    In vitro irradiation with long wavelength ultraviolet light (UV-A), in clinically relevant dosages, of a natural killer cell line containing cell preparations from 17 control subjects reduced natural killer cell cytotoxicity with the cell line K562 as target. The spontaneous function of natural killer cells from 12 patients with systematic lupus erythematosus (SLE) correlated inversely with the one hour erythrocyte sedimentation rate, but not with glucocorticoid doses. After UV-A exposure, natural killer cells from patients with SLE exert either increased or decreased cytotoxicity, and the direction of change is inversely correlated with the spontaneous natural killer cell function. (Author)

  15. Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism

    Directory of Open Access Journals (Sweden)

    Velez-Pardo Carlos

    2011-06-01

    Full Text Available Abstract Background Secondary therapy-related acute lymphoblastic leukemia might emerge following chemotherapy and/or radiotherapy for primary malignancies. Therefore, other alternatives should be pursued to treat leukemia. Results It is shown that vitamin K3- or vitamin C- induced apoptosis in leukemia cells by oxidative stress mechanism involving superoxide anion radical and hydrogen peroxide generation, activation of NF-κB, p53, c-Jun, protease caspase-3 activation and mitochondria depolarization leading to nuclei fragmentation. Cell death was more prominent when Jurkat and K562 cells are exposed to VC and VK3 in a ratio 1000:1 (10 mM: 10 μM or 100:1 (300 μM: 3 μM, respectively. Conclusion We provide for the first time in vitro evidence supporting a causative role for oxidative stress in VK3- and VC-induced apoptosis in Jurkat and K562 cells in a domino-like mechanism. Altogether these data suggest that VK3 and VC should be useful in the treatment of leukemia.

  16. Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

    International Nuclear Information System (INIS)

    Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

    1987-01-01

    The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2 + , CD3 + , CD4 + or CD2 + , CD3 + , CD8 + ) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by 51 Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype

  17. A Metabolic Biofuel Cell: Conversion of Human Leukocyte Metabolic Activity to Electrical Currents

    Directory of Open Access Journals (Sweden)

    Cui X Tracy

    2011-05-01

    Full Text Available Abstract An investigation of the electrochemical activity of human white blood cells (WBC for biofuel cell (BFC applications is described. WBCs isolated from whole human blood were suspended in PBS and introduced into the anode compartment of a proton exchange membrane (PEM fuel cell. The cathode compartment contained a 50 mM potassium ferricyanide solution. Average current densities between 0.9 and 1.6 μA cm-2 and open circuit potentials (Voc between 83 and 102 mV were obtained, which were both higher than control values. Cyclic voltammetry was used to investigate the electrochemical activity of the activated WBCs in an attempt to elucidate the mechanism of electron transfer between the cells and electrode. Voltammograms were obtained for the WBCs, including peripheral blood mononuclear cells (PBMCs - a lymphocyte-monocyte mixture isolated on a Ficoll gradient, a B lymphoblastoid cell line (BLCL, and two leukemia cell lines, namely K562 and Jurkat. An oxidation peak at about 363 mV vs. SCE for the PMA (phorbol ester activated primary cells, with a notable absence of a reduction peak was observed. Oxidation peaks were not observed for the BLCL, K562 or Jurkat cell lines. HPLC confirmed the release of serotonin (5-HT from the PMA activated primary cells. It is believed that serotonin, among other biochemical species released by the activated cells, contributes to the observed BFC currents.

  18. Antiproliferative Effects of Bacillus coagulans Unique IS2 in Colon Cancer Cells.

    Science.gov (United States)

    Madempudi, Ratna Sudha; Kalle, Arunasree M

    2017-10-01

    In the present study, the in vitro anticancer (antiproliferative) effects of Bacillus coagulans Unique IS2 were evaluated on human colon cancer (COLO 205), cervical cancer (HeLa), and chronic myeloid leukemia (K562) cell lines with a human embryonic kidney cell line (HEK 293T) as noncancerous control cells. The Cytotoxicity assay (MTT) clearly demonstrated a 22%, 31.7%, and 19.5% decrease in cell proliferation of COLO 205, HeLa, and K562 cells, respectively, when compared to the noncancerous HEK 293T cells. Normal phase-contrast microscopic images clearly suggested that the mechanism of cell death is by apoptosis. To further confirm the induction of apoptosis by Unique IS2, the sub-G0-G1 peak of the cell cycle was quantified using a flow cytometer and the data indicated 40% of the apoptotic cells in Unique IS2-treated COLO cells when compared with their untreated control cells. The Western blot analysis showed an increase in pro-apoptotic protein BAX, decrease in antiapoptotic protein, Bcl2, decrease in mitochondrial membrane potential, increase in cytochrome c release, increase in Caspase 3 activity, and cleavage of poly(ADP-ribose) polymerase. The present study suggests that the heat-killed culture supernatant of B. coagulans can be more effective in inducing apoptosis of colon cancer cells and that can be considered for adjuvant therapy in the treatment of colon carcinoma.

  19. Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Santosh Podder

    2015-01-01

    Full Text Available Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF at 5 mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5 mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50 μM or 1 mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5 mM NaF together with radiation (1 Gy induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

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

    Directory of Open Access Journals (Sweden)

    Djihad Hadjadj

    2016-09-01

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

  1. Isolation and purification of a novel anticancer 60 K daltons protein from the Persian Gulf sea hare, Aplysia Dactylomela

    Directory of Open Access Journals (Sweden)

    Keyvan Zandi

    2004-02-01

    Full Text Available Sea hares have attracted the interest of many workers investigating chemical defense substances. Most of these substances are low molecular weight compounds derived from algal diets. Anticancer effects of a novel protein isolated from purple fluid of A. dactylomela are reported. The purification procedure consisted basically of ammonium sulphate fractionation, ion exchange and ultrafiltration techniques. For cytotoxicity effects, L929, K562, HL60 and NB4 cell lines and MTT assay were used. A protein of 60000 Da of the purple fluid of A. dactylomela had antiproliferative effects on the cell lines it was maximally active at 0.5-1.5 microgram/ml on NB4 cell line. Therefore, the purple fluid of A. dactylomela has a novel antiproliferative agent.

  2. The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers

    International Nuclear Information System (INIS)

    Azam, Philippe; Peiffer, Jean-Luc; Chamousset, Delphine; Tissier, Marie-Helene; Bonnet, Pierre-Antoine; Vian, Laurence; Fabre, Isabelle; Ourlin, Jean-Claude

    2006-01-01

    Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Only MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers

  3. Down-regulation of human endogenous retrovirus type K (HERV-K) viral env RNA in pancreatic cancer cells decreases cell proliferation and tumor growth

    Science.gov (United States)

    Li, Ming; Radvanyi, Laszlo; Yin, Bingnan; Li, Jia; Chivukula, Raghavender; Lin, Kevin; Lu, Yue; Shen, JianJun; Chang, David Z.; Li, Donghui; Johanning, Gary L.; Wang-Johanning, Feng

    2017-01-01

    Purpose We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer (PC). Experimental Design shRNA was employed to knockdown (KD) the expression of HERV-K in PC cells. Results HERV-K env expression was detected in seven PC cell lines and in 80% of PC patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several PC cell lines. RT activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in PC patient sera (N=106) than in normal donor sera (N=40). Importantly, the in vitro and in vivo growth rates of three PC cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several PC cells or tumors. Conclusion These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in PC. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of PC, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis and immunotherapy of PC. PMID:28679769

  4. 31 CFR 56.2 - Sales price.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at prices offered through the competitive...

  5. The role of a new CD44st in increasing the invasion capability of the human breast cancer cell line MCF-7

    International Nuclear Information System (INIS)

    Fang, Xin Jian; Jiang, Hua; Zhao, Xv Peng; Jiang, Wei Mei

    2011-01-01

    CD44, a hyaluronan (HA) receptor, is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival. The CD44 gene contains 20 exons that are alternatively spliced, giving rise to many CD44 isoforms, perhaps including tumor-specific sequences. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to detect CD44st mRNA and CD44 protein in sensitive MCF-7, Lovo, K562 and HL-60 cell lines as well as their parental counterparts, respectively. The full length cDNA encoding CD44st was obtained from the total RNA isolated from MCF-7/Adr cells by RT-PCR, and subcloned into the pMD19-T vector. The CD44st gene sequence and open reading frame were confirmed by restriction enzyme analysis and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44st was transfected into MCF-7 cells using Lipofectamine. After transfection, the positive clones were obtained by G418 screening. The changes of the MMP-2 and MMP-9 genes and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells penetrating through the artificial matrix membrane in each group (MCF-7, MCF-7+HA, MCF-7/neo, MCF-7/neo+HA, MCF-7/CD44st, MCF-7/CD44st+HA and MCF-7/CD44st+Anti-CD44+HA) was counted to compare the change of the invasion capability regulated by the CD44st. Erk and P-Erk were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by the CD44st. Sensitive MCF-7, Lovo, K562 and HL-60 cells did not contain CD44st mRNA and CD44 protein. In contrast, the multidrug resistance MCF-7/Adr, Lovo/Adr, K562/Adr and HL-60/Adr cells expressed CD44st mRNA and CD44 protein. The CD44st m

  6. Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector

    International Nuclear Information System (INIS)

    Balamotis, Michael Andrew; Huang, Katie; Mitani, Kohnosuke

    2004-01-01

    Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Δ) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected ∼50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Δ Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Δ Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism

  7. Asynchronous DNA replication within the human β-globin gene locus

    International Nuclear Information System (INIS)

    Epner, E.; Forrester, W.C.; Groudine, M.

    1988-01-01

    The timing of DNA replication of the human β-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span >120 kilobases across the human β-globin gene locus, the authors show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-γ-globin gene region and approximately 20 kilobases 5' to the ε-globin gene and 20 kilobases 3' to the β-globin gene, replicate later and throughout S phase. A similar area is also present in the α-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks

  8. Inhibition of H3K9me2 Reduces Hair Cell Regeneration after Hair Cell Loss in the Zebrafish Lateral Line by Down-Regulating the Wnt and Fgf Signaling Pathways

    Science.gov (United States)

    Tang, Dongmei; Lin, Qin; He, Yingzi; Chai, Renjie; Li, Huawei

    2016-01-01

    The activation of neuromast (NM) supporting cell (SC) proliferation leads to hair cell (HC) regeneration in the zebrafish lateral line. Epigenetic mechanisms have been reported that regulate HC regeneration in the zebrafish lateral line, but the role of H3K9me2 in HC regeneration after HC loss remains poorly understood. In this study, we focused on the role of H3K9me2 in HC regeneration following neomycin-induced HC loss. To investigate the effects of H3K9me2 in HC regeneration, we took advantage of the G9a/GLP-specific inhibitor BIX01294 that significantly reduces the dimethylation of H3K9. We found that BIX01294 significantly reduced HC regeneration after neomycin-induced HC loss in the zebrafish lateral line. BIX01294 also significantly reduced the proliferation of NM cells and led to fewer SCs in the lateral line. In situ hybridization showed that BIX01294 significantly down-regulated the Wnt and Fgf signaling pathways, which resulted in reduced SC proliferation and HC regeneration in the NMs of the lateral line. Altogether, our results suggest that down-regulation of H3K9me2 significantly decreases HC regeneration after neomycin-induced HC loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus H3K9me2 plays a critical role in HC regeneration. PMID:27303264

  9. Inhibition of H3K9me2 Reduces Hair Cell Regeneration after Hair Cell Loss in the Zebrafish Lateral Line by Down-Regulating the Wnt and Fgf Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Dongmei eTang

    2016-05-01

    Full Text Available The activation of neuromast supporting cell (SC proliferation leads to hair cell (HC regeneration in the zebrafish lateral line. Epigenetic mechanisms have been reported that regulate HC regeneration in the zebrafish lateral line, but the role of H3K9me2 in HC regeneration after HC loss remains poorly understood. In this study, we focused on the role of H3K9me2 in HC regeneration following neomycin-induced HC loss. To investigate the effects of H3K9me2 in HC regeneration, we took advantage of the G9a/GLP-specific inhibitor BIX01294 that significantly reduces the dimethylation of H3K9. We found that BIX01294 significantly reduced HC regeneration after neomycin-induced HC loss in the zebrafish lateral line. BIX01294 also significantly reduced the proliferation of neuromast cells and led to fewer SCs in the lateral line. In situ hybridization showed that BIX01294 significantly down-regulated the Wnt and Fgf signaling pathways, which resulted in reduced SC proliferation and HC regeneration in the neuromasts of the lateral line. Altogether, our results suggest that down-regulation of H3K9me2 significantly decreases HC regeneration after neomycin-induced HC loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus H3K9me2 plays a critical role in HC regeneration.

  10. A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function

    Directory of Open Access Journals (Sweden)

    Snehal Shabrish

    2016-01-01

    Full Text Available Natural killer (NK cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA and calcium ionophore (Ca2+-ionophore instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p<0.0001. It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells.

  11. 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.

  12. Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia

    Science.gov (United States)

    Heydari, Nasrin; Shariati, Laleh; Khanahmad, Hossein; Hejazi, Zahra; Shahbazi, Mansoureh; Salehi, Mansoor

    2016-01-01

    Objective(s): β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites to the K562 cell line. Materials and Methods: A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on β-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 μg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 μg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells. Results: A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones. Conclusion: The findings demonstrate that the spongy effect of KLF1-binding site on BCL11A and β-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease. PMID:27872702

  13. Two new meroterpenoids produced by the endophytic fungus Penicillium sp. SXH-65.

    Science.gov (United States)

    Sun, Xinhua; Kong, Xianglan; Gao, Huquan; Zhu, Tianjiao; Wu, Guangwei; Gu, Qianqun; Li, Dehai

    2014-08-01

    Two new meroterpenoids, arisugacins I (1) and J (2), together with five known meroterpenoids including arisugacin B (3), arisugacin F (4), arisugacin G (5), territrem B (6) and territrem C (7) were isolated from an endophytic fungus Penicillium sp. SXH-65. Their structures were determined by extensive spectroscopic experiments and comparison with literature data. Their cytotoxicities were evaluated against Hela, HL-60 and K562 cell lines, and only 3 and 4 exhibited weak cytotoxicities against Hela, HL-60 and K562 cell lines with IC50 values ranging from 24 to 60 μM.

  14. Effect of verapamil on cellular uptake of Tc-99m MIBI and tetrofosmin on several cancer cells

    International Nuclear Information System (INIS)

    Kim, Dae Hyun; Yoo, Jung Ah; Bae, Jin Ho; Jeong, Shin Young; Suh, Myung Rang; Ahn, Byeong Cheol; Lee, Kyu Bo; Lee, Jae Tae

    2004-01-01

    Cellular uptake of 99 mTc-sestamibi (MIBI) and 99 mTc-tetrofosmin (TF) is low in cancer cells expressing multidrug resistance(MDR) by p-glycoprotein(Pgp) or multidrug related protein(MRP). Verapamil is known to increase cellular uptake of MIBI in MDR cancer cells, but is recently reported to have different effects on tracer uptake in certain cancer cells. This study was prepared to evaluate effects of verapamil on cellular uptake of MIBI and TF in several cancer cells. Cellular uptakes of Tc-99m MIBI and TF were measured in erythroleukemia K562 cell, breast cancer MCF7 cell, and human ovarian cancer SK-OV-3 cells, and data were compared with those of doxorubicin-resistant K562(Ad) cells. RT-PCR and Western blot analysis were used for the detection of mdr1 mRNA and Pgp expression, and to observe changes in isotypes of PKC enzyme. Effects of verapamil on MIBI and TF uptake were evaluated at different concentrations upto 200 μM at 1*10 6 cells/ m l at 37.deg.C. Radioactivity in supernatant and pellet was measured with gamma counter to calculate cellular uptake ratio. Toxicity of verapamil was measured with MTT assay. Cellular uptakes of MIBI and TF were increased by time in four cancer cells studied. Co-incubation with verapamil resulted in an increase in uptake of MIBI and TF in K562(Adr) cell at a concentration of 100 μM and the maximal increase at 50 μM was 10-times to baseline. In contrast, uptakes of MIBI and TF in K562, MCF7m SK-OV3 cells were decreased with verapamil treatment at a concentration over 1 μM. With a concentration of 200 μM verapamil, respectively. Cellular uptakes of MIBI and TF in MCF7 and SK-OV-3 cells were not changed with 10μM, but were also decreased with verapamil higher than 10μM, resulting 40% and 5% of baseline at 50 μM. MTT assay of four cells revealed that K562, MCF7, SK-OV3 were not damaged with verapamil at 200 μM. Although verapamil increases uptake of MIBI and TF in MDR cancer cells, cellular uptakes were further decreased

  15. A comparison of cytotoxicity of some phosphoramides against K562 cell line

    Directory of Open Access Journals (Sweden)

    niloufar Dorosti

    2012-03-01

    Conclusion: Since hydrogen bonds play a key role in biology processes, these results suggest that increase in the hydrogen bonds of derivatives bearing urea moiety (4-6 may be increase cytotoxicity of these compounds. Moreover, the 3-NO2 group showed higher anti-cancer activity than two other positions owing to possibility electronic and steric effects.

  16. Human primary erythroid cells as a more sensitive alternative in vitro hematological model for nanotoxicity studies: Toxicological effects of silver nanoparticles.

    Science.gov (United States)

    Rujanapun, Narawadee; Aueviriyavit, Sasitorn; Boonrungsiman, Suwimon; Rosena, Apiwan; Phummiratch, Duangkamol; Riolueang, Suchada; Chalaow, Nipon; Viprakasit, Vip; Maniratanachote, Rawiwan

    2015-12-01

    Although immortalized cells established from cancerous cells have been widely used for studies in nanotoxicology studies, the reliability of the results derived from immortalized cells has been questioned because of their different characteristics from normal cells. In the present study, human primary erythroid cells in liquid culture were used as an in vitro hematological cell model for investigation of the nanotoxicity of silver nanoparticles (AgNPs) and comparing the results to the immortalized hematological cell lines HL60 and K562. The AgNPs caused significant cytotoxic effects in the primary erythroid cells, as shown by the decreased cell viability and induction of intracellular ROS generation and apoptosis, whereas they showed much lower cytotoxic and apoptotic effects in HL60 and K562 cells and did not induced ROS generation in these cell lines. Scanning electron microcopy revealed an interaction of AgNPs to the cell membrane in both primary erythroid and immortalized cells. In addition, AgNPs induced hemolysis in the primary erythroid cells in a dose-dependent manner, and transmission electron microcopy analysis revealed that AgNPs damaged the erythroid cell membrane. Taken together, these results suggest that human primary erythroid cells in liquid culture are a more sensitive alternative in vitro hematological model for nanotoxicology studies. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Biosynthesis of 10 kDa and 7.5 kDa insulin-like growth factor II in a human rhabdomyosarcoma cell line

    DEFF Research Database (Denmark)

    Nielsen, F C; Haselbacher, G; Christiansen, Jan

    1993-01-01

    In the present study we have analysed the expression of insulin-like growth factor II (IGF-II) in the human rhabdomyosarcoma cell line IN157.IN157 cells express high levels of three IGF-II mRNAs of 6.0 kb, 4.8 kb and 4.2 kb. In contrast, normal skeletal muscle expresses a negligible amount of IGF......-II mRNA. Two forms of IGF-II with molecular masses of 7.5 kDa and 10 kDa, corresponding to the mature IGF-II and IGF-II with a C-terminal extension of 21 amino acids (IGF-IIE21), were secreted into the culture medium at amounts of 17 ng/ml (2.3 nM) and 15 ng/ml (1.5 nM), respectively. IN157 cells also......-II and IGF-IIE21 with Kd values of 0.5 nM and 2 nM, respectively, and IGF-I with about 500 times lower affinity. IGF-II and IGF-IIE21 stimulated DNA synthesis via the IGF-I receptor, whereas the IGF-II/Man 6-P receptor mediated their rapid internalization and inactivation. During culture of IN157 cells about...

  18. Comparison of Cytotoxic Activity in Leukemic Lineages Reveals Important Features of β-Hairpin Antimicrobial Peptides.

    Science.gov (United States)

    Buri, Marcus V; Torquato, Heron F Vieira; Barros, Carlos Castilho; Ide, Jaime S; Miranda, Antonio; Paredes-Gamero, Edgar J

    2017-07-01

    Several reports described different modes of cell death triggered by antimicrobial peptides (AMPs) due to direct effects on membrane disruption, and more recently by apoptosis and necrosis-like patterns. Cytotoxic curves of four β-hairpin AMPs (gomesin, protegrin, tachyplesin, and polyphemusin) were obtained from several human leukemic lineages and normal monocytes and Two cell lines were then selected based on their cytotoxic sensitivity. One was sensitive to AMPs (K562) and the other resistant (KG-1) and their effect compared between these lineages. Thus, these lineages were chosen to further investigate biological features related with their cytotoxicities to AMPs. Stimulation with AMPs produced cell death, with activation of caspase-3, in K562 lineage. Increase on the fluidity of plasmatic membrane by reducing cholesterol potentiated cytotoxicity of AMPs in both lineages. Quantification of internal and external gomesin binding to the cellular membrane of both K562 and KG-1 cells showed that more peptide is accumulated inside of K562 cells. Additionally, evaluation of multi-drug resistant pumps activity showed that KG-1 has more activity than K562 lineage. A comparison of intrinsic gene patterns showed great differences between K562 and KG-1, but stimulation with gomesin promoted few changes in gene expression patterns. Differences in internalization process through the plasma membrane, multidrug resistance pumps activity, and gene expression pattern are important features to AMPs regulated cell death. J. Cell. Biochem. 118: 1764-1773, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  19. 40 CFR 721.562 - Substituted alkylamine salt.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted alkylamine salt. 721.562 Section 721.562 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.562 Substituted alkylamine salt...

  20. 32 CFR 562.3 - Definitions.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 3 2010-07-01 2010-07-01 true Definitions. 562.3 Section 562.3 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS... area. Region commanders are located at: (1) US Army First ROTC Region, Fort Bragg, NC 28307. (2) US...

  1. Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Edwards Jeremy

    2003-07-01

    Full Text Available Abstract Background The identification of known mutations in a cell population is important for clinical applications and basic cancer research. In this work an immobilized form of the polymerase chain reaction, referred to as polony technology, was used to detect mutations as well as gene deletions, resulting in loss of heterozygosity (LOH, in cancer cell lines. Specifically, the mutational hotspots in p53, namely codons 175, 245, 248, 249, 273, and 282, and K-ras2, codons 12, 13 and 61, were genotyped in the pancreatic cell line, Panc-1. In addition LOH analysis was also performed for these same two genes in Panc-1 by quantifying the relative gene copy number of p53 and K-ras2. Results Using polony technology, Panc-1 was determined to possess only one copy of p53, which possessed a mutation in codon 273, and two copies of K-ras2, one wildtype and one with a mutation in codon 12. To further demonstrate the general approach of this method, polonies were also used to detect K-ras2 mutations in the pancreatic cell lines, AsPc-1 and CAPAN-1. Conclusions In conclusion, we have developed an assay that can detect mutations in hotspots of p53 and K-ras2 as well as diagnose LOH in these same genes.

  2. Characterization of histone H3K27 modifications in the β-globin locus

    International Nuclear Information System (INIS)

    Kim, Yea Woon; Kim, AeRi

    2011-01-01

    Research highlights: → The β-globin locus control region is hyperacetylated and monomethylated at histone H3K27. → Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. → Association of PRC2 subunits is comparable with H3K27me3 pattern. → Modifications of histone H3K27 are established in an enhancer-dependent manner. -- Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human β-globin locus using the ChIP assay. The LCR of the human β-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the β-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer.

  3. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    DEFF Research Database (Denmark)

    Jensen, P O; Larsen, J K; Christensen, I J

    1994-01-01

    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogonal...... light scatter. The method was optimized using the human leukemia cell lines HL-60 and K-562. Samples of 10(5) ethanol-fixed cells were treated with pepsin/HCl and stained as a nuclear suspension with anti-BrdUrd antibody, FITC-conjugated secondary antibody, and propidium iodide. Labelled mitoses could...

  4. Antibacterial and Antiproliferative Activities of Plumericin, an Iridoid Isolated from Momordica charantia Vine

    Directory of Open Access Journals (Sweden)

    Jutamas Saengsai

    2015-01-01

    Full Text Available Plumericin, an iridoid lactone, was isolated with relatively high yield from Momordica charantia vine using the supercritical fluid extraction (SFE and the separation box (Sepbox comprising dual combination of high-performance liquid chromatography and solid phase extraction. This compound showed antibacterial activity against Enterococcus faecalis and Bacillus subtilis with minimum inhibitory concentration (MIC values better than cloxacillin. Plumericin potently inhibited proliferation of two leukemic cancer cell lines: they were acute and chronic leukemic cancer cell lines, NB4 and K562, with the effective doses (ED50 of 4.35 ± 0.21 and 5.58 ± 0.35 μg/mL, respectively. In addition, the mechanism of growth inhibition in both cell lines was induced by apoptosis, together with G2/M arrest in K562 cells.

  5. 16 CFR 5.62 - Hearing rights of respondent.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Hearing rights of respondent. 5.62 Section 5.62 Commercial Practices FEDERAL TRADE COMMISSION ORGANIZATION, PROCEDURES AND RULES OF PRACTICE STANDARDS OF CONDUCT Disciplinary Actions Concerning Postemployment Conflict of Interest § 5.62 Hearing...

  6. Limiting-dilution analysis for the determination of leukemic cell frequencies after bone marrow decontamination with mafosfamide or merocyanine 540

    Energy Technology Data Exchange (ETDEWEB)

    Porcellini, A.; Talevi, N.; Marchetti-Rossi, M.T.; Palazzi, M.; Manna, A.; Sparaventi, G.; Delfini, C.; Valentini, M.

    1987-11-01

    To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation.

  7. Limiting-dilution analysis for the determination of leukemic cell frequencies after bone marrow decontamination with mafosfamide or merocyanine 540

    International Nuclear Information System (INIS)

    Porcellini, A.; Talevi, N.; Marchetti-Rossi, M.T.; Palazzi, M.; Manna, A.; Sparaventi, G.; Delfini, C.; Valentini, M.

    1987-01-01

    To stimulate a leukemia remission marrow, cell suspensions of normal human bone marrow were mixed with human acute lymphoblastic or myelogenous leukemic cells of the CCRF-SF, Nalm-6, and K-562 lines. The cell mixtures were incubated in vitro with mafosfamide (AZ) or with the photoreactive dye merocyanine 540 (MC-540). A quantity of 10(4) cells of the treated suspensions was dispensed into microculture plates, and graded cell numbers of the line used to contaminate the normal marrow were added. Limiting-dilution analysis was used to estimate the frequency of leukemia cells persisting after treatment with the decontaminating agents. Treatment with AZ or MC-540 produced a total elimination (ie, 6 logs or 5.3 logs respectively) of B cell acute leukemia cells (CCRF-SB), whereas nearly 1.7 logs and 2 logs of K-562 acute myelogenous blasts were still present in the cell mixtures after treatment with MC-540 and AZ, respectively. Treatment of the Nalm-6-contaminated cell mixtures with AZ resulted in 100% elimination of clonogenic cells, whereas nearly 80% decontamination was obtained with MC-540. Our results suggest that treatment with AZ could be an effective method of eliminating clonogenic tumor cells from human bone marrow. MC-540, shown by previous studies to spare sufficient pluripotential stem cells to ensure hemopoietic reconstitution in the murine model and in clinical application, has comparable effects and merits trials for possible clinical use in autologous bone marrow transplantation

  8. 46 CFR 154.562 - Cargo hose: Hydrostatic test.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Cargo hose: Hydrostatic test. 154.562 Section 154.562 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY... Hose § 154.562 Cargo hose: Hydrostatic test. Each cargo hose must pass a hydrostatic pressure test at...

  9. Two-dimensional analysis of metabolically and cell surface radiolabeled proteins of some human lymphoid and myeloid leukemia cell lines. II. Glycosylated and phosphorylated proteins

    Energy Technology Data Exchange (ETDEWEB)

    Chorvath, B; Duraj, J; Sedlak, J; Pleskova, I

    1986-01-01

    Cell surface glycoproteins, radiolabelled by the sodium metaperiodate/tritiated borohydride technique, and cell phosphoproteins, metabolically radiolabelled with /sup 32/P-orthophosphate were analyzed by two-dimensional electrophoretic analysis in some myeloid and lymphoid leukemia cell lines. Some markedly expressed major glycoproteins were predominant in some of the cell lines (such as 95k and 100k glycoproteins with marked charge heterogeneity in non-T, non-B acute lymphoblastic leukemia cell lines NALM 6 and NALM 16), but markedly quantitatively reduced in other examined cell lines, such as lymphoblastoid cell line UHKT 34/2. /sup 32/P-orthophosphate radiolabelled phosphoprotein two-dimensional patterns of the examined lymphoid leukemia cell lines were essentially similar, with some minor differences, in examined lymphoid and myeloid leukemia cell lines, such as marked expression of a series of large phosphoproteins in the molecular weight range 80-100k in lymphoid cell lines and almost complete absence of these phosphoproteins on the examined myeloid leukemia cell lines. Another configuration of acidic phosphoproteins (30-35k) exhibited individual cell line variability and differences between both individual myeloid leukemia cell lines and between the lymphoid and myeloid cell lines examined. (author) 2 figs., 15 refs.

  10. Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

    International Nuclear Information System (INIS)

    Hristov, K.; Mitev, V.; Knox, K.

    2006-01-01

    Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

  11. C-reactive protein bearing cells are a subpopulation of natural killer cell precursors

    International Nuclear Information System (INIS)

    Baum, L.L.; Krueger, N.X.

    1986-01-01

    Cell surface C-reactive protein (S-CRP) is expressed on the surface membrane of a small percentage of lymphocytes. Anti-CRP inhibits natural killer (NK) function. Since NK effectors are heterogeneous, they suspected that the cells expressing S-CRP (CRP + ) might respond differently to stimulation than the NK effectors lacking S-CRP (CRP - ). Methods were developed to separate CRP + and CRP - lymphocytes and their functional responses were examined and compared. These techniques are dependent upon the binding of CRP to its ligands, C-polysaccharide (CPS) or Phosphocholine (PC). The first method involves rosette formation with CPS coupled autologous red blood cells; the second method utilizes the binding of CRP + lymphocytes to PC-sepharose. Lymphocytes separated using either of these techniques yield similar results. CRP - lymphocytes respond to 3 day incubation with PHA or Il-2 by producing effectors which kill 51 Cr labeled K562 tumor cells, CRP + precursors do not. CRP + lymphocytes respond to a 5 day incubation with inactivated K562 by producing effectors which kill K562; CRP - precursors do not. NK functional activity of both is increased by incubation with interferon. This ability to respond differently to stimulation suggests that CRP + and CRP - cells are functionally distinct

  12. Antiradical and cytotoxic activity of different Helichrysum plicatum flower extracts.

    Science.gov (United States)

    Bigović, Dubravka; Savikin, Katarina; Janković, Teodora; Menković, Nebojsa; Zdunić, Gordana; Stanojković, Tatjana; Djurić, Zorica

    2011-06-01

    Flowers of Helichrysum plicatum were extracted under different experimental conditions, and their antioxidant activity was determined by DPPH radical scavenging assay. Extracts obtained with higher concentration of ethyl acetate (90% or 100%) were found to contain the greatest amount of total phenolics (> 250 mg gallic acid equivalents/g of dried extract), and high correlation between total phenolic content and antiradical activity was observed (r = -0.79). Based on the total phenolic content and antiradical activity, some extracts were selected for investigation of cytotoxic activity toward PC3, HeLa and K562 human cancer cell lines in vitro. All tested extracts exhibited moderate activity against HeLa cells (41.9-42.1 microg/mL), whereas the extract obtained with 100% ethyl acetate was the most active against K562 and PC3 cell lines (25.9 and 39.2 microg/mL, respectively). Statistical analysis revealed significant correlation between total phenolic content and cytotoxic activity against PC3 and K562 cells. HPLC identification of phenolic compounds from the extracts indicated the presence of apigenin, naringenin and kaempferol as free aglycones, and glycosides of apigenin, naringenin, quercetin and kaempferol. Among aglycones, kaempferol displayed moderate cytostatic activity against all cell lines (24.8-64.7 microM).

  13. Cytoskeleton, endoplasmic reticulum and nucleus alterations in CHO-K1 cell line after Crotalus durissus terrificus (South American rattlesnake venom treatment

    Directory of Open Access Journals (Sweden)

    B. P. Tamieti

    2007-01-01

    Full Text Available Snake venoms are toxic to a variety of cell types. However, the intracellular damages and the cell death fate induced by venom are unclear. In the present work, the action of the South American rattlesnake Crotalus durissus terrificus venom on CHO-K1 cell line was analyzed. The cells CHO-K1 were incubated with C. d. terrificus venom (10, 50 and 100g/ml for 1 and 24 hours, and structural alterations of actin filaments, endoplasmic reticulum and nucleus were assessed using specific fluorescent probes and agarose gel electrophoresis for DNA fragmentation. Significant structural changes were observed in all analyzed structures. DNA fragmentation was detected suggesting that, at the concentrations used, the venom induced apoptosis.

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

    Science.gov (United States)

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

    2013-03-01

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

  15. [Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

    Science.gov (United States)

    Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

    2004-01-01

    To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P ras4B can enhance the expression of ERalpha and

  16. Synergistic apoptosis of CML cells by buthionine sulfoximine and hydroxychavicol correlates with activation of AIF and GSH-ROS-JNK-ERK-iNOS pathway.

    Directory of Open Access Journals (Sweden)

    Avik Acharya Chowdhury

    Full Text Available BACKGROUND: Hydroxychavicol (HCH, a constituent of Piper betle leaf has been reported to exert anti-leukemic activity through induction of reactive oxygen species (ROS. The aim of the study is to optimize the oxidative stress -induced chronic myeloid leukemic (CML cell death by combining glutathione synthesis inhibitor, buthionine sulfoximine (BSO with HCH and studying the underlying mechanism. MATERIALS AND METHODS: Anti-proliferative activity of BSO and HCH alone or in combination against a number of leukemic (K562, KCL22, KU812, U937, Molt4, non-leukemic (A549, MIA-PaCa2, PC-3, HepG2 cancer cell lines and normal cell lines (NIH3T3, Vero was measured by MTT assay. Apoptotic activity in CML cell line K562 was detected by flow cytometry (FCM after staining with annexin V-FITC/propidium iodide (PI, detection of reduced mitochondrial membrane potential after staining with JC-1, cleavage of caspase- 3 and poly (ADP-ribose polymerase proteins by western blot analysis and translocation of apoptosis inducing factor (AIF by confocal microscopy. Intracellular reduced glutathione (GSH was measured by colorimetric assay using GSH assay kit. 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM were used as probes to measure intracellular increase in ROS and nitric oxide (NO levels respectively. Multiple techniques like siRNA transfection and pharmacological inhibition were used to understand the mechanisms of action. RESULTS: Non-apoptotic concentrations of BSO significantly potentiated HCH-induced apoptosis in K562 cells. BSO potentiated apoptosis-inducing activity of HCH in CML cells by caspase-dependent as well as caspase-independent but apoptosis inducing factor (AIF-dependent manner. Enhanced depletion of intracellular GSH induced by combined treatment correlated with induction of ROS. Activation of ROS- dependent JNK played a crucial role in ERK1/2 activation which subsequently induced the

  17. Silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway.

    Science.gov (United States)

    Li, Yumei; Zhang, Chunmei; Cai, Danfeng; Chen, Congde; Mu, Dongmei

    2017-12-01

    Rhabdoid tumors, which tend to occur prior to the age of 2 years, are one of the most aggressive malignancies and have a poor prognosis due to the frequency of metastasis. Silibinin, a natural extract, has been approved as a potential tumor suppressor in various studies, however, whether or not it also exerts its antitumor capacity in rhabdoid tumors, particularly with regards to tumor migration and invasion, is unclear. The rhabdoid tumor G401 cell line was used in the present in vitro study. An MTT assay was used to assess the cytotoxicity of silibinin on G401 cells, cell migration was studied using a wound healing assay and a Transwell migration assay, and cell invasion was determined using a Transwell invasion assay. The underlying mechanism in silibinin inhibited cell migration and invasion was investigated by western blot analysis and further confirmed using a specific inhibitor. Experimental results demonstrated that high doses of silibinin suppressed cell viability, and that low doses of silibinin inhibited cell migration and invasion without affecting cell proliferation. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway was involved in the silibinin-induced inhibition of metastasis. Silibinin inactivated the PI3K/Akt pathway, and inhibited cell migration and invasion, an effect that was further enhanced when LY294002, a classic PI3K inhibitor, was used concurrently. In general, silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway and may be a potential chemotherapeutic drug to combat rhabdoid tumors in the future.

  18. Cytotoxicity and Antiproliferative Activity Assay of Clove Mistletoe (Dendrophthoe pentandra (L. Miq. Leaves Extracts

    Directory of Open Access Journals (Sweden)

    Vida Elsyana

    2016-01-01

    Full Text Available Clove mistletoe (Dendrophthoe pentandra (L. Miq. is a semiparasitic plant that belongs to Loranthaceae family. Clove mistletoe was traditionally used for cancer treatment in Indonesia. In the present study, we examined cytotoxicity of clove mistletoe leaves extracts against brine shrimps and conducted their antiproliferative activity on K562 (human chronic myelogenous leukemia and MCM-B2 (canine benign mixed mammary cancer cell lines in vitro. The tested samples were water extract, ethanol extract, ethanol fraction, ethyl acetate fraction, and n-hexane fraction. Cytotoxicity was screened using Brine Shrimp Lethality Test (BSLT. Antiproliferative activity was conducted using Trypan Blue Dye Method and cells were counted using haemocytometer. The results showed that n-hexane fraction exhibited significant cytotoxicity with LC50 value of 55.31 μg/mL. The n-hexane fraction was then considered for further examination. The n-hexane fraction of clove mistletoe could inhibit growth of K562 and MCM-B2 cancer cell lines in vitro. The inhibition activity of clove mistletoe n-hexane fraction at concentration of 125 μg/mL on K562 cancer cell lines was 38.69%, while on MCM-B2 it was 41.5%. Therefore, it was suggested that clove mistletoe had potential natural anticancer activity.

  19. K-line photometry

    International Nuclear Information System (INIS)

    Henry, R.C.

    1979-01-01

    A brief review is given of more than a decade of work involving measurement of the strength of the K line of calcium in A-type stars. The effects of interstellar reddening are reexamined, and an improved estimate for the range in calcium abundance among field stars is obtained. (Auth.)

  20. The combinatorial PP1-binding consensus Motif (R/Kx( (0,1V/IxFxx(R/Kx(R/K is a new apoptotic signature.

    Directory of Open Access Journals (Sweden)

    Angélique N Godet

    Full Text Available BACKGROUND: Previous studies established that PP1 is a target for Bcl-2 proteins and an important regulator of apoptosis. The two distinct functional PP1 consensus docking motifs, R/Kx((0,1V/IxF and FxxR/KxR/K, involved in PP1 binding and cell death were previously characterized in the BH1 and BH3 domains of some Bcl-2 proteins. PRINCIPAL FINDINGS: In this study, we demonstrate that DPT-AIF(1, a peptide containing the AIF(562-571 sequence located in a c-terminal domain of AIF, is a new PP1 interacting and cell penetrating molecule. We also showed that DPT-AIF(1 provoked apoptosis in several human cell lines. Furthermore, DPT-APAF(1 a bi-partite cell penetrating peptide containing APAF-1(122-131, a non penetrating sequence from APAF-1 protein, linked to our previously described DPT-sh1 peptide shuttle, is also a PP1-interacting death molecule. Both AIF(562-571 and APAF-1(122-131 sequences contain a common R/Kx((0,1V/IxFxxR/KxR/K motif, shared by several proteins involved in control of cell survival pathways. This motif combines the two distinct PP1c consensus docking motifs initially identified in some Bcl-2 proteins. Interestingly DPT-AIF(2 and DPT-APAF(2 that carry a F to A mutation within this combinatorial motif, no longer exhibited any PP1c binding or apoptotic effects. Moreover the F to A mutation in DPT-AIF(2 also suppressed cell penetration. CONCLUSION: These results indicate that the combinatorial PP1c docking motif R/Kx((0,1V/IxFxxR/KxR/K, deduced from AIF(562-571 and APAF-1(122-131 sequences, is a new PP1c-dependent Apoptotic Signature. This motif is also a new tool for drug design that could be used to characterize potential anti-tumour molecules.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  2. Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication.

    Directory of Open Access Journals (Sweden)

    Silvia Dotti

    Full Text Available The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field.

  3. Dihydroartemisinin inhibits the human erythroid cell differentiation by altering the cell cycle

    International Nuclear Information System (INIS)

    Finaurini, Sara; Basilico, Nicoletta; Corbett, Yolanda; D’Alessandro, Sarah; Parapini, Silvia; Olliaro, Piero; Haynes, Richard K.; Taramelli, Donatella

    2012-01-01

    Artemisinin derivatives such as dihydroartemisinin (DHA) induce significant depletion of early embryonic erythroblasts in animal models. We have reported previously that DHA specifically targets pro-erythroblasts and basophilic erythroblasts, when human CD34+ stem cells are differentiated toward the erythroid lineage, indicating that a window of susceptibility to artemisinins may exist also in human developmental erythropoiesis during pregnancy. To better investigate the toxicity of artemisinin derivatives, the structure–activity relationship was evaluated against the K562 leukaemia cell line, used as a model for differentiating early human erythroblasts. All artemisinins derivatives, except deoxyartemisinin, inhibited both spontaneous and induced erythroid differentiation, confirming that the peroxide bridge is responsible for the erythro-toxicity. On the contrary, cell growth was markedly reduced by DHA, artemisone and artesunate but not by artemisinin, 10-deoxoartemisinin or deoxy-artemisinin. The substituent at position C-10 is responsible only for the anti-proliferative effect, since 10-deoxoartemisinin did not reduce cell growth but arrested the differentiation of K562 cells. In particular, the results showed that DHA resulted the most potent and rapidly acting compound of the drug family, causing (i) the decreased expression of GpA surface receptors and the down regulation the γ-globin gene; (ii) the alteration of S phase of cell cycle and (iii) the induction of programmed cell death of early erythroblasts in a dose dependent manner within 24 h. In conclusion, these findings confirm that the active metabolite DHA is responsible for the erythro-toxicity of most of artemisinins used in therapy. Thus, as long as no further clinical data are available, current WHO recommendations of avoiding malaria treatment with artemisinins during the first trimester of pregnancy remain valid.

  4. A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds.

    Directory of Open Access Journals (Sweden)

    Marta Durlak

    Full Text Available The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562 to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies.

  5. Kelussia odoratissima potentiates cytotoxic effects of radiation in HeLa cancer cell line

    Directory of Open Access Journals (Sweden)

    Azar Hosseini

    2017-02-01

    Full Text Available Objective: Cervical cancer is the second most common cause of death from cancer in women throughout the world. The aim of this study was to evaluate the cytotoxic activity of Kelussia odoratissima (K. odoratissima extract associated with radiotherapy in cervical cancer cells (HeLa cell line.Materials and Methods: Different concentration of the extract (25-500µg/ml was tested in HeLa cell lines. Cell cytotoxicity of the extract and the effects of the extract on radiation (2Gy/min-induced damages were assessed by MTT assay. Apoptosis was assessed using flow cytometric analysis.Result: K. odoratissima decreased cell viability in HeLa cell line in a concentration and time-dependent manner. When compared to the control,K. odoratissima induced a sub-G1 peak in the flow cytometry histogram of treated cells, indicating that apoptotic cell death is involved in K. odoratissima-induced toxicity. It was also shown that K. odoratissima sensitizes cells to radiation-induced toxicity.Conclusion: Our result showed the extract increased the radiation effect. This observation may be related to the presence of active compounds such as phthalides and ferulic acid.

  6. Inhibition of p70S6K2 down-regulates Hedgehog/GLI pathway in non-small cell lung cancer cell lines

    Directory of Open Access Journals (Sweden)

    Kotani Hidehito

    2009-07-01

    Full Text Available Abstract Background The Hedgehog (HH pathway promotes tumorigenesis in a diversity of cancers. Activation of the HH signaling pathway is caused by overexpression of HH ligands or mutations in the components of the HH/GLI1 cascade, which lead to increased transactivation of GLI transcription factors. Although negative kinase regulators that antagonize the activity of GLI transcription factors have been reported, including GSK3β, PKA and CK1s, little is known regarding positive kinase regulators that are suitable for use on cancer therapeutic targets. The present study attempted to identify kinases whose silencing inhibits HH/GLI signalling in non-small cell lung cancer (NSCLC. Results To find positive kinase regulators in the HH pathway, kinome-wide siRNA screening was performed in a NSCLC cell line, A549, harboring the GLI regulatory reporter gene. This showed that p70S6K2-silencing remarkably reduced GLI reporter gene activity. The decrease in the activity of the HH pathway caused by p70S6K2-inhibition was accompanied by significant reduction in cell viability. We next investigated the mechanism for p70S6K2-mediated inhibition of GLI1 transcription by hypothesizing that GSK3β, a negative regulator of the HH pathway, is activated upon p70S6K2-silencing. We found that phosphorylated-GSK3β (Ser9 was reduced by p70S6K2-silencing, causing a decreased level of GLI1 protein. Finally, to further confirm the involvement of p70S6K2 in GLI1 signaling, down-regulation in GLI-mediated transcription by PI3KCA-inhibition was confirmed, establishing the pivotal role of the PI3K/p70S6K2 pathway in GLI1 cascade regulation. Conclusion We report herein that inhibition of p70S6K2, known as a downstream effector of the PI3K pathway, remarkably decreases GLI-mediated transactivation in NSCLC by reducing phosphorylated-GSK3β followed by GLI1 degradation. These results infer that p70S6K2 is a potential therapeutic target for NSCLC with hyperactivated HH/GLI pathway.

  7. Protolichesterinic acid enhances doxorubicin-induced apoptosis in HeLa cells in vitro.

    Science.gov (United States)

    Brisdelli, Fabrizia; Perilli, Mariagrazia; Sellitri, Doriana; Bellio, Pierangelo; Bozzi, Argante; Amicosante, Gianfranco; Nicoletti, Marcello; Piovano, Marisa; Celenza, Giuseppe

    2016-08-01

    The aim of this study was to investigate the effect of protolichesterinic acid, a lichen secondary metabolite, on anti-proliferative activity of doxorubicin in three human cancer cell lines, HeLa, SH-SY5Y and K562 cells. The data obtained from MTT assays, performed on cells treated with protolichesterinic acid and doxorubicin alone and in combination, were analysed by the median-effect method as proposed by Chou and Talalay and the Bliss independence model. Apoptosis rate was evaluated by fluorescence microscopy, caspase-3, 8 and 9 activities were detected by spectrofluorimetric analysis and protein expression of Bim, Bid, Bax and Mcl-2 was analysed by Western blotting. The interaction of protolichesterinic acid with thioesterase domain of human fatty acid synthase (hFAS) was investigated by a molecular docking study. The in vitro activity of doxorubicin against HeLa cancer cell line, but not against SH-SY5Y and K562 cells, was synergically increased by protolichesterinic acid. The increased cytotoxicity caused by protolichesterinic acid in HeLa cells was due to a pro-apoptotic effect and was associated to caspase-3, 8 and 9 activation. The simultaneous treatment for 24h with protolichesterinic acid plus doxorubicin caused an increase of Bim protein expression and the appearance of cleaved form of Bid protein. The molecular modelling analysis showed that protolichesterinic acid seemed to behave as a competitive inhibitor of hFAS. These results suggest that protolichesterinic acid could be envisaged as an useful tool against certain types of tumor cells in combination with anticancer drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. RAC3 nuclear receptor co-activator has a protective role in the apoptosis induced by different stimuli

    International Nuclear Information System (INIS)

    Colo, Georgina P.; Rubio, Maria F.; Alvarado, Cecilia V.; Costas, Monica A.

    2007-01-01

    RAC3 belongs to the family of p160 nuclear receptors co activators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-κB co activator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H 2 O 2 in the human embryonic kidney cell line (HEK293), and tumor necrosis factor-related apoptosis inducing ligand (TRAIL) in a human chronic myeloid leukemia cell line (K562) naturally resistant to TRAIL. We observed that the tumoral K562 cells have high levels of RAC3 if compared with the non-tumoral HEK293 cells. The normal or transfected co activator over-expression inhibits apoptosis through a diminished caspase activity and AIF nuclear translocation, increased NF--κB, AKT and p38, and decreased ERK activities. In contrast, inhibition of RAC3 by siRNA induced sensitivity of K562 to TRAIL-induced apoptosis. Such results suggest that over-expression of RAC3 contributes to tumor development through molecular mechanisms that do not depend strictly on acetylation and/or steroid hormones, which control cell death. This could be a possible target for future tumor therapies. (author) [es

  9. A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

    International Nuclear Information System (INIS)

    Dao, T.; Holan, V.; Minowada, J.

    1993-01-01

    A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

  10. Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study.

    Science.gov (United States)

    Beck, Raphaël; Pedrosa, Rozangela Curi; Dejeans, Nicolas; Glorieux, Christophe; Levêque, Philippe; Gallez, Bernard; Taper, Henryk; Eeckhoudt, Stéphane; Knoops, Laurent; Calderon, Pedro Buc; Verrax, Julien

    2011-10-01

    Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

  11. Experiment list: SRX069159 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available | cell lineage=The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural effusi...on of a 53-year-old female with chronic myelogenous leukemia in terminal blast cris

  12. Experiment list: SRX037116 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available | cell lineage=The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural effusi...on of a 53-year-old female with chronic myelogenous leukemia in terminal blast cris

  13. Experiment list: SRX069222 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available | cell lineage=The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural effusi...on of a 53-year-old female with chronic myelogenous leukemia in terminal blast cris

  14. Anti-proliferative, Cytotoxic and NF-ĸB Inhibitory Properties of Spiro(Lactone-Cyclohexanone) Compounds in Human Leukemia.

    Science.gov (United States)

    Bouhenna, Mustapha M; Orlikova, Barbora; Talhi, Oualid; Schram, Ben; Pinto, Diana C G A; Taibi, Nadia; Bachari, Khaldoun; Diederich, Marc; Silva, Artur M S; Mameri, Nabil

    2017-09-01

    NF-ĸB affects most aspects of cellular physiology. Deregulation of NF-ĸB signaling is associated with inflammatory diseases and cancer. In this study, we evaluated the cytotoxic and NF-ĸB inhibition potential of new spiro(lactone-cyclohexanone) compounds in two different human leukemia cell lines (U937 and K562). The anti-proliferative effects of the spiro(lactone-cyclohexanone) compounds on human K562 and U937 cell lines was evaluated by trypan blue staining, as well as their involvement in NF-kB regulation were analyzed by luciferase reporter gene assay, Caspase-3/7 activities were evaluated to analyze apoptosis induction. Both spiro(coumarin-cyclohexanone) 4 and spiro(6- methyllactone-cyclohexanone) 9 down-regulated cancer cell viability and proliferation. Compound 4 inhibited TNF-α-induced NF-ĸB activation in a dose-dependent manner and induced caspase-dependent apoptosis in both leukemia cell lines. Results show that compound 4 and compound 9 have potential as anti-cancer agents. In addition, compound 4 exerted NF-kB inhibition activity in leukemia cancer cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. Photoionization Modeling and the K Lines of Iron

    Science.gov (United States)

    Kallman, T. R.; Palmeri, P.; Bautista, M. A.; Mendoza, C.; Krolik, J. H.

    2004-01-01

    We calculate the efficiency of iron K line emission and iron K absorption in photoionized models using a new set of atomic data. These data are more comprehensive than those previously applied to the modeling of iron K lines from photoionized gases, and allow us to systematically examine the behavior of the properties of line emission and absorption as a function of the ionization parameter, density and column density of model constant density clouds. We show that, for example, the net fluorescence yield for the highly charged ions is sensitive to the level population distribution produced by photoionization, and these yields are generally smaller than those predicted assuming the population is according to statistical weight. We demonstrate that the effects of the many strongly damped resonances below the K ionization thresholds conspire to smear the edge, thereby potentially affecting the astrophysical interpretation of absorption features in the 7-9 keV energy band. We show that the centroid of the ensemble of K(alpha) lines, the K(beta) energy, and the ratio of the K(alpha(sub 1)) to K(alpha(sub 2)) components are all diagnostics of the ionization parameter of our model slabs.

  16. Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines

    International Nuclear Information System (INIS)

    Maroschik, Belinda; Gürtler, Anne; Krämer, Anne; Rößler, Ute; Gomolka, Maria; Hornhardt, Sabine; Mörtl, Simone; Friedl, Anna A

    2014-01-01

    Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h. So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels

  17. Experiment list: SRX189948 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ozzio from the pleural effusion of a 53-year-old female with chronic myelogenous leukemia in terminal blast ... organism=human || cell description=leukemia, The continuous cell line K-562 was established by Lozzio and L

  18. Experiment list: SRX150436 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Lozzio from the pleural effusion of a 53-year-old female with chronic myelogenous leukemia in terminal blast...l organism=human || cell description=leukemia, The continuous cell line K-562 was established by Lozzio and

  19. Experiment list: SRX190011 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ozzio from the pleural effusion of a 53-year-old female with chronic myelogenous leukemia in terminal blast ... organism=human || cell description=leukemia, The continuous cell line K-562 was established by Lozzio and L

  20. Experiment list: SRX150506 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ozzio from the pleural effusion of a 53-year-old female with chronic myelogenous leukemia in terminal blast ... organism=human || cell description=leukemia, The continuous cell line K-562 was established by Lozzio and L

  1. Synthesis, characterization and anti cancer activity of some fluorinated 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles

    Directory of Open Access Journals (Sweden)

    Deepak Chowrasia

    2017-05-01

    Full Text Available A series of fluorinated 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles (2a–2i was synthesized by condensation of various substituted 4-amino-5-phenyl-4H-1,2,4-triazole-3-thiols (1a–1i with penta fluoro benzoic acid in good yields (60–80%. The synthesized compounds were screened for anticancer activity against three cancerous cell lines MCF7 (human breast cancer, SaOS-2 (human osteosarcoma and K562 (human myeloid leukemia. The compounds showed moderate to good antiproliferative potency against the studied cell lines. Among these, compound 2b showed higher antiproliferative activity (IC50 22.1, 19 and 15 μM against MCF7, SaOS-2 and K562, respectively while 2a exhibited least antiproliferative activity (IC50 30.2, 39 and 29.4 μM against MCF7, SaOS-2 and K562 cells, respectively. Therefore, the present study demonstrates that fluorine substituted 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles would be a better prospective in the development of anticancer drugs.

  2. Experiment list: SRX150512 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available usion of a 53-year-old female with chronic myelogenous leukemia in terminal blast c...organism=human || cell description=leukemia, The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural eff

  3. Experiment list: SRX058651 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available anism=human || cell description=leukemia, The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural effusi...on of a 53-year-old female with chronic myelogenous leukemia in terminal blast cris

  4. Experiment list: SRX058656 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available usion of a 53-year-old female with chronic myelogenous leukemia in terminal blast c...organism=human || cell description=leukemia, The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural eff

  5. Experiment list: SRX190085 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available usion of a 53-year-old female with chronic myelogenous leukemia in terminal blast c...organism=human || cell description=leukemia, The continuous cell line K-562 was established by Lozzio and Lozzio from the pleural eff

  6. INTERNALIZATION OF ANTIMICROBIAL PEPTIDE ACIPENSIN 1 INTO HUMAN TUMOR CELLS

    Directory of Open Access Journals (Sweden)

    E. S. Umnyakova

    2016-01-01

    Full Text Available Search for new compounds providing delivery of drugs into infected or neoplastic cells, is an important direction of biomedical research. Cell-penetrating peptides are among those compounds, due to their ability to translocate through membranes of eukaryotic cells, serving as potential carriers of various therapeutic agents to the target cells. The aim of present work was to investigate the ability of acipensin 1, an antimicrobial peptide of innate immune system, for in vitro penetration into human tumor cells. Acipensin 1 is a cationic peptide that we have previously isolated from leukocytes of the Russian sturgeon, Acipenser gueldenstaedtii. Capability of acipensin 1 to enter the human erytroleukemia K-562 cells has been investigated for the first time. A biotechnological procedure for producing a recombinant acipensin 1 peptide has been developed. The obtained peptide was conjugated with a fluorescent probe BODIPY FL. By means of confocal microscopy, we have shown that the tagged acipensin 1 rapidly enters into K-562 cells and can be detected in the intracellular space within 5 min after its addition to the cell culture. Using flow cytometry technique, penetration kinetics of the labeled peptide into K-562 cells (at nontoxic micromolar concentrations has been studied. We have observed a rapid internalization of the peptide to the target cells, thus confirming the results of microscopic analysis, i.e, the labeled acipensin was detectable in K-562 cells as soon as wihin 2-3 seconds after its addition to the incubation medium. The maximum of fluorescence was reached within a period of approx. 45 seconds, with further “plateau” at the terms of >100 seconds following cell stimulation with the test compound. These data support the concept, that the antimicrobial peptides of innate immunity system possess the features of cell-penetrating peptides, and allow us to consider the studied sturgeon peptide a promising template for development of new

  7. 2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate promotes megakaryocytic differentiation of myeloid leukaemia cells and primary human CD34⁺ haematopoietic stem cells.

    Science.gov (United States)

    Limb, Jin-Kyung; Song, Doona; Jeon, Mijeong; Han, So-Yeop; Han, Gyoonhee; Jhon, Gil-Ja; Bae, Yun Soo; Kim, Jaesang

    2015-04-01

    In this study we showed that 2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient differentiation of megakaryocytes. Specifically, (R)-TEMOSPho induces cell cycle arrest, cell size increase and polyploidization from K562 and HEL cells, which are used extensively to model megakaryocytic differentiation. In addition, megakaryocyte-specific cell surface markers showed a dramatic increase in expression in response to (R)-TEMOSPho treatment. Importantly, we demonstrated that such megakaryocytic differentiation can also be induced from primary human CD34(+) haematopoietic stem cells. Activation of the PI3K-AKT pathway and, to a lesser extent, the MEK-ERK pathway appears to be required for this process, as blocking with specific inhibitors interferes with the differentiation of K562 cells. A subset of (R)-TEMOSPho-treated K562 cells undergoes spontaneous apoptosis and produces platelets that are apparently functional, as they bind to fibrinogen, express P-selectin and aggregate in response to SFLLRN and AYPGFK, the activating peptides for the PAR1 and PAR4 receptors, respectively. Taken together, these results indicate that (R)-TEMOSPho will be useful for dissecting the molecular mechanisms of megakaryocytic differentiation, and that this class of compounds represents potential therapeutic reagents for thrombocytopenia. Copyright © 2012 John Wiley & Sons, Ltd.

  8. Anti-leishmanial and Anti-cancer Activities of a Pentacyclic ...

    African Journals Online (AJOL)

    Erah

    against promastigotes of Leishmania donovani, and anti-cancer activity on K562 leukaemic cell line. Results: A .... crisis of chronic myeloid leukemia was used for this test. The cells ... containing 1×106 cells/ml, 2 mM L-glutamine and 50 µg/ml ...

  9. Measurement of Nitrogen Hyperfine Structure on the 53 CM (562 MHz) Butyronitrile Line

    Science.gov (United States)

    Dewberry, Christopher T.; Grubbs, Garry S. Grubbs, II; Raphelt, Andrew; Cooke, Stephen A.

    2009-06-01

    Recent improvements to our cavity-based Fourier transform radiofrequency spectrometer will be presented. Amongst other improvements use of Miteq amp, model AMF-6F-00100400-10-10P (0.1 GHz to 4 GHz, 65 dB gain minimum, 1 dB noise figure maximum) together with shielding from an improved Faraday cage have significantly helped us in this regard. Electromagnetic fields within our near-spherical cavity have been modeled and results will be presented. We have been able to easily resolve the nitrogen hyperfine structure on the ^aQ_{0,-1} transition 1_{1,0} ← 1_{1,1} located at 562 MHz. This result will be discussed.

  10. Cytotoxic Activity of the Leaf and Stem Extracts of Hibiscus rosa ...

    African Journals Online (AJOL)

    Methods: The crude petroleum ether, ethyl acetate and methanol extracts of the leaf and stem of Hibiscus rosa sinensis were prepared using cold extraction method. The in vitro cytotoxic activity of the extracts (20 - 100 μg/ml) was evaluated on leukaemic cancer cell line (K-562) and Mardin-Darby kidney cell line (MDBK) ...

  11. Nicotine promotes cervical carcinoma cell line HeLa migration and invasion by activating PI3k/Akt/NF-κB pathway in vitro.

    Science.gov (United States)

    Wang, Chengze; Gu, Weiting; Zhang, Yunpeng; Ji, Yawen; Wen, Yong; Xu, Xin

    2017-07-05

    Cigarette smoking is one of highly risk factors of cervical cancer. Recently nicotine has been reported to increase proliferation and invasion in some smoking related cancers, like non-small cell lung cancer and esophageal squamous cell cancer. However, the effects and mechanisms of nicotine stimulation on cervical cancer cells are not clear. Here, we investigated the effects and mechanisms of nicotine stimulation on HeLa cells in vitro. In our study, we found that nicotine could accelerate HeLa cells migration and invasion, activate PI3K/Akt and NF-κB pathways and increase the expression of Vimentin in vitro. Moreover, we demonstrated that the specific PI3K inhibitor LY294002 could reverse nicotine-induced cell migration and invasion, NF-κB activation and up-regulation of Vimentin. Inhibition of NF-κB by Pyrrolidine dithiocarbamate (PDTC) also antagonized nicotine-induced cell migration, invasion and up-regulation of Vimentin. Simply put, these findings suggest that nicotine promotes cervical carcinoma cell line HeLa migration and invasion by activating PI3k/Akt/NF-κB pathway in vitro. Copyright © 2017 Elsevier GmbH. All rights reserved.

  12. Factors mediating lipofection potency of a series of cationic phosphonolipids in human cell lines.

    Science.gov (United States)

    Koumbi, Daphne; Clement, Jean-Claude; Sideratou, Zili; Yaouanc, Jean-Jacques; Loukopoulos, Dimitris; Kollia, Panagoula

    2006-08-01

    A series of cationic liposomes known as cationic phosphonolipids (CPs) were evaluated as vehicles for in vitro gene transfer in K562 erythroleukemia cells and 5637 epithelial carcinoma cells. For each CP and target cell type examined, detailed analyses were performed to determine optimal transfection conditions (lipid/ DNA (+/-) charge ratio, amount of complexed episomal DNA, liposomal and lipoplex size, complexation medium and duration of complex-cell exposure time). Lipofection conditions were determined to be both cell- and lipid-type specific. Complexation medium critically affected transfection competence. The initial size of the liposome was not always predictive of lipofection potency. The lipid chemical composition had a strong impact upon lipofection efficiency; DOPE inclusion in the liposome formulations was found to affect the levels of transgene expression in a cell-dependent way. Notably, effective transgene expression was characterized by prominent plasmid nuclear incorporation. Human A gamma- and epsilon-globin transgene nuclear incorporation and expression in 5637 cells post GLB.391-mediated lipofection lends credence to its use as a vehicle of therapeutic transgene delivery.

  13. Induction of erythroid differentiation in human erythroleukemia cells by depletion of malic enzyme 2.

    Directory of Open Access Journals (Sweden)

    Jian-Guo Ren

    2010-09-01

    Full Text Available Malic enzyme 2 (ME2 is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel

  14. Stimulation of granulocytic cell iodination by pine cone antitumor substances

    International Nuclear Information System (INIS)

    Unten, S.; Sakagami, H.; Konno, K.

    1989-01-01

    Antitumor substances (Fractions VI and VII) prepared from the NaOH extract of pine cone significantly stimulated the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood adherent mononuclear cells, polymorphonuclear cells (PMN), and human promyelocytic leukemic HL-60 cells. In contrast, these fractions did not significantly increase the iodination of nonadherent mononuclear cells, red blood cells, other human leukemic cell lines (U-937, THP-1, K-562), human diploid fibroblast (UT20Lu), or mouse cell lines (L-929, J774.1). Iodination of HL-60 cells, which were induced to differentiate by treatment with either retinoic acid or tumor necrosis factor, were stimulated less than untreated cells. The stimulation of iodination of both PMN and HL-60 cells required the continuous presence of these fractions and was almost completely abolished by the presence of myeloperoxidase inhibitors. The stimulation activity of these fractions was generally higher than that of various other immunopotentiators. Possible mechanisms of extract stimulation of myeloperoxidase-containing cell iodination are discussed

  15. The effect of lysate of spleen cells after low dose radiation (LDR) on NK activity

    International Nuclear Information System (INIS)

    Lu Duicai; Su Liaoyuan

    2003-01-01

    To find effect of lysate of spleen cells after LDR on NK activity of CD 57 cells or non-CD 57 cells, lysate of spleen cells after LDR were extracted. McAb (anti CD 57 cells) was used to separate CD 57 cells from human peripheral blood by Panning direct method. The CD 57 cells and non-CD 57 cells were used as effective cells. K 562 cells labelled by 3 H-TdR were used as target cells. The ratio of effect cells to target cells was 10:1. NK activity of CD 57 cells or non-CD 5 -7 cells with the lysate of spleen cells after LDR was reflected by the efficiency of anti tumor cells. The 3 H-TdR incorporation in K 562 cells cultured with non-CD 57 cells was significantly lower than that with CD 57 cells. After use of the lysate of spleen cells after LDR, NK activities of CD 57 cells and non-CD 57 cells were 1.24 and 1.58 respectively. They were both increased obviously compared with control groups. The effect of anti K 562 cells of non-CD 57 cells is even greater than that of CD 57 cells. The lysate of spleen cells after LDR can enhance the effect of both non-CD 57 cells and CD 57 cells

  16. KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity.

    Science.gov (United States)

    Suck, Garnet; Branch, Donald R; Smyth, Mark J; Miller, Richard G; Vergidis, Joanna; Fahim, Soad; Keating, Armand

    2005-10-01

    To compare the cytotoxicity of KHYG-1 with other natural killer (NK)/NK T-cell lines and identify molecules that may be associated with enhanced cytotoxicity, thereby eventually leading to improved NK cell-mediated cancer immunotherapy. NK/NK T-cell lines KHYG-1, NK-92, YT, and SNT-8 were compared with a novel flow cytometric cytotoxicity assay under different culture conditions. Transcription, expression, and phosphorylation studies were performed using polymerase chain reaction sequence-specific primers, reverse transcription polymerase chain reaction, immunoblotting, and flow cytometry. KHYG-1 is a highly cytotoxic cell line, exceeding the cytolytic capacity of the other cell lines against K562. KHYG-1 is also highly cytotoxic against the leukemia cell lines EM2, EM3, and HL60. The novel activation receptor NKp44 and its adaptor, DAP12, NKG2D, and constitutively phosphorylated ERK2 may be associated with the enhanced cytotoxicity of KHYG-1. This cell line most likely mediates cytolysis by granzyme M (but not granzymes A and B) together with perforin, which is constitutively fully cleaved to the 60-kD form, in contrast to the other cell lines. KHYG-1 is a valuable model for the study of enhanced cytotoxicity by NK cells. In addition to the activation of NKp44, KHYG-1 may induce apoptosis of tumor cells by the newly described granzyme M/perforin pathway. Targeted modifications of effector molecules demonstrated in this model could generate NK cells with even greater killing ability that may be particularly attractive for clinical application. Moreover, our demonstration of greater cytotoxicity of KHYG-1 versus NK-92 cells, already in clinical trials, suggests a direct therapeutic role for KHYG-1.

  17. One of the 10 cells of AA Injection Kicker K4

    CERN Multimedia

    CERN PhotoLab

    1979-01-01

    The 3.5 GeV/c Antiproton Accumulator (AA) had 2 delay-line type injection kickers, K3 (12 cells) and K4 (10 cells). Here we see one of the K4 cells, with ferrite between stainless-steel plates. Pulse voltage: 61 kV; rise/fall-time 86 ns; flat-top 460 ns; top flatness +-2%. During injection, the open side of the C-shaped kickers was closed off with a fast shutter, so that their stray field would not perturb the stack of already accumulated antiprotons.

  18. [SP600125-induced polyploidization of megakaryocytic leukemia cell lines by ribosomal protein S6 kinase 1 depends on the degree of cell differentiation].

    Science.gov (United States)

    Wang, Lili; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Zhao, Song; Ma, Dongchu

    2016-10-01

    Objective To investigate regulatory role of ribosomal protein S6 kinase 1 (S6K1) in the polyploidization of different megakaryocytic leukemia cell lines at the different differentiation stages. Methods Megakaryocytic leukemia cell lines (Dami, Meg-01 and HEL cells) were induced towards polyploidization by SP600125, a c-Jun N-terminal kinase (JNK) inhibitor. The SP600125-inducing process was blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor. The phenotype (CD41a, CD42a and CD42b) and DNA ploidy were detected by flow cytometry. The expression and phosphorylation of S6K1 and related proteins were detected by Western blotting. Results SP600125 induced polyploidization and increased the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) in Dami, Meg-01 and HEL cells. However, the effect of SP600125 on polyploidization of the three cell lines was different, with the strongest effect on Dami cells and the weakest on Meg-01 cells. Moreover, SP600125 increased the phosphorylation of S6K1 Thr421/Ser424 and decreased the phosphorylation of Thr389 in Dami cells. However, it only increased the phosphorylation of Thr389 in HEL cells and had no effect on the phosphorylation of S6K1 in Meg-01 cells. Interestingly, H-89 only partially blocked the polyploidization of Dami cells, although it decreased the phosphorylation of 4E-BP1 in all SP600125-induced three cell lines. Noticeably, H-89 decreased the phosphorylation of S6K1 Thr421/Ser424 and increased the phosphorylation of Thr389 in Dami cells. However, H-89 had no effect on the phosphorylation of Thr421/Ser424, although it increased the phosphorylation of Thr389 in Meg-01 and HEL cells. Phenotypic analysis showed that the three cell lines were at different levels of differentiation in megakaryocytic lineage, with the highest differentiation in Dami and the lowest in Meg-01 cells. Conclusion SP600125-induced polyploidization of megakaryocytic leukemia cell lines is dependent on the effect

  19. Natural killer (NK)-cell activity in sorted subsets of peripheral blood mononuclear cells from patients with severe combined immunodeficiency

    NARCIS (Netherlands)

    ten Berge, R. J.; Schellekens, P. T.; Budding-Koppenol, A.; Dooren, L. J.; Vossen, J. M.

    1987-01-01

    Natural killer-cell activity for K562 target cells was measured in 13 patients with severe combined immunodeficiency before bone marrow transplantation. Both unseparated peripheral blood mononuclear cells and sorted cell subsets (B73.1 positive, B73.1 negative, OKT3 positive, OKT3 negative) were

  20. Contribution of Protein Tyrosine Phosphateses to the Ontogeny and Progression of Chronic Myeloid Leukemia

    National Research Council Canada - National Science Library

    Tremblay, Michel

    2006-01-01

    ...). Inappropriate STAT1 and STAT5 activation have been observed in the Philadelphia chromosome-positive CML cell lines K562 and BV17, yet low levels of JAK1 tyrosine phosphorylation were observed...

  1. LET effects on normal and radiosensitive cell lines

    International Nuclear Information System (INIS)

    Geard, C.R.; Travisano, M.

    1986-01-01

    Charged particles in the track segment mode were produced by the RARAF Van de Graaff accelerator and used to irradiate two CHO cell lines, a radiosensitive hypermutable line EM9 and its normal parent AA8. Asynchronous cells were irradiated attached to 6 micrometer thick Mylar with protons, deuterons and helium-3 particles at LETs ranging from 10 to 150 keV per micrometer. A 50 kVp x-ray tube integrated into the track segment facility provided a low LET comparison. Following irradiation cells were monitored for clonogenicity, and in a separate series of experiments frequencies of sister chromatid exchanges. Up to 9 experiments were carried out at each LET, with a total of 8 radiations of different LETs being compared. The optimally effective LET for cell survival was between 80 and 120 keV per micrometer, with the 150 keV per micrometer particles indicating energy wastage. The differential between the normal and radiosensitive cell lines was maintained at all LETs

  2. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

    International Nuclear Information System (INIS)

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B.

    2007-01-01

    The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells

  3. CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.

    Science.gov (United States)

    Pardanani, A; Lasho, T; Smith, G; Burns, C J; Fantino, E; Tefferi, A

    2009-08-01

    Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

  4. 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.

  5. Cytotoxic Properties of Three Isolated Coumarin-hemiterpene Ether Derivatives from Artemisia armeniaca Lam.

    Science.gov (United States)

    Mojarrab, Mahdi; Emami, Seyed Ahmad; Delazar, Abbas; Tayarani-Najaran, Zahra

    2017-01-01

    Considering multiple reports on cytotoxic activity of the Artemisia genus and its phytochemicals, in the current study A. armeniaca Lam. and the three components isolated from the plant were subjected to cytotoxic studies. Analytical fractionation of A. armeniaca aerial parts for the first time was directed to the isolation of 7-hydroxy-8-(4-hydroxy-3-methylbutoxy) comarin (armenin), 8-hydroxy-7-(4-hydroxy-3-methylbutoxy) comarin (isoarmenin) and deoxylacarol. Cytotoxicity assessed with alamalBlue® assay and apoptosis was detected by PI staining and western blot analysis of Bax and PARP proteins. Extracts and all compounds exhibited cytotoxic activity against apoptosis-proficient HL-60 and apoptosis-resistant K562 cells, with the lowest cytotoxic activity on J774 cell line as non-malignant cell. Armenin as the most potent component decreased the viability of cell with IC50 of 22.5 and 71.1 µM for K562 and HL-60 cells respectively and selected for further mechanistic study. Armenin increased the sub-G1 peak in flow cytometry histogram of HL-60 and K562 treated cells and increase in the amount of Bax protein and the cleavage of PARP in comparison with the control after treatment for 48 h in K562 treated cells verified the apoptotic activity of the armenin. Taken together, according to the finding of this study armenin was introduced as a novel cytotoxic compound with apoptotic activity, which is encouraging for further mechanistic and clinical studies.

  6. Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

    Science.gov (United States)

    Hirasawa, Kazuhiro; Moriya, Shota; Miyahara, Kana; Kazama, Hiromi; Hirota, Ayako; Takemura, Jun; Abe, Akihisa; Inazu, Masato; Hiramoto, Masaki; Tsukahara, Kiyoaki

    2016-01-01

    Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”. PMID

  7. Difference in membrane repair capacity between cancer cell lines and a normal cell line

    DEFF Research Database (Denmark)

    Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

    2016-01-01

    repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

  8. High basal Wnt signaling is further induced by PI3K/mTor inhibition but sensitive to cSRC inhibition in mammary carcinoma cell lines with HER2/3 overexpression

    International Nuclear Information System (INIS)

    Timmermans-Sprang, Elpetra P. M.; Gracanin, Ana; Mol, Jan A.

    2015-01-01

    Elevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, β-Catenin or GSK3β but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling. Twelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level. High basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of β-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased β-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity. High basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated

  9. Antioxidant activity and cytotoxic profie of Chuquiraga spinosa Lessing on human tumor cell lines: A promissory plant from Peruvian flra

    Directory of Open Access Journals (Sweden)

    Oscar Herrera-Calderon

    2017-05-01

    Full Text Available Objective: To determine the phytochemical content, antioxidant activity in vitro and cytotoxicity of crude ethanol extract (CEE, n-hexane fraction (NHF, petroleum ether fraction (PEF, chloroform fraction (CLF and ethyl acetate fraction (EAF of aerial parts of Chuquiraga spinosa (C. spinosa Lessing. Methods: Phytochemical screening was developed by color and precipitated formation. The evaluation of antioxidant activity was assessed using hydroxyl and nitric oxide radical. Total phenolic content (TPC and total flavonoids content (TFC were measured by using standard methods by spectrophotometry. The cytotoxic effect was determined on human tumor cell lines including MCF-7, H-460, HT-29, M-14, HUTU-80, K-562 and DU-145. Results: Phytochemical analysis confirmed the presence of phenols, flavonoids in crude extract and its all fractions. The CEE showed the highest antioxidant activity, for OH and NO radical scavenging tests (IC50 = 15.16 ± 3.45 μg/mL and IC50 = 18.91 ± 1.13 μg/mL, respectively. TPC was found to be the highest in the CEE (121.36 mg of gallic acid equivalent/g of dried extract compared to other fractions. The ranking order of NHF, PEF, CLF, EAF and CEE for TFC was 21.17 < 35.20 < 62.19 < 70.25 < 78.25 mg quercetin equivalent/g of dried extract. The crude ethanolic extract (μg/mL showed a high cytotoxicity on MCF-7 (IC50 = 9.25 ± 0.81, K-562 (IC50 = 7.34 ± 1.00, HT-29 (IC50 = 8.52 ± 2.69, H-460 (IC50 = 5.32 ± 1.05, M-14 (IC50 = 8.30 ± 0.60, DU-145 (IC50 = 7.09 ± 0.09, HUTU-80 (IC50 = 6.20 ± 0.50. Conclusions: The study showed that CEE of the aerial parts of C. spinosa can be measured as a natural source of antioxidant which might be effective towards preventing or slowing oxidative stress related to chronic diseases as well as cytotoxic agent.

  10. Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line.

    Science.gov (United States)

    Goldoni, Dana; Zhao, YouYou; Green, Brian D; McDermott, Barbara J; Collins, Anthony

    2010-11-01

    HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100 µM) inhibited 44 ± 0.05% (mean ± s.e.m., n = 11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15 µM at -150  mV to 148 µM at -75  mV in 120  mM external K(+). This current was insensitive to 10 µM glybenclamide. A component of whole-cell current was sensitive to 150 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype. © 2010 Wiley-Liss, Inc.

  11. Three sesquiterpene compounds biosynthesised from artemisinic acid using suspension-cultured cells of Averrhoa carambola (Oxalidaceae).

    Science.gov (United States)

    Yang, Li; Zhu, Jianhua; Song, Liyan; Shi, Xiaojian; Li, Xingyi; Yu, Rongmin

    2012-01-01

    A new sesquiterpene glycoside, artemisinic acid 3-β-O-β-D-glucopyranoside (3, 31.24%) and other two biotransformation products, 3-β-hydroxyartemisinic acid (2, 36.69%) and 3-β-hydroxyartemisinic acid β-D-glucopyranosyl ester (4, 7.03%), were biosynthesised after artemisinic acid (1) was administered to the cultured cells of Averrhoa carambola. The three biotransformation products were obtained for the first time by using the suspension-cultured cells of A. carambola as a new biocatalyst system, and their structures were identified on the basis of the physico-chemical properties, NMR and mass spectral analyses. The results indicate that the cultured cells of A. carambola have the abilities to hydroxylate and glycosylate sesquiterpene compounds in a regio- and stereoselective manner. Furthermore, the anti-tumour activity of compounds 3 and 4 was evaluated against K562 and HeLa cell lines. Compound 4 showed strong activity against HeLa cell line, with the IC₅₀ value of 0.56 µmol mL⁻¹.

  12. The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

    Science.gov (United States)

    Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

    2016-06-22

    Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML.

  13. NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

    International Nuclear Information System (INIS)

    Wang, Yi; Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing

    2011-01-01

    Highlights: → Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. → Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. → P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. → Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the regulation of foam

  14. NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yi, E-mail: wangyi2004a@126.com [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China); Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China)

    2011-08-05

    Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the

  15. Cyclin K and cyclin D1b are oncogenic in myeloma cells

    Directory of Open Access Journals (Sweden)

    Renoir Jack-Michel

    2010-05-01

    Full Text Available Abstract Background Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM and always associated with mantle cell lymphoma (MCL. CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive. Results To test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis. Conclusions Our study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions.

  16. 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.)

  17. Radiosensitivity of mesothelioma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

    1996-10-01

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

  18. IGF-1R tyrosine kinase inhibitors and Vitamin K1 enhance the antitumor effects of Regorafenib in HCC cell lines.

    Science.gov (United States)

    Refolo, Maria Grazia; D'Alessandro, Rosalba; Lippolis, Catia; Carella, Nicola; Cavallini, Aldo; Messa, Caterina; Carr, Brian Irving

    2017-11-28

    The recent RESORCE trial showed that treatment with Regorafenib after Sorafenib failure provided a significant improvement in overall survival in HCC patients. Preclinical and clinical trial data showed that Regorafenib is a more potent drug than Sorafenib. In this study we aimed at improving Regorafenib actions and at reducing its toxicity, by targeting parallel pathways or by combination with Vitamins K (VKs). We investigated the effects of Regorafenib administrated at low concentrations and in combination with either VK1 and/or with GSK1838705A or OSI-906, two IGF1-R inhibitors, on HCC cell growth and motility. Our results showed that both IGF1-R inhibitors potentiated the antiproliferative and pro-apoptotic effects of Regorafenib and/or VK1 in HCC cell lines. Moreover we provide evidence that the combined treatment with IG1-R antagonists and Regorafenib (and/or VK1) also caused a significant reduction and depolymerization of actin resulting in synergistic inhibition exerted on cell migration. Thus, simultaneous blocking of MAPK and PI3K/Akt cascades with IGF1-R inhibitors plus Regorafenib could represent a more potent approach for HCC treatment.

  19. Eurycomanone and Eurycomanol from Eurycoma longifolia Jack as Regulators of Signaling Pathways Involved in Proliferation, Cell Death and Inflammation

    Directory of Open Access Journals (Sweden)

    Shéhérazade Hajjouli

    2014-09-01

    Full Text Available Eurycomanone and eurycomanol are two quassinoids from the roots of Eurycoma longifolia Jack. The aim of this study was to assess the bioactivity of these compounds in Jurkat and K562 human leukemia cell models compared to peripheral blood mononuclear cells from healthy donors. Both eurycomanone and eurycomanol inhibited Jurkat and K562 cell viability and proliferation without affecting healthy cells. Interestingly, eurycomanone inhibited NF-κB signaling through inhibition of IκBα phosphorylation and upstream mitogen activated protein kinase (MAPK signaling, but not eurycomanol. In conclusion, both quassinoids present differential toxicity towards leukemia cells, and the presence of the α,β-unsaturated ketone in eurycomanone could be prerequisite for the NF-κB inhibition.

  20. Research on Line Patrol Strategy of 110kV Transmission Line after Lightning Strike

    Directory of Open Access Journals (Sweden)

    Li Mingjun

    2016-01-01

    Full Text Available Lightning faults occupy in the majority of instantaneous fault and reclosing can usually be successful, so power supply can be restored without immediate patrol in many cases. Firstly, this paper introduces the lightning fault positioning and identifying method. Then test electrical performance of insulators after lightning strike from 110kV lines. Data shows that lightning strike has little effect on the electric performance of insulator. Finally, illustrating disposal process of the 110 kV transmission line after lightning fault, certifying that the power supply reliability be ensured without line patrol.

  1. Vitamins K2, K3 and K5 exert antitumor effects on established colorectal cancer in mice by inducing apoptotic death of tumor cells.

    Science.gov (United States)

    Ogawa, Mutsumi; Nakai, Seiji; Deguchi, Akihiro; Nonomura, Takako; Masaki, Tsutomu; Uchida, Naohito; Yoshiji, Hitoshi; Kuriyama, Shigeki

    2007-08-01

    Although a number of studies have shown that vitamin K possesses antitumor activities on various neoplastic cell lines, there are few reports demonstrating in vivo antitumor effects of vitamin K, and the antitumor effect on colorectal cancer (CRC) remains to be examined. Therefore, antitumor effects of vitamin K on CRC were examined both in vitro and in vivo. Vitamins K2, K3 and K5 suppressed the proliferation of colon 26 cells in a dose-dependent manner, while vitamin K1 did not. On flow cytometry, induction of apoptosis by vitamins K2, K3 and K5 was suggested by population in sub-G1 phase of the cell cycle. Hoechst 33342 staining and a two-color flow cytometric assay using fluorescein isothiocyanate-conjugated annexin V and propidium iodide confirmed that vitamins K2, K3 and K5 induced apoptotic death of colon 26 cells. Enzymatic activity of caspase-3 in colon 26 cells was significantly up-regulated by vitamins K2, K3 and K5. The pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, substantially prevented vitamin K-mediated apoptosis. In vivo study using syngeneic mice with subcutaneously established colon 26 tumors demonstrated that intravenous administration of vitamins K2, K3 and K5 significantly suppressed the tumor growth. The number of apoptotic tumor cells was significantly larger in the vitamin K-treated groups than in the control group. These results suggest that vitamins K2, K3 and K5 exerted effective antitumor effects on CRC in vitro and in vivo by inducing caspase-dependent apoptotic death of tumor cells, suggesting that these K vitamins may be promising agents for the treatment of patients with CRC.

  2. The Supportive Role of Insulin-Like Growth Factor-I in the Differentiation of Murine Mesenchymal Stem Cells into Corneal-Like Cells

    Czech Academy of Sciences Publication Activity Database

    Trošan, Peter; Javorková, Eliška; Zajícová, Alena; Hájková, Michaela; Heřmánková, Barbora; Kössl, Jan; Holáň, Vladimír

    2016-01-01

    Roč. 7, č. 17 (2016), s. 23156-23169 ISSN 1547-3287 R&D Projects: GA ČR(CZ) GA14-12580S; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1309; GA MŠk(CZ) LO1508 Institutional support: RVO:68378041 Keywords : mesenchymal stem cells * corneal-like cells * insulin -like growth factor-I * differentiation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.562, year: 2016

  3. Comparison at the peptide level with post-translational modification consideration reveals more differences between two unenriched samples.

    Science.gov (United States)

    Yin, Jianrui; Shao, Chen; Jia, Lulu; Gao, Youhe

    2014-06-30

    In shotgun strategies, peptide sequences are first identified from tandem mass (MS/MS) spectra, and the existence and abundance of the proteins are then inferred from the peptide information. However, the protein inference step can produce errors and a loss of information. To identify the information that is lost using the traditional approaches, this study compared the proteomic data of two leukemia cell lines (Jurkat and K562) at the peptide level with consideration of post-translational modifications (PTMs). The raw files from the two cell lines were searched against the decoy IPI-human database version 3.68, which contains forward and reverse sequences. Then the observed modification name in the results was matched with the modification classification on the Unimod website by a manual search. Only the peptides with 'post-translational' modifications were compared between the two cell lines. After searching the database with consideration of PTMs, a total of 44046 non-redundant peptides were identified in both the Jurkat and K562 cell lines. Of these peptides, even without specific PTM enrichment, 11.43% of them (with at least two spectra in one cell line) existed in different PTM forms between the two cell lines, and 1.73% of the peptides were modified in both cell lines, but with different modifications or possibly on different sites. Comparing proteomic data at the peptide level with consideration of PTMs can reveal more differences between two unenriched samples. Copyright © 2014 John Wiley & Sons, Ltd.

  4. Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment.

    Science.gov (United States)

    Zhou, Zhuo Long; Ma, Jing; Tong, Ming-Hui; Chan, Barbara Pui; Wong, Alice Sze Tsai; Ngan, Alfonso Hing Wan

    The adhesion and traction behavior of leukemia cells in their microenvironment is directly linked to their migration, which is a prime issue affecting the release of cancer cells from the bone marrow and hence metastasis. In assessing the effectiveness of phorbol 12-myristate 13-acetate (PMA) treatment, the conventional batch-cell transwell-migration assay may not indicate the intrinsic effect of the treatment on migration, since the treatment may also affect other cellular behavior, such as proliferation or death. In this study, the pN-level adhesion and traction forces between single leukemia cells and their microenvironment were directly measured using optical tweezers and traction-force microscopy. The effects of PMA on K562 and THP1 leukemia cells were studied, and the results showed that PMA treatment significantly increased cell adhesion with extracellular matrix proteins, bone marrow stromal cells, and human fibroblasts. PMA treatment also significantly increased the traction of THP1 cells on bovine serum albumin proteins, although the effect on K562 cells was insignificant. Western blots showed an increased expression of E-cadherin and vimentin proteins after the leukemia cells were treated with PMA. The study suggests that PMA upregulates adhesion and thus suppresses the migration of both K562 and THP1 cells in their microenvironment. The ability of optical tweezers and traction-force microscopy to measure directly pN-level cell-protein or cell-cell contact was also demonstrated.

  5. PI3K/PKB signaling in Rhipicephalus (Boophilus) microplus tick embryo cell line BME26

    International Nuclear Information System (INIS)

    Abreu, L.; Fabres, A.; Logullo, C.; Esteves, E.; Daffre, S.

    2008-01-01

    Full text: Ticks are obligatory blood-sucking arthropods and important vectors of both human and animal diseases. In order to study the insulin triggered pathway and its possible roles during embryogenesis we are using a culture of embryonic Rhipicephalus (Boophilus) micro plus cells (BME26). Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis and development. In such cascade Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). Exogenous insulin is able to increase the expression level of PI3K's regulatory sub unity (p85), as determined by Real Time RT-PCR. In the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were reversed. This correlates well with the activation of PKB by phosphorylation, as it appears to be PI3K-dependent. Additionally, PI3K inhibition increased the expression level of two insulin-regulated downstream targets from glycogen metabolism (GSK3b) and gluneogenesis (PEPCK) pathways. GSK3b inhibition by phosphorylation diminished in cells treated with PI3K inhibitors. These results strongly suggest the presence of an insulin sensitive PI3K-PKB axis in BME26 cells. The further study of PI3K and PKB activity in egg homogenates during embryogenesis may help us to understand the role of ISP for R. micro plus development

  6. Effect of Allium sativum (garlic) methanol extract on viability and ...

    African Journals Online (AJOL)

    diphenyltetrazolium bromide (MTT) assay at concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 ug/mL of Allium sativum extract following 48-h treatment on U-937, Jurkat Clone E6-1 and K-562 cell lines. The mode of cell death was ...

  7. Alkaloid-rich fraction of Himatanthus lancifolius contains anti-tumor agents against leukemic cells

    Directory of Open Access Journals (Sweden)

    Melissa Pires de Lima

    2010-06-01

    Full Text Available The effects of the alkaloid-rich fraction of Himatanthus lancifolius (Müll. Arg Woodson on normal marrow cells and leukemic cell lines were investigated. After 48 h exposure, the proliferation assay showed significant cell growth inhibition for Daudi (0.1-10 µg/mL, K-562 (1-10 µg/mL, and REH cells (10-100 µg/mL, yet was inert for normal marrow cells. A similar inhibition profile was observed in clonogenic assays. This alkaloid-rich fraction, in which uleine is the main compound, showed no signs of toxicity to any cells up to 10 µg/mL. Cell feature analyses after induction of differentiation showed maintenance of the initial phenotype. Flow cytometric expression of Annexin-V and 7-AAD in K-562 and Daudi cells has indicated that the cells were not undergoing apoptosis or necrosis, suggesting cytostatic activity for tumor cellsOs efeitos da fração rica em alcalóides indólicos de Himatanthus lancifolius (Müll. Arg Woodson sobre células normais de medula óssea e linhagens celulares leucêmicas foram investigados. Após 48 horas de exposição, os ensaios de proliferação demonstraram efeitos inibitórios significativos para as linhagens Daudi (0,1-10 µg/mL, K-562 (1-10 µg/mL e REH (10-100 µg/mL, enquanto mostrou-se inerte sobre células normais de medula óssea. Os perfis de inibição se repetiram nos ensaios clonogênicos. A fração rica em alcalóides, na qual a uleína é a substância majoritária, não demonstrou toxicidade até a dose de 10 µg/mL para nenhuma das células incluídas no estudo. Da mesma forma, não se observou influência dessa fração sobre a diferenciação celular dessas linhagens, mas manutenção de seu estado maturacional inicial. O conjunto de dados descritos associado à baixa co-expressão de anexina-V e 7-AAD sugerem que esta fração exerce atividade citostática para células tumorais.

  8. EM23, a natural sesquiterpene lactone from Elephantopus mollis H.B.K., induces apoptosis in human myeloid leukemia cells through thioredoxin- and reactive oxygen species-mediated signaling pathways

    Directory of Open Access Journals (Sweden)

    Hongyu eLi

    2016-03-01

    Full Text Available Elephantopus mollis H.B.K. (EM is a traditional herbal medicine with multiple pharmacological activities. However, the efficacy of EM in treating human leukemia is currently unknown. In the current study, we report that EM23, a natural sesquiterpene lactone isolated from EM, inhibits the proliferation of human chronic myeloid leukemia K562 cells and acute myeloid leukemia HL-60 cells by inducing apoptosis. Translocation of membrane-associated phospholipid phosphatidylserines, changes in cell morphology, activation of caspases and cleavage of PARP were concomitant with this inhibition. The involvement of the mitochondrial pathway in EM23-mediated apoptosis was suggested by observed disruptions in mitochondrial membrane potential (MMP. Mechanistic studies indicated that EM23 caused a marked increase in the level of reactive oxygen species (ROS. Pretreatment with N-acetyl-L-cysteine (NAC, a ROS scavenger, almost fully reversed EM23-mediated apoptosis. In EM23-treated cells, the expression levels of thioredoxin (Trx and thioredoxinreductase (TrxR, two components of the Trx system involved in maintaining cellular redox homeostasis, were significantly down-regulated. Concomitantly, Trx regulated the activation of apoptosis signal-regulating kinase 1 (ASK1 and its downstream regulatory targets, the p38, JNK, and ERK MAPKs. EM23-mediated activation of ASK1/MAPKs was significantly inhibited in the presence of NAC. Furthermore, tumor necrosis factor alpha (TNF-α-mediated activation of nuclear factor-κB (NF-κB was suppressed by EM23, as suggested by the observed blockage of p65 nuclear translocation, phosphorylation and reversion of IκBα degradation following EM23 treatment. Taken together, these results provide important insights into the anticancer activities of the EM component EM23 against human chronic myeloid leukemia K562 cells and acute myeloid leukemia HL-60 cells.

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

    Directory of Open Access Journals (Sweden)

    Julian Biau

    2016-08-01

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

  10. Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

    Science.gov (United States)

    Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

    1989-04-01

    Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

  11. Characterization of miRNomes in Acute and Chronic Myeloid

    Directory of Open Access Journals (Sweden)

    Qian Xiong

    2014-04-01

    Full Text Available Myeloid leukemias are highly diverse diseases and have been shown to be associated with microRNA (miRNA expression aberrations. The present study involved an in-depth miRNome analysis of two human acute myeloid leukemia (AML cell lines, HL-60 and THP-1, and one human chronic myeloid leukemia (CML cell line, K562, via massively parallel signature sequencing. mRNA expression profiles of these cell lines that were established previously in our lab facilitated an integrative analysis of miRNA and mRNA expression patterns. miRNA expression profiling followed by differential expression analysis and target prediction suggested numerous miRNA signatures in AML and CML cell lines. Some miRNAs may act as either tumor suppressors or oncomiRs in AML and CML by targeting key genes in AML and CML pathways. Expression patterns of cell type-specific miRNAs could partially reflect the characteristics of K562, HL-60 and THP-1 cell lines, such as actin filament-based processes, responsiveness to stimulus and phagocytic activity. miRNAs may also regulate myeloid differentiation, since they usually suppress differentiation regulators. Our study provides a resource to further investigate the employment of miRNAs in human leukemia subtyping, leukemogenesis and myeloid development. In addition, the distinctive miRNA signatures may be potential candidates for the clinical diagnosis, prognosis and treatment of myeloid leukemias.

  12. RBE of neutrons for induction of cell reproductive death and chromosome aberrations in three cell lines

    International Nuclear Information System (INIS)

    Zoetelief, J.; Kuijpers, W.C.; Baten-Wittwer, A.; Barendsen, G.W.

    1983-01-01

    The authors have compared the RBE values for induction of dicentrics and centric rings with those for cell inactivation and with the mean or effective quality factors (Q) recommended for radiation protection. The induction of cell reproductive death and chromosome aberrations has been investigated in plateau phase cultures of established lines of a rat rhabdomyosarcoma, a rat ureter carcinoma and Chinese hamster cells for single doses of 300 kV X-rays and 0.5, 4.2 and 15 MeV neutrons. The different cell lines show considerable variations in sensitivity and the RBE values obtained are presented in tabular form. The mean RBE values for the rat rhabdomyosarcoma cells are lower than those for the other two relatively resistant cell lines. Those for the Chinese hamster cells extrapolated to levels according to low doses of X-rays are in good agreement with the quoted Q values. (Auth./C.F.)

  13. PI3K/PKB signaling in Rhipicephalus (Boophilus) microplus tick embryo cell line BME26

    Energy Technology Data Exchange (ETDEWEB)

    Abreu, L.; Fabres, A.; Logullo, C. [Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, RJ (Brazil). Centro de Biociencias e Biotecnologia. Lab. de Quimica e Funcao de Proteinas e Peptideos (LQFPP)]. E-mail: leoabreu@uenf.br; Esteves, E.; Daffre, S. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Quimica. Dept. de Bioquimica

    2008-07-01

    Full text: Ticks are obligatory blood-sucking arthropods and important vectors of both human and animal diseases. In order to study the insulin triggered pathway and its possible roles during embryogenesis we are using a culture of embryonic Rhipicephalus (Boophilus) micro plus cells (BME26). Besides its metabolic role, insulin signaling pathway (ISP) is widely described as crucial for vertebrate and invertebrate embryogenesis and development. In such cascade Phosphatidylinositol 3-OH Kinase (PI3K) is hierarchically located upstream Protein Kinase B (PKB). Exogenous insulin is able to increase the expression level of PI3K's regulatory sub unity (p85), as determined by Real Time RT-PCR. In the presence of PI3K inhibitors (Wortmannin or LY294002) these effects were reversed. This correlates well with the activation of PKB by phosphorylation, as it appears to be PI3K-dependent. Additionally, PI3K inhibition increased the expression level of two insulin-regulated downstream targets from glycogen metabolism (GSK3b) and gluneogenesis (PEPCK) pathways. GSK3b inhibition by phosphorylation diminished in cells treated with PI3K inhibitors. These results strongly suggest the presence of an insulin sensitive PI3K-PKB axis in BME26 cells. The further study of PI3K and PKB activity in egg homogenates during embryogenesis may help us to understand the role of ISP for R. micro plus development.

  14. Activation of the PI3K/AKT pathway in Merkel cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Christian Hafner

    Full Text Available Merkel cell carcinoma (MCC is a highly aggressive skin cancer with an increasing incidence. The understanding of the molecular carcinogenesis of MCC is limited. Here, we scrutinized the PI3K/AKT pathway, one of the major pathways activated in human cancer, in MCC. Immunohistochemical analysis of 41 tumor tissues and 9 MCC cell lines revealed high levels of AKT phosphorylation at threonine 308 in 88% of samples. Notably, the AKT phosphorylation was not correlated with the presence or absence of the Merkel cell polyoma virus (MCV. Accordingly, knock-down of the large and small T antigen by shRNA in MCV positive MCC cells did not affect phosphorylation of AKT. We also analyzed 46 MCC samples for activating PIK3CA and AKT1 mutations. Oncogenic PIK3CA mutations were found in 2/46 (4% MCCs whereas mutations in exon 4 of AKT1 were absent. MCC cell lines demonstrated a high sensitivity towards the PI3K inhibitor LY-294002. This finding together with our observation that the PI3K/AKT pathway is activated in the majority of human MCCs identifies PI3K/AKT as a potential new therapeutic target for MCC patients.

  15. Proliferation-dependent changes in amino acid transport and glucose metabolism in glioma cell lines

    International Nuclear Information System (INIS)

    Sasajima, Toshio; Miyagawa, Tadashi; Oku, Takamitsu; Gelovani, Juri G.; Finn, Ronald; Blasberg, Ronald

    2004-01-01

    Amino acid imaging is increasingly being used for assessment of brain tumor malignancy, extent of disease, and prognosis. This study explores the relationship between proliferative activity, amino acid transport, and glucose metabolism in three glioma cell lines (U87, Hs683, C6) at different phases of growth in culture. Growth phase was characterized by direct cell counting, proliferation index determined by flow cytometry, and [ 3 H]thymidine (TdR) accumulation, and was compared with the uptake of two non-metabolized amino acids ([ 14 C]aminocyclopentane carboxylic acid (ACPC) and [ 14 C]aminoisobutyric acid (AIB)), and [ 18 F]fluorodeoxyglucose (FDG). Highly significant relationships between cell number (density), proliferation index, and TdR accumulation rate were observed in all cell lines (r>0.99). Influx (K 1 ) of both ACPC and AIB was directly related to cell density, and inversely related to the proliferation index and TdR accumulation in all cell lines. The volume of distribution (V d ) for ACPC and AIB was lowest during rapid growth and highest during the near-plateau growth phase in all cell lines. FDG accumulation in Hs683 and C6 cells was unaffected by proliferation rate, growth phase, and cell density, whereas FDG accumulation was correlated with TdR accumulation, growth phase, and cell density in U87 cells. This study demonstrates that proliferation rate and glucose metabolism are not necessarily co-related in all glioma cell lines. The values of K 1 and V d for ACPC and AIB under different growth conditions suggest that these tumor cell lines can up-regulate amino acid transporters in their cell membranes when their growth conditions become adverse and less than optimal. (orig.)

  16. Burkholderia cenocepacia K56-2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation.

    Science.gov (United States)

    Mil-Homens, Dalila; Pinto, Sandra N; Matos, Rute G; Arraiano, Cecília; Fialho, Arsenio M

    2017-04-01

    Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence. © 2016 John Wiley & Sons Ltd.

  17. Na+, K+-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms

    Directory of Open Access Journals (Sweden)

    Ali Mobasheri

    2012-04-01

    Full Text Available Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na+, K+-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons. Studying the distribution of Na+, K+-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na+, K+-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na+, K+-ATPase in a human chondrocyte cell line (C-20/A4 using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na+, K+-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na+, K+-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na+, K+-ATPase expression and regulation in chondrocytes.

  18. Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

    Science.gov (United States)

    Corral-Vázquez, C; Aguilar-Quesada, R; Catalina, P; Lucena-Aguilar, G; Ligero, G; Miranda, B; Carrillo-Ávila, J A

    2017-06-01

    Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

  19. Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Feuser, Paulo Emilio [Federal University of Santa Catarina, Department of Chemical Engineering and Food Engineering (Brazil); Jacques, Amanda Virtuoso [Federal University of Santa Catarina, Department of Clinical Analyses (Brazil); Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin [Federal University of Paraná, Department of Biochemistry and Molecular Biology (Brazil); Santos-Silva, Maria Claudia dos [Federal University of Santa Catarina, Department of Clinical Analyses (Brazil); Sayer, Claudia; Araújo, Pedro H. Hermes de, E-mail: pedro.h.araujo@ufsc.br [Federal University of Santa Catarina, Department of Chemical Engineering and Food Engineering (Brazil)

    2016-04-15

    The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

  20. Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

    International Nuclear Information System (INIS)

    Feuser, Paulo Emilio; Jacques, Amanda Virtuoso; Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin; Santos-Silva, Maria Claudia dos; Sayer, Claudia; Araújo, Pedro H. Hermes de

    2016-01-01

    The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

  1. Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

    Science.gov (United States)

    Feuser, Paulo Emilio; Jacques, Amanda Virtuoso; Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin; dos Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H. Hermes

    2016-04-01

    The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

  2. Do endothelial cells belong to the primitive stem leukemic clone in CML? Role of extracellular vesicles.

    Science.gov (United States)

    Ramos, Teresa L; Sánchez-Abarca, Luis Ignacio; López-Ruano, Guillermo; Muntión, Sandra; Preciado, Silvia; Hernández-Ruano, Montserrat; Rosado, Belén; de las Heras, Natalia; Chillón, M Carmen; Hernández-Hernández, Ángel; González, Marcos; Sánchez-Guijo, Fermín; Del Cañizo, Consuelo

    2015-08-01

    The expression of BCR-ABL in hematopoietic stem cells is a well-defined primary event in chronic myeloid leukemia (CML). Some reports have described the presence of BCR-ABL on endothelial cells from CML patients, suggesting the origin of the disease in a primitive hemangioblastic cell. On the other hand, extracellular vesicles (EVs) released by CML leukemic cells are involved in the angiogenesis modulation process. In the current work we hypothesized that EVs released from BCR-ABL(+) cells may carry inside the oncogene that can be transferred to endothelial cells leading to the expression of both BCR-ABL transcript and the oncoprotein. EVs from K562 cells and plasma of newly diagnosed CML patients were isolated by ultracentrifugation. RT-PCR analysis detected the presence of BCR-ABL RNA in the EVs isolated from both K562 cells and plasma of CML patients. The incorporation of these EVs into endothelial cells was demonstrated by flow cytometry and fluorescence microscopy showed that after 24h of incubation most EVs were incorporated. BCR-ABL transcripts were detected in all experiments on endothelial cells incubated with EVs from both sources. The presence of BCR-ABL on endothelial cells incubated with Philadelphia(+) EVs was also confirmed by Western blot assays. In summary, endothelial cells acquire BCR-ABL RNA and the oncoprotein after incubation with EVs released from Ph(+) positive cells (either from K562 cells or from plasma of newly diagnosed CML patients). This results challenge the hypothesis that endothelial cells may be part of the Philadelphia(+) clone in CML. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. [PHI regulates histone methylation and acetylation in Burkitt lymphoma Daudi cell line].

    Science.gov (United States)

    Hong, Ling-Ling; Ma, Xu-Dong; Huang, Yi-Qun

    2011-02-01

    This study was purposed to investigate the effects of phenylhexyl isothiocyanate (PHI) on Burkitt lymphoma Daudi cell line and regulation of histone acetylation and methylation in Daudi cells, and to explore the potential mechanism. The apoptotic rate of Daudi cells treated with PHI was measured by flow cytometry, the changes of histone H3 and H4 acetylation, histone H3K9 and H3K4 methylation in Daudi cells treated with PHI were detected by Western blot. The results showed that PHI could induce apoptosis of Daudi cells, increased the acetylation level of H3 and H4, enhanced the methylation of H3K4, but reduced the methylation of H3K9. It is concluded that the PHI can up-regulate the acetylation level of histone H3 associated with transcription stimulation and the methylation of histone H3K4, down-regulate the methylation on histone H3K9 associated with transcription inhibition, promotes the apoptosis of Daudi cells. PHI may be a potential agent for target therapy of lymphoma.

  4. Maslinic acid inhibits proliferation of renal cell carcinoma cell lines and suppresses angiogenesis of endothelial cells

    Directory of Open Access Journals (Sweden)

    Parth Thakor

    2017-03-01

    Full Text Available Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC remains a treatment-re-sistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1, endothelial cells (human umbilical vein endothelial cell line [HUVEC], and primary cultures of kidney proximal tubular epithelial cells (PTEC were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy.

  5. Molecular characterization of breast cancer cell lines through multiple omic approaches.

    Science.gov (United States)

    Smith, Shari E; Mellor, Paul; Ward, Alison K; Kendall, Stephanie; McDonald, Megan; Vizeacoumar, Frederick S; Vizeacoumar, Franco J; Napper, Scott; Anderson, Deborah H

    2017-06-05

    Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated

  6. JS-K, a nitric oxide-releasing prodrug, induces breast cancer cell death while sparing normal mammary epithelial cells.

    Science.gov (United States)

    McMurtry, Vanity; Saavedra, Joseph E; Nieves-Alicea, René; Simeone, Ann-Marie; Keefer, Larry K; Tari, Ana M

    2011-04-01

    Targeted therapy with reduced side effects is a major goal in cancer research. We investigated the effects of JS-K, a nitric oxide (NO) prodrug designed to release high levels of NO when suitably activated, on human breast cancer cell lines, on non-transformed human MCF-10A mammary cells, and on normal human mammary epithelial cells (HMECs). Cell viability assay, flow cytometry, electron microscopy, and Western blot analysis were used to study the effects of JS-K on breast cancer and on mammary epithelial cells. After a 3-day incubation, the IC50s of JS-K against the breast cancer cells ranged from 0.8 to 3 µM. However, JS-K decreased the viability of the MCF-10A cells by only 20% at 10-µM concentration, and HMECs were unaffected by 10 µM JS-K. Flow cytometry indicated that JS-K increased the percentages of breast cancer cells under-going apoptosis. Interestingly, flow cytometry indicated that JS-K increased acidic vesicle organelle formation in breast cancer cells, suggesting that JS-K induced autophagy in breast cancer cells. Electron microscopy confirmed that JS-K-treated breast cancer cells underwent autophagic cell death. Western blot analysis showed that JS-K induced the expression of microtubule light chain 3-II, another autophagy marker, in breast cancer cells. However, JS-K did not induce apoptosis or autophagy in normal human mammary epithelial cells. These data indicate that JS-K selectively induces programmed cell death in breast cancer cells while sparing normal mammary epithelial cells under the same conditions. The selective anti-tumor activity of JS-K warrants its further investigation in breast tumors.

  7. Characterization of human mucin (MUC15) and identification of ovine and caprine orthologs

    DEFF Research Database (Denmark)

    Pallesen, Lone Tjener; Pedersen, Lise Refstrup Linnebjerg; Petersen, Torben Ellebæk

    2008-01-01

    expression in placenta, salivary gland, thyroid gland, trachea, esophagus, kidney, testis, and the leukemia K-562 cell line. Furthermore, moderate expression was seen in the pancreas, adult and fetal lung, fetal kidney, lymph node, adult and fetal thymus, and parietal lobe. Structural motifs for interactions...

  8. Effects of Voltage-Gated K+ Channel on Cell Proliferation in Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Wei Wang

    2014-01-01

    Full Text Available Objective. To study the effects and underlying mechanisms of voltage-gated K+ channels on the proliferation of multiple myeloma cells. Methods. RPMI-8226 MM cell line was used for the experiments. Voltage-gated K+ currents and the resting potential were recorded by whole-cell patch-clamp technique. RT-PCR detected Kv channel mRNA expression. Cell viability was analyzed with MTT assay. Cell counting system was employed to monitor cell proliferation. DNA contents and cell volume were analyzed by flow cytometry. Results. Currents recorded in RPMI-8226 cells were confirmed to be voltage-gated K+ channels. A high level of Kv1.3 mRNA was detected but no Kv3.1 mRNA was detected in RPMI-8226 cells. Voltage-gated K+ channel blocker 4-aminopyridine (4-AP (2 mM depolarized the resting potential from −42 ± 1.7 mV to −31.8 ± 2.8 mV (P0.05. Conclusions. In RPMI-8226, voltage-gated K+ channels are involved in proliferation and cell cycle progression its influence on the resting potential and cell volume may be responsible for this process; the inhibitory effect of the voltage-gated K+ channel blocker on RPMI-8226 cell proliferation is a phase-specific event.

  9. Characterization of a mutant rat kangaroo cell line with alterations in the cell cycle and DNA repair

    OpenAIRE

    Miyaji, E.N.; Johnson, R.T.; Downes, C.S.; Eveno, E.; Mezzina, M.; Sarasin, A.; Menck, C.F.M.

    2000-01-01

    Using a positive selection system for isolating DNA replication and repair related mutants, we isolated a clone from a rat kangaroo cell line (PtK2) that has increased sensitivity to UV light. Characterization of this clone indicated normal post-replication repair after UV irradiation, and normal removal rates of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, po...

  10. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-07-30

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

  11. Radiation sensitivity of Merkell cell carcinoma cell lines

    International Nuclear Information System (INIS)

    Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

    1995-01-01

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

  12. Dendritic cells decreased the concomitant expanded Tregs and Tregs related IL-35 in cytokine-induced killer cells and increased their cytotoxicity against leukemia cells.

    Directory of Open Access Journals (Sweden)

    Ying Pan

    Full Text Available Regulatory T cells (Tregs are potent immunosuppressive cells and essential for inducing immune tolerance. Recent studies have reported that Tregs and Tregs related cytokines can inhibit the antitumor activity of cytokine-induced killer (CIK cells, but dendritic cells co-cultured CIK (DC-CIK cells can be used for induction of a specific immune response by blocking of Tregs and TGF-β, IL-10. As a novel identified cytokine, IL-35 is specially produced by Tregs and plays an essential role in immune regulation. However, it remains unknown whether IL-35 roles in tumor immunotherapy mediated by CIK and DC-CIK cells. In this study, we cultured CIK and DC-CIK cells from the same healthy adult samples, and investigated their phenotype, proliferation, cytotoxic activity against leukemia cell lines K562 and NB4 by FCM and CCK-8, measured IL-35, TGF-β and IL-10 protein by ELISA, detected Foxp3, IL-35 and IL-35 receptor mRNA by Real-time PCR, respectively. We found Tregs and IL-35 concomitantly expanded by a time-dependent way during the generation of CIK cells, but DC significantly down-regulated the expression of them and simultaneously up-regulated the proliferation ability as well as cytotoxic activity of CIK cells against leukemia cell lines. Therefore, our data suggested that DC decreased concomitant expanded Tregs and Tregs related IL-35 in CIK cells and might contribute to improve their cytotoxicity against leukemia cells in vitro.

  13. Interaction of leukemic cells with proteins of the extracellular matrix Interações de células leucêmicas com proteínas da matriz extracelular

    Directory of Open Access Journals (Sweden)

    Adriana Rodrigues-Anjos

    2004-01-01

    Full Text Available The interaction of neoplastic cells with basement membrane molecules is the first step for the dissemination of tumor cells in vivo. Leukemic cells have a great ability to spread in the host, since cells are released from the bone marrow to the circulation. In this study we analysed whether CEM, U937, K562 and HL-60 cells were able to attach to different concentrations of laminin and/or fibronectin and/or type IV collagen. Attachment to type IV collagen was low, but it increased with the addition of laminin and occurred in all four leukemic cell lines. On the other hand, attachment to fibronectin was higher, but it decreased with the addition of laminin in the assays using U937 and HL-60 cells. The combination of type IV collagen and fibronectin was a good substratum for cellular attachment. However, the addition of laminin to this substratum impaired its attachment activity in U937, HL-60 and K562. These data suggest that laminin may control cellular attachment to the extracellular matrix during leukemic dissemination in hosts in different ways.A interação de células neoplásicas com moléculas da membrana basal é a primeira etapa para a disseminação, in vivo, de células tumorais in vivo. As células leucêmicas possuem grande capacidade de espraiamento e disseminação no organismo uma vez que as mesmas são liberadas da medula óssea para a circulação. Neste trabalho avaliamos a capacidade das linhagens celulares CEM, U937, K562 and HL-60 em aderirem a uma matriz extracelular constituída por diferentes concentrações de laminina e,ou fibronectina e sobre colágeno IV. A adesão de todas a linhagens leucêmicas a colágeno IV foi baixa, mas aumentou com a associação à laminina. Por outro lado, as células U937 e HL-60 apresentaram alta ligação à fibronectina porém, foi reduzida com a adição de laminina. A associação de colágeno IV e fibronectina possibilitou um bom substrato para a adesão celular. Entretanto, a adi

  14. Effect of low dose radiation (LDR) on biological activity of NK cell

    International Nuclear Information System (INIS)

    Yang Liyun; Lin Meixiong; Luo Min; Ran Min; Liang Xuefei

    2006-01-01

    Objective: To study the in vitro and in vivo effect of LDR on the proliferation and killing activity of mouse NK cells with exploitation of the related mechanism of signal transduction. The effect of infused NK cells on inhibiton of oncogenesis and tumor burden regression was also studied. Methods: Mononuclear cells extracted from mouse spleen were treated with immunomagnetic bead for the isolation of CD3 - /CD16 + , CD56 + cells. After verified with flowcytometry, these NK cells were cultured with mice splenic cells (irradiated with 20Gy 60 Co gamma ray) as feeder cells and rhIL-2 as induction factor for 3 rounds (5 days each round). Specimens of cultured NK cells were treated with different doses of radiation (25mGy, 75mGy, 200mGy, 500mGy), the proliferation index (PI) with tumoreidal activity on K562 cells (with 3 H-TdR) incorporation was examined at 4h, 24h, 48h, 72h after irradiation respectively. The role of P38MAPK signal pathway in the LDR effect was examined with adding either inhibitor (SB203580) or activator (P79350) of P38MAPK into the culture and measuring the PI, Killing activity (as expression of the related factors IFN-gamma, FasL, perforin) of NK cells thereafter. The in vivo test involved exposing mice to whole body 25mGy irradiation, harvesting splenic NK cells at 4h, 24h, 48h, 72h later respectively and performing the above-described in vitro procedures. Inhibition of oncogenesis was examined in vivo with infusion of cultured NK cells (LDR treated vs LDR non-treated) 10 days after infusion of K562 cells into mice and examination of hepatic/splenic CD 13+ , S-stage cells and peripheral blood tumor cells in the sacrificed animal another 10 days later. Also, K562 cells were innoculated subcutaneously into mice. After tumor nodule formation (2.0 x 2.0 mm), NK cells (LDR treated vs non-treated) were infused and regression of the tumor nodule with the weight of hepatic tumor mass was noticed in sacrificed animals on d 8 and the survival rate on d 40

  15. Synthesis and Characterization of New Palladium(II) Thiosemicarbazone Complexes and Their Cytotoxic Activity against Various Human Tumor Cell Lines

    Science.gov (United States)

    Hernández, Wilfredo; Paz, Juan; Carrasco, Fernando; Spodine, Evgenia; Manzur, Jorge; Sieler, Joachim; Blaurock, Steffen; Beyer, Lothar

    2013-01-01

    The palladium(II) bis-chelate complexes of the type [Pd(TSC1-5)2] (6–10), with their corresponding ligands 4-phenyl-1-(acetone)-thiosemicarbazone, HTSC1 (1), 4-phenyl-1-(2′-chloro-benzaldehyde)-thiosemicarbazone, HTSC2 (2), 4-phenyl-1-(3′-hydroxy-benzaldehyde)-thiosemicarbazone, HTSC3 (3), 4-phenyl-1-(2′-naphthaldehyde)-thiosemicarbazone, HTSC4 (4), and 4-phenyl-1-(1′-nitro-2′-naphthaldehyde)-thiosemicarbazone, HTSC5 (5), were synthesized and characterized by elemental analysis and spectroscopic techniques (IR and 1H- and 13C-NMR). The molecular structure of HTSC3, HTSC4, and [Pd(TSC1)2] (6) have been determined by single crystal X-ray crystallography. Complex 6 shows a square planar geometry with two deprotonated ligands coordinated to PdII through the azomethine nitrogen and thione sulfur atoms in a cis arrangement. The in vitro cytotoxic activity measurements indicate that the palladium(II) complexes (IC50 = 0.01–9.87 μM) exhibited higher antiproliferative activity than their free ligands (IC50 = 23.48–70.86 and >250 μM) against different types of human tumor cell lines. Among all the studied palladium(II) complexes, the [Pd(TSC3)2] (8) complex exhibited high antitumor activity on the DU145 prostate carcinoma and K562 chronic myelogenous leukemia cells, with low values of the inhibitory concentration (0.01 and 0.02 μM, resp.). PMID:24391528

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

    Science.gov (United States)

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

    2009-01-01

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

  17. 12 CFR 562.2 - Regulatory reports.

    Science.gov (United States)

    2010-01-01

    ... § 562.2 Regulatory reports. (a) Definition and scope. This section applies to all regulatory reports, as... (TFR) are examples of regulatory reports. Regulatory reports are regulatory documents, not accounting... limited to, the accounting instructions provided in the TFR, guidance contained in OTS regulations...

  18. Cloning of a glutathione S-transferase decreasing during differentiation of HL60 cell line

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae Chul; Park, In Kyu; Lee, Kyu Bo; Sohn, Sang Kyun; Kim, Moo Kyu; Kim, Jung Chul [College of Medicine, Kyungpook National Univ., Taegu (Korea, Republic of)

    1999-06-01

    By sequencing the Expressed Sequence Tags of human dermal papilla cDNA library, we identified a clone named K872 of which the expression decreased during differentiation of HL60 cell line. K872 plasmid DNA was isolated according to QIA plasmid extraction kit (Qiagen GmbH, Germany). The nucleotide sequencing was performed by Sanger's method with K872 plasmid DNA. The most updated GenBank EMBL necleic acid banks were searched through the internet by using BLAST (Basic Local Alignment Search Tools) program. Northern bots were performed using RNA isolated from various human tissues and cancer cell lines. The gene expression of the fusion protein was achieved by His-Patch Thiofusion expression system and the protein product was identified on SDS-PAGE. K872 clone is 1006 nucleotides long, and has a coding region of 675 nucleotides and a 3' non-coding region of 280 nucleotides. The presumed open reading frame starting at the 5' terminus of K872 encodes 226 amino acids, including the initiation methionine residue. The amino acid sequence deduced from the open reading frame of K872 shares 70% identity with that of rat glutathione S-transferase kappa 1 (rGSTK1). The transcripts were expressed inh a variety of human tissues and cancer cells. The levels of transcript were relatively high in those tissues such as heart, skeletal muscle, and peripheral blood leukocyte. It is noteworthy that K872 was found to be abundantly expressed in colorectal cancer and melanoma cell lines. Homology search result suggests that K872 clone is the human homolog of the rGSTK1 which is known to be involved in the resistance of cytotoxic therapy. We propose that meticulous functional analysis should be followed to confirm that.

  19. 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.

  20. 40 CFR 80.562-80.569 - [Reserved

    Science.gov (United States)

    2010-07-01

    ... (CONTINUED) REGULATION OF FUELS AND FUEL ADDITIVES Motor Vehicle Diesel Fuel; Nonroad, Locomotive, and Marine Diesel Fuel; and ECA Marine Fuel Other Hardship Provisions §§ 80.562-80.569 [Reserved] Labeling...

  1. Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

    Science.gov (United States)

    Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

    2013-12-16

    Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

  2. Nuclear scaffold organization in the X-ray sensitive Chinese hamster mutant cell line, xrs-5

    International Nuclear Information System (INIS)

    Yasui, L.S.; Fink, T.J.; Enrique, A.M.

    1994-01-01

    Nuclear organization was probed in the radiation-sensitive Chinese hamster ovary (CHO) cell line, xrs-5, and compared with parental CHO K1 cells using the resinless section technique and DNase I digestions. The resinless section data showed no gross morphological differences in core filaments from the nuclear scaffolds of unirradiated CHO K1 and xrs-5 cells. However, the nuclear scaffolds of irradiated xrs-5 cells (1 Gy) had significantly increased ground substance. Irradiated and unirradiated CHO K1 cell nuclear scaffolds were morphologically identical. These data suggest that both CHO K1 and xrs-5 cell nuclear scaffolds had internal nuclear scaffolding networks that could provide DNA attachment sites. (author)

  3. A novel imidazopyridine PI3K inhibitor with anticancer activity in non-small cell lung cancer cells.

    Science.gov (United States)

    Lee, Hyunseung; Kim, Soo Jung; Jung, Kyung Hee; Son, Mi Kwon; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

    2013-08-01

    Lung cancer is the leading cause of cancer-related mortality in the world, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases. Since more than 60% of NSCLC cases express the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors are used to treat NSCLC. However, due to the acquired resistance associated with EGFR-targeted therapy, other strategies for the treatment of NSCLC are urgently needed. Therefore, we investigated the anticancer effects of a novel phosphatidylinositol 3-kinase α (PI3Kα) inhibitor, HS-173, in human NSCLC cell lines. HS-173 demonstrated anti-proliferative effects in NSCLC cells and effectively inhibited the PI3K signaling pathway in a dose‑dependent manner. In addition, it induced cell cycle arrest at G2/M phase as well as apoptosis. Taken together, our results demonstrate that HS-173 exhibits anticancer activities, including the induction of apoptosis, by blocking the PI3K/Akt/mTOR pathway in human NSCLC cell lines. We, therefore, suggest that this novel drug could potentially be used for targeted NSCLC therapy.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  5. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho, E-mail: kihos@catholic.ac.kr

    2014-01-17

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and

  6. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    International Nuclear Information System (INIS)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho

    2014-01-01

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1 + -K cells) was established. Induction of Neurogenin3 expression in Nkx6.1 + -K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1 + -K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation

  7. 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

  8. Inhibitors of pan PI3K signaling synergize with BRAF or MEK inhibitors to prevent BRAF-mutant melanoma cell growth

    Directory of Open Access Journals (Sweden)

    Melanie eSweetlove

    2015-06-01

    Full Text Available BRAF and MEK inhibitors have improved outcomes for patients with BRAF-mutant melanoma, but their efficacy is limited by both intrinsic and acquired resistance. Activation of the PI3K pathway can mediate resistance to these agents, providing a strong rationale for combination therapy in melanoma. Here, a panel of 9 low passage human metastatic melanoma cell lines with BRAF mutations were tested in cell proliferation and protein expression assays for sensitivity to inhibitors of MEK (selumetinib and BRAF (vemurafenib as single agents and in combination with inhibitors of pan-PI3K (ZSTK474, pan-PI3K/mTOR (BEZ235, individual PI3K isoforms (p110α, A66; p110β, TGX-221; p110γ, AS-252424; p110δ, idelalisib, or mTORC1/2 (KU-0063794. Selumetinib and vemurafenib potently inhibited cell proliferation in all cell lines, especially in those that expressed low levels of pAKT. ZSTK474 and BEZ235 also inhibited cell proliferation in all cell lines and enhanced the antitumor activity of selumetinib and vemurafenib in the majority of lines by either interacting synergistically or additively to increase potency or by inducing cytotoxicity by significantly increasing the magnitude of cell growth inhibition. Furthermore, ZSTK474 or BEZ235 combined with selumetinib to produce robust inhibition of pERK, pAKT and pS6 expression and synergistic inhibition of NZM20 tumor growth. The inhibitors of individual PI3K isoforms or mTORC1/2 were less effective at inhibiting cell proliferation either as single agents or in combination with selumetinib or vemurafenib, although KU-0063794 synergistically interacted with vemurafenib and increased the magnitude of cell growth inhibition with selumetinib or vemurafenib in certain cell lines. Overall, these results suggest that the sensitivity of BRAF-mutant melanoma cells to BRAF or MEK inhibitors is at least partly mediated by activation of the PI3K pathway and can be enhanced by combined inhibition of the BRAF/MEK and PI3K

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Zhang Ping

    2006-09-01

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

  11. Prostaglandins with antiproliferative activity induce the synthesis of a heat shock protein in human cells

    International Nuclear Information System (INIS)

    Santoro, M.G.; Garaci, E.; Amici, C.

    1989-01-01

    Prostaglandins (PGs)A 1 and J 2 were found to potently suppress the proliferation of human K562 erythroleukemia cells and to induce the synthesis of a 74-kDa protein (p74) that was identified as a heat shock protein related to the major 70-kDa heat shock protein group. p74 synthesis was stimulated at doses of PGA 1 and PGJ 2 that inhibited cell replication, and its accumulation ceased upon removal of the PG-induced proliferation block. PGs that did not affect K562 cell replication did not induce p74 synthesis. p74 was found to be localized mainly in the cytoplasm of PG-treated cells, but moderate amounts were found also in dense areas of the nucleus after PGJ 2 treatment. p74 was not necessarily associated with cytotoxicity or with inhibition of cell protein synthesis. The results described support the hypothesis that synthesis of the 70-kDa heat shock proteins is associated with changes in cell proliferation. The observation that PGs can induce the synthesis of heat shock proteins expands our understanding of the mechanism of action of these compounds whose regulatory role is well known in many physiological phenomena, including the control of fever production

  12. Dynamic iodide trapping by tumor cells expressing the thyroidal sodium iodide symporter

    International Nuclear Information System (INIS)

    Dingli, David; Bergert, Elizabeth R.; Bajzer, Zeljko; O'Connor, Michael K.; Russell, Stephen J.; Morris, John C.

    2004-01-01

    The thyroidal sodium iodide symporter (NIS) in combination with various radioactive isotopes has shown promise as a therapeutic gene in various tumor models. Therapy depends on adequate retention of the isotope in the tumor. We hypothesized that in the absence of iodide organification, isotope trapping is a dynamic process either due to slow efflux or re-uptake of the isotope by cells expressing NIS. Iodide efflux is slower in ARH-77 and K-562 cells expressing NIS compared to a thyroid cell line. Isotope retention half times varied linearly with the number of cells expressing NIS. With sufficient NIS expression, iodide efflux is a zero-order process. Efflux kinetics in the presence or absence of perchlorate also supports the hypothesis that iodide re-uptake occurs and contributes to the retention of the isotope in tumor cells. Iodide organification was insignificant. In vivo studies in tumors composed of mixed cell populations confirmed these observations

  13. Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line

    Science.gov (United States)

    Korch, Christopher; Hall, Erin M.; Dirks, Wilhelm G.; Ewing, Margaret; Faries, Mark; Varella‐Garcia, Marileila; Robinson, Steven; Storts, Douglas; Turner, Jacqueline A.; Wang, Ying; Burnett, Edward C.; Healy, Lyn; Kniss, Douglas; Neve, Richard M.; Nims, Raymond W.; Reid, Yvonne A.; Robinson, William A.

    2017-01-01

    A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. PMID:28940260

  14. Establishment, characterization and chemosensitivity of three mismatch repair deficient cell lines from sporadic and inherited colorectal carcinomas.

    Directory of Open Access Journals (Sweden)

    Claudia Maletzki

    Full Text Available BACKGROUND: Colorectal cancer (CRC represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113 along with their corresponding xenografts. METHODOLOGY: MMR-D cell lines (HROC24, HROC87, and HROC113 were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total. Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo. PRINCIPAL FINDINGS: Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-ras(wt, B-raf(mut, HROC87: K-ras(wt, B-raf(mut, whereas the HROC113 cell line (K-ras(mut, B-raf(wt was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM(+ tumor cells, showing surface tumor marker expression (CEACAM(+. MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity. CONCLUSIONS: These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the

  15. Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells.

    Directory of Open Access Journals (Sweden)

    Gábor Balogh

    Full Text Available Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations in membrane fluidity achieved by heat stress (HS resulted in pronounced membrane organization alterations which could be intimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-induced membrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, in contrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantly elevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtained by anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membrane changes upon HS. However, the structurally different probes revealed substantially distinct alterations in membrane heterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtle changes in membrane microstructure in the decision-making of thermal cell killing could have potential application in cancer therapy.

  16. Vitamin K2, a menaquinone present in dairy products targets castration-resistant prostate cancer cell-line by activating apoptosis signaling.

    Science.gov (United States)

    Dasari, Subramanyam; Samy, Angela Lincy Prem Antony; Kajdacsy-Balla, Andre; Bosland, Maarten C; Munirathinam, Gnanasekar

    2018-05-01

    The aim of this study was to evaluate the therapeutic effects of vitamin K2 (VK2) on castration-resistant prostate cancer (CRPC) and its anti-cancer mechanisms in a pre-clinical study using a VCaP cell line (ATCC ® CRL-2876™) which was established from a vertebral bone metastasis from a patient with hormone refractory prostate cancer. Our data showed that VK2 significantly inhibited CRPC VCaP cell proliferation in a dose-dependent manner at 48 h treatment in vitro. In addition, VK2 reduced the migration potential of VCaP cells and inhibited anchorage-independent growth of these cells. Our results also showed that VK2 induces apoptosis in VCaP cells. Furthermore, VK2 enforced growth arrest in VCaP cells by activating cellular senescence. Notably, VK2 treatment elevated the levels of reactive oxygen species in VCaP cells. Western blot analysis revealed that VK2 downregulated the expression of androgen receptor, BiP, survivin, while activating caspase-3 and -7, PARP-1 cleavage, p21 and DNA damage response marker, phospho-H2AX in VCaP cells. In conclusion, our study suggests that VK2 might be a potential anti-cancer agent for CRPC by specifically targeting key anti-apoptotic, cell cycle progression and metastasis-promoting signaling molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Nucleoli in human early erythroblasts (K2, K1, K1/2 cells).

    Science.gov (United States)

    Smetana, K; Jirásková, I; Klamová, H

    2005-01-01

    Human early erythroid precursors classified according to the nuclear size were studied to provide information on nucleoli in these cells using simple cytochemical procedures for demonstration of RNA and proteins of silver-stained nucleolar organizers. K2 cells with nuclear diameter larger than 13 microm and K1 cells with nuclear diameter larger than 9 microm corresponding to proerythroblasts and macroblasts (large basophilic erythroblasts) mostly possessed large irregularly shaped nucleoli with multiple fibrillar centres representing "active nucleoli". K1/2 cells with nuclear diameter smaller than 9 microm corresponding to small basophilic erythroblasts were usually characterized by the presence of micronucleoli representing "inactive nucleolar types". On the other hand, a few K1/2 cells contained large nucleoli with multiple fibrillar centres similar to those present in K2 cells and thus appeared as "microproerythroblasts". The nucleolar asynchrony expressed by the presence of large irregularly shaped nucleoli with multiple nucleoli (active nucleoli) and ring-shaped nucleoli (resting nucleoli) in one and the same nucleus of K2 or K1 cells was not exceptional and might reflect a larger resistance of these cells to negative factors influencing the erythropoiesis. The intranucleolar translocation of silver-stained nucleolus organized regions was noted in K2 cells and might indicate the premature aging of these cells without further differentiation. More studies, however, are required in this direction.

  18. Marys Lake 69/115-kV transmission line upgrade and substation expansion projects

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-05-01

    Western Area Power Administration (Western) and the Platte River Power Authority (Platte River) propose to upgrade portions of the existing electric transmission and substation system that serves the Town of Estes Park, Colorado. The existing transmission lines between the Estes Power Plant Switchyard and the Marys Lake Substation include a 115,000 volt (115-kV) line and 69,000 volt (69-kV) line. Approximately one mile is a double-circuit 115/69-kV line on steel lattice structures, and approximately two miles consists of separate single-circuit 115-kV and a 69-kV lines, constructed on wood H-Frame structures. Both lines were constructed in 1951 by the US Bureau of Reclamation. The existing transmission lines are on rights-of-way (ROW) that vary from 75 feet to 120 feet and are owned by Western. There are 48 landowners adjacent to the existing ROW. All of the houses were built adjacent to the existing ROW after the transmission lines were constructed. Upgrading the existing 69-kV transmission line between the Marys Lake Substation and the Estes Power Plant Switchyard to 115-kV and expanding the Marys Lake Substation was identified as the most effective way in which to improve electric service to Estes Park. The primary purpose and need of the proposed project is to improve the reliability of electric service to the Town of Estes Park. Lack of reliability has been a historical concern, and reliability will always be less than desired until physical improvements are made to the electrical facilities serving Estes Park.

  19. Marys Lake 69/115-kV transmission line upgrade and substation expansion projects

    International Nuclear Information System (INIS)

    1996-05-01

    Western Area Power Administration (Western) and the Platte River Power Authority (Platte River) propose to upgrade portions of the existing electric transmission and substation system that serves the Town of Estes Park, Colorado. The existing transmission lines between the Estes Power Plant Switchyard and the Marys Lake Substation include a 115,000 volt (115-kV) line and 69,000 volt (69-kV) line. Approximately one mile is a double-circuit 115/69-kV line on steel lattice structures, and approximately two miles consists of separate single-circuit 115-kV and a 69-kV lines, constructed on wood H-Frame structures. Both lines were constructed in 1951 by the US Bureau of Reclamation. The existing transmission lines are on rights-of-way (ROW) that vary from 75 feet to 120 feet and are owned by Western. There are 48 landowners adjacent to the existing ROW. All of the houses were built adjacent to the existing ROW after the transmission lines were constructed. Upgrading the existing 69-kV transmission line between the Marys Lake Substation and the Estes Power Plant Switchyard to 115-kV and expanding the Marys Lake Substation was identified as the most effective way in which to improve electric service to Estes Park. The primary purpose and need of the proposed project is to improve the reliability of electric service to the Town of Estes Park. Lack of reliability has been a historical concern, and reliability will always be less than desired until physical improvements are made to the electrical facilities serving Estes Park

  20. Paraptosis cell death induction by the thiamine analog benfotiamine in leukemia cells.

    Directory of Open Access Journals (Sweden)

    Naomi Sugimori

    Full Text Available Benfotiamine is a synthetic thiamine analogue that stimulates transketolase, a cellular enzyme essential for glucose metabolism. Currently, benfotiamine is used to treat diabetic neuropathy. We recently reported that oral benfotiamine induced a temporary but remarkable recovery from acute myeloid leukemia in an elderly patient who was ineligible for standard chemotherapy due to dementia and renal failure. In the present study we present evidences that benfotiamine possess antitumor activity against leukemia cells. In a panel of nine myeloid leukemia cell lines benfotiamine impaired the viability of HL-60, NB4, K562 and KG1 cells and also inhibited the growing of primary leukemic blasts. The antitumor activity of benfotiamine is not mediated by apoptosis, necrosis or autophagy, but rather occurs though paraptosis cell death induction. Mechanistic studies revealed that benfotiamine inhibited the activity of constitutively active ERK1/2 and concomitantly increased the phosphorylation of JNK1/2 kinase in leukemic cells. In addition, benfotiamine induced the down regulation of the cell cycle regulator CDK3 which resulted in G1 cell cycle arrest in the sensitive leukemic cells. Moreover, combination index studies showed that benfotiamine enhanced the antiproliferative activities of cytarabine against leukemia cells. These findings suggest that benfotiamine has antitumor therapeutic potential.

  1. Paraptosis cell death induction by the thiamine analog benfotiamine in leukemia cells.

    Science.gov (United States)

    Sugimori, Naomi; Espinoza, J Luis; Trung, Ly Quoc; Takami, Akiyoshi; Kondo, Yukio; An, Dao Thi; Sasaki, Motoko; Wakayama, Tomohiko; Nakao, Shinji

    2015-01-01

    Benfotiamine is a synthetic thiamine analogue that stimulates transketolase, a cellular enzyme essential for glucose metabolism. Currently, benfotiamine is used to treat diabetic neuropathy. We recently reported that oral benfotiamine induced a temporary but remarkable recovery from acute myeloid leukemia in an elderly patient who was ineligible for standard chemotherapy due to dementia and renal failure. In the present study we present evidences that benfotiamine possess antitumor activity against leukemia cells. In a panel of nine myeloid leukemia cell lines benfotiamine impaired the viability of HL-60, NB4, K562 and KG1 cells and also inhibited the growing of primary leukemic blasts. The antitumor activity of benfotiamine is not mediated by apoptosis, necrosis or autophagy, but rather occurs though paraptosis cell death induction. Mechanistic studies revealed that benfotiamine inhibited the activity of constitutively active ERK1/2 and concomitantly increased the phosphorylation of JNK1/2 kinase in leukemic cells. In addition, benfotiamine induced the down regulation of the cell cycle regulator CDK3 which resulted in G1 cell cycle arrest in the sensitive leukemic cells. Moreover, combination index studies showed that benfotiamine enhanced the antiproliferative activities of cytarabine against leukemia cells. These findings suggest that benfotiamine has antitumor therapeutic potential.

  2. Lactic Acid Bacteria from Kefir Increase Cytotoxicity of Natural Killer Cells to Tumor Cells

    OpenAIRE

    Takuya Yamane; Tatsuji Sakamoto; Takenori Nakagaki; Yoshihisa Nakano

    2018-01-01

    The Japanese fermented beverage, homemade kefir, contains six lactic acid bacteria: Lactococcus. lactis subsp. Lactis, Lactococcus. lactis subsp. Cremoris, Lactococcus. Lactis subsp. Lactis biovar diacetylactis, Lactobacillus plantarum, Leuconostoc meseuteroides subsp. Cremoris and Lactobacillus casei. In this study, we found that a mixture of the six lactic acid bacteria from kefir increased the cytotoxicity of human natural killer KHYG-1 cells to human chronic myelogenous leukemia K562 cell...

  3. Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules.

    Science.gov (United States)

    Kuriyama, Shigeki; Hitomi, Misuzu; Yoshiji, Hitoshi; Nonomura, Takako; Tsujimoto, Tatsuhiro; Mitoro, Akira; Akahane, Takami; Ogawa, Mutsumi; Nakai, Seiji; Deguchi, Akihiro; Masaki, Tsutomu; Uchida, Naohito

    2005-08-01

    A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.

  4. Opioid binding site in EL-4 thymoma cell line

    International Nuclear Information System (INIS)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of [ 3 H] bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10 6 cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of [ 3 H] bremazocine with an IC 50 value = 0.57μM. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, [D-Pen 2 , D-Pen 5 ] enkephalin and β-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC 50 = 60μM, that was similar to naloxone. 32 references, 3 figures, 2 tables

  5. Characterization of a mutant rat kangaroo cell line with alterations in the cell cycle and DNA repair

    Directory of Open Access Journals (Sweden)

    Miyaji E.N.

    2000-01-01

    Full Text Available Using a positive selection system for isolating DNA replication and repair related mutants, we isolated a clone from a rat kangaroo cell line (PtK2 that has increased sensitivity to UV light. Characterization of this clone indicated normal post-replication repair after UV irradiation, and normal removal rates of cyclobutane pyrimidine dimers and pyrimidine(6-4pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, possibly indicating a defect in preferential repair of actively transcribed genes, and a slower cell proliferation rate, including a longer S-phase. This phenotype reinforces the present notion that control of key mechanisms in cell metabolism, such as cell cycle control, repair, transcription and cell death, can be linked.

  6. Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

    International Nuclear Information System (INIS)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-01-01

    Aldosterone and insulin stimulate Na + transport through mechanisms involving protein synthesis. Na + -K + -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na + -K + -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na + -K + -[ 32 P]ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na + -K + -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na + entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na + -K + -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/

  7. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  8. K basin sandfilter backwash line characterization project, analytical results for campaign 15

    International Nuclear Information System (INIS)

    Steen, F.H.

    1999-01-01

    Sample 183KWBMF was taken from the K West Sandfilter Backwash Pit on August 28, 1998 and received by 222-S Laboratory on August 28, 1998. Analyses were performed in accordance with ''Letter of Instruction for K Basins Sandfilter Backwash Line Samples'' (LOI) in support of the K Basin Sandfilter Backwash Line Characterization Project

  9. OP9 Feeder Cells Are Superior to M2-10B4 Cells for the Generation of Mature and Functional Natural Killer Cells from Umbilical Cord Hematopoietic Progenitors

    Directory of Open Access Journals (Sweden)

    Lara Herrera

    2017-06-01

    Full Text Available Adoptive natural killer (NK cell therapy relies on the acquisition of large numbers of mature and functional NK cells. An option for future immunotherapy treatments is to use large amounts of NK cells derived and differentiated from umbilical cord blood (UCB CD34+ hematopoietic stem cells (HSCs, mainly because UCB is one of the most accessible HSC sources. In our study, we compared the potential of two stromal cell lines, OP9 and M2-10B4, for in vitro generation of mature and functional CD56+ NK cells from UCB CD34+ HSC. We generated higher number of CD56+ NK cells in the presence of the OP9 cell line than when they were generated in the presence of M2-10B4 cells. Furthermore, higher frequency of CD56+ NK cells was achieved earlier when cultures were performed with the OP9 cells than with the M2-10B4 cells. Additionally, we studied in detail the maturation stages of CD56+ NK cells during the in vitro differentiation process. Our data show that by using both stromal cell lines, CD34+ HSC in vitro differentiated into the terminal stages 4–5 of maturation resembled the in vivo differentiation pattern of human NK cells. Higher frequencies of more mature NK cells were reached earlier by using OP9 cell line than M2-10B4 cells. Alternatively, we observed that our in vitro NK cells expressed similar levels of granzyme B and perforin, and there were no significant differences between cultures performed in the presence of OP9 cell line or M2-10B4 cell line. Likewise, degranulation and cytotoxic activity against K562 target cells were very similar in both culture conditions. The results presented here provide an optimal strategy to generate high numbers of mature and functional NK cells in vitro, and point toward the use of the OP9 stromal cell line to accelerate the culture procedure to obtain them. Furthermore, this method could establish the basis for the generation of mature NK cells ready for cancer immunotherapy.

  10. Microchip screening platform for single cell assessment of NK cell cytotoxicity

    Directory of Open Access Journals (Sweden)

    Karolin eGuldevall

    2016-04-01

    Full Text Available Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75% were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3 target cells within the 12 hours long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

  11. Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

    Science.gov (United States)

    Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

    2016-01-01

    Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

  12. Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

    Directory of Open Access Journals (Sweden)

    Marcela Cristina Correa de Freitas

    2014-06-01

    Full Text Available OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO and baby hamster kidney cells (BHK. Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

  13. Combination therapy with vemurafenib (PLX4032/RG7204 and metformin in melanoma cell lines with distinct driver mutations

    Directory of Open Access Journals (Sweden)

    Recio Juan A

    2011-05-01

    Full Text Available Abstract Background A molecular linkage between the MAPK and the LKB1-AMPK energy sensor pathways suggests that combined MAPK oncogene inhibition and metabolic modulation of AMPK would be more effective than either manipulation alone in melanoma cell lines. Materials and methods The combination of the BRAF inhibitor vemurafenib (formerly PLX4032 and metformin were tested against a panel of human melanoma cell lines with defined BRAF and NRAS mutations for effects on viability, cell cycle and apoptosis. Signaling molecules in the MAPK, PI3K-AKT and LKB1-AMPK pathways were studied by Western blot. Results Single agent metformin inhibited proliferation in 12 out of 19 cell lines irrespective of the BRAF mutation status, but in one NRASQ61K mutant cell line it powerfully stimulated cell growth. Synergistic anti-proliferative effects of the combination of metformin with vemurafenib were observed in 6 out of 11 BRAFV600E mutants, including highly synergistic effects in two BRAFV600E mutant melanoma cell lines. Antagonistic effects were noted in some cell lines, in particular in BRAFV600E mutant cell lines resistant to single agent vemurafenib. Seven out of 8 BRAF wild type cell lines showed marginally synergistic anti-proliferative effects with the combination, and one cell line had highly antagonistic effects with the combination. The differential effects were not dependent on the sensitivity to each drug alone, effects on cell cycle or signaling pathways. Conclusions The combination of vemurafenib and metformin tended to have stronger anti-proliferative effects on BRAFV600E mutant cell lines. However, determinants of vemurafenib and metformin synergism or antagonism need to be understood with greater detail before any potential clinical utility of this combination.

  14. Promoter, transgene, and cell line effects in the transfection of mammalian cells using PDMAEMA-based nano-stars

    Directory of Open Access Journals (Sweden)

    Alexander Raup

    2016-09-01

    Full Text Available Non-viral transfection protocols are typically optimized using standard cells and reporter proteins, potentially underestimating cellular or transgene effects. Here such effects were studied for two human (Jurkat, HEK-293 and two rodent (CHO-K1, L929 cell lines and three fluorescent reporter proteins. Expression of the enhanced green fluorescent protein (EGFP was studied under the control of the human elongation factor 1 alpha promoter and three viral promoters (SV40, SV40/enhancer, CMV, that of ZsYellow1 (yellow fluorescence and mCherry (red fluorescence for the CMV promoter. Results varied with the cell line, in particular for the Jurkat cells. Pair-wise co-transfection of the CMV controlled transgenes resulted in a significant fraction of monochromatic cells (EGFP for EGFP/YFP and EGFP/RFP co-transfections, YFP in case of YFP/RFP co-transfections. Only Jurkat cells were almost incapable of expressing YFP. Dilution of the plasmid DNA with a non-expressed plasmid showed cell line dependent effects on transfection efficiency and/or expression levels.

  15. K Basin sandfilter backwash line characterization project, analytical results for Campaign 20

    International Nuclear Information System (INIS)

    STEEN, F.H.

    1999-01-01

    Sample 112KWBMF was taken from the K West Sandfilter Backwash Pit on June 1 , 1999, and received by 222-S Laboratory on June 2,1999. Analyses were performed on sample 112KWBMF in accordance with Letter of Instruction for K Basins Sandfilter Backwash Line Samples (LOI) in support of the K Basin Sandfilter Backwash Line Characterization Project

  16. Live imaging of H3K9 acetylation in plant cells

    Science.gov (United States)

    Kurita, Kazuki; Sakamoto, Takuya; Yagi, Noriyoshi; Sakamoto, Yuki; Ito, Akihiro; Nishino, Norikazu; Sako, Kaori; Yoshida, Minoru; Kimura, Hiroshi; Seki, Motoaki; Matsunaga, Sachihiro

    2017-01-01

    Proper regulation of histone acetylation is important in development and cellular responses to environmental stimuli. However, the dynamics of histone acetylation at the single-cell level remains poorly understood. Here we established a transgenic plant cell line to track histone H3 lysine 9 acetylation (H3K9ac) with a modification-specific intracellular antibody (mintbody). The H3K9ac-specific mintbody fused to the enhanced green fluorescent protein (H3K9ac-mintbody-GFP) was introduced into tobacco BY-2 cells. We successfully demonstrated that H3K9ac-mintbody-GFP interacted with H3K9ac in vivo. The ratio of nuclear/cytoplasmic H3K9ac-mintbody-GFP detected in quantitative analysis reflected the endogenous H3K9ac levels. Under chemically induced hyperacetylation conditions with histone deacetylase inhibitors including trichostatin A, Ky-2 and Ky-14, significant enhancement of H3K9ac was detected by H3K9ac-mintbody-GFP dependent on the strength of inhibitors. Conversely, treatment with a histone acetyltransferase inhibitor, C646 caused a reduction in the nuclear to cytoplasmic ratio of H3K9ac-mintbody-GFP. Using this system, we assessed the environmental responses of H3K9ac and found that cold and salt stresses enhanced H3K9ac in tobacco BY-2 cells. In addition, a combination of H3K9ac-mintbody-GFP with 5-ethynyl-2′-deoxyuridine labelling confirmed that H3K9ac level is constant during interphase. PMID:28418019

  17. SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

    Directory of Open Access Journals (Sweden)

    Zaborski Margarete

    2009-01-01

    Full Text Available Abstract Background SET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9(q34.11q34.13 has recently been described in T-cell acute lymphoblastic leukemia (T-ALL and in one case of acute myeloid leukemia (AML. The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene. Results Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF-Iβ-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH and array-based copy number analysis were both consistent with del(9(q34.11q34.13 as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein. Conclusion Cell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.

  18. In-vitro singlet oxygen threshold dose at PDT with Radachlorin photosensitizer

    Science.gov (United States)

    Klimenko, V. V.; Shmakov, S. V.; Kaydanov, N. E.; Knyazev, N. A.; Kazakov, N. V.; Rusanov, A. A.; Bogdanov, A. A.; Dubina, M. V.

    2017-07-01

    In this present study we investigate the Radachlorin photosensitizer accumulation in K562 cells and Hela cells and determined the cell viability after PDT. Using the macroscopic singlet oxygen modeling and cellular photosensitizer concentration the singlet oxygen threshold doses for K562 cells and Hela cells were calculated.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  20. PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

    Directory of Open Access Journals (Sweden)

    Diana Marklein

    Full Text Available We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX. We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.

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

    Directory of Open Access Journals (Sweden)

    Dongseob Tark

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

  2. In vitro effects of PCDDs/Fs on NK-like cell activity of Eisenia andrei earthworms

    Directory of Open Access Journals (Sweden)

    Hayet Belmeskine

    2012-02-01

    Full Text Available In this study, we assessed in vitro the effects of PCDD/Fs on the NK-like cell activity in Eisenia andrei earthworms using flow cytometry for analysis. NK-like coelomocytes isolated from E. andrei and used as effectors were exposed to various concentrations of PCDDs/Fs mixture, C1 (6.25x10-3 ng 2378- TCDD/mL, C2 (12.5x10-3 ng 2378-TCDD/mL and C3 (25x10-3 ng 2378-TCDD/mL, before adding them to human tumoral cells (K562 used as targets. We evaluated the percentage of targets lysed by Nk-like cells. The results showed a significant stimulation of the NKlike activity at C3 when PCDD/Fs were not removed from effectors before contact with targets, while no effects were noted when the effectors were washed (PCDD/Fs removed or fixed. Assessment of the viability of the targets (K562, exposed alone and separately from effectors, to the three concentrations of PCDD/Fs, C1, C2 and C3, showed that all these concentrations were cytotoxic for K562. Results suggest that PCDD/Fs concentrations tested in this assay may be considered too low to induce suppressive effects on the immune function such as the NK-like activity in E. andrei earthworms.

  3. Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells.

    Science.gov (United States)

    Kalle, Arunasree M; Mallika, A; Badiger, Jayasree; Alinakhi; Talukdar, Pinaki; Sachchidanand

    2010-10-08

    Overexpression of SIRT1, a NAD+-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Inhibition of H3K27me3 Histone Demethylase Activity Prevents the Proliferative Regeneration of Zebrafish Lateral Line Neuromasts

    Science.gov (United States)

    Bao, Beier; He, Yingzi; Tang, Dongmei; Li, Wenyan; Li, Huawei

    2017-01-01

    The H3K27 demethylases are involved in a variety of biological processes, including cell differentiation, proliferation, and cell death by regulating transcriptional activity. However, the function of H3K27 demethylation in the field of hearing research is poorly understood. Here, we investigated the role of H3K27me3 histone demethylase activity in hair cell regeneration using an in vivo animal model. Our data showed that pharmacologic inhibition of H3K27 demethylase activity with the specific small-molecule inhibitor GSK-J4 decreased the number of regenerated hair cells in response to neomycin damage. Furthermore, inhibition of H3K27me3 histone demethylase activity dramatically suppressed cell proliferation and activated caspase-3 levels in the regenerating neuromasts of the zebrafish lateral line. GSK-J4 administration also increased the expression of p21 and p27 in neuromast cells and inhibited the ERK signaling pathway. Collectively, our findings indicate that H3K27me3 demethylation is a key epigenetic regulator in the process of hair cell regeneration in zebrafish and suggest that H3K27me3 histone demethylase activity might be a novel therapeutic target for the treatment of hearing loss. PMID:28348517

  5. Opioid binding site in EL-4 thymoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  6. Comparison of growth factor signalling pathway utilisation in cultured normal melanocytes and melanoma cell lines

    International Nuclear Information System (INIS)

    Kim, Ji Eun; Stones, Clare; Joseph, Wayne R; Leung, Euphemia; Finlay, Graeme J; Shelling, Andrew N; Phillips, Wayne A; Shepherd, Peter R; Baguley, Bruce C

    2012-01-01

    The phosphatidylinositol-3-kinase (PI3K-PKB), mitogen activated protein kinase (MEK-ERK) and the mammalian target of rapamycin (mTOR- p70S6K), are thought to regulate many aspects of tumour cell proliferation and survival. We have examined the utilisation of these three signalling pathways in a number of cell lines derived from patients with metastatic malignant melanoma of known PIK3CA, PTEN, NRAS and BRAF mutational status. Western blotting was used to compare the phosphorylation status of components of the PI3K-PKB, MEK-ERK and mTOR-p70S6K signalling pathways, as indices of pathway utilisation. Normal melanocytes could not be distinguished from melanoma cells on the basis of pathway utilisation when grown in the presence of serum, but could be distinguished upon serum starvation, where signalling protein phosphorylation was generally abrogated. Surprisingly, the differential utilisation of individual pathways was not consistently associated with the presence of an oncogenic or tumour suppressor mutation of genes in these pathways. Utilisation of the PI3K-PKB, MEK-ERK and mTOR-p70S6K signalling pathways in melanoma, as determined by phosphorylation of signalling components, varies widely across a series of cell lines, and does not directly reflect mutation of genes coding these components. The main difference between cultured normal melanocytes and melanoma cells is not the pathway utilisation itself, but rather in the serum dependence of pathway utilisation

  7. Luteoloside Inhibits Proliferation of Human Chronic Myeloid ...

    African Journals Online (AJOL)

    Purpose: To investigate the effects of luteoloside on the proliferation of human chronic myeloid leukemia K562 cells and whether luteoloside induces cell cycle arrest and apoptosis in K562 cells. Methods: Luteoloside's cytotoxicity was assessed using a cell counting kit. Cell cycle distribution was analysed by flow cytometry ...

  8. Characterization of dengue virus 2 growth in megakaryocyte–erythrocyte progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Clark, Kristina B. [Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA (United States); Hsiao, Hui-Mien; Bassit, Leda [Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, GA (United States); Crowe, James E. [Departments of Pediatrics, Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN (United States); Schinazi, Raymond F. [Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, GA (United States); Perng, Guey Chuen [Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan (China); Villinger, Francois [Division of Microbiology and Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA (United States); New Iberia Research Center, University of Louisiana at Lafayette, New Iberia, LA (United States)

    2016-06-15

    Megakaryocyte–erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis. - Highlights: • DenV replicates efficiently in undifferentiated megakaryocyte–erythrocyte progenitors. • MEP produced DenV differs in protein content from Vero produced DenV. • MEP produced DenV may be more difficult to neutralize relative to Vero DenV.

  9. Characterization of dengue virus 2 growth in megakaryocyte–erythrocyte progenitor cells

    International Nuclear Information System (INIS)

    Clark, Kristina B.; Hsiao, Hui-Mien; Bassit, Leda; Crowe, James E.; Schinazi, Raymond F.; Perng, Guey Chuen; Villinger, Francois

    2016-01-01

    Megakaryocyte–erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis. - Highlights: • DenV replicates efficiently in undifferentiated megakaryocyte–erythrocyte progenitors. • MEP produced DenV differs in protein content from Vero produced DenV. • MEP produced DenV may be more difficult to neutralize relative to Vero DenV.

  10. Actinide L-line ED-XRF and Hybrid K-edge Densitometer Spectra Processing

    International Nuclear Information System (INIS)

    Esbelin, E.

    2015-01-01

    The analysis laboratory in the CEA Atalante complex at Marcoule (France) performs numerous R and D studies carried out in glove-boxes or in hot cells. Most of the samples are measured in liquid phase, aqueous or organic. The concentration of the main actinides of interest (U, Np, Pu, Am and Cm) are determined by XRF in a hot cell via their L-line X-ray between 13 and 15 keV. In order to limit the counting rate of many radioactive emitters (X-ray and gamma emitters) in the analysis solution and the continuous spectrum, a graphite monochromator is placed between the sample and detector. Commercial or free, the software packages available for processing X-ray spectra are designed and dedicated to a specific instrument and/or do not take into account the specific feature of our system, in other words, the presence of a monochromator. Therefore, a new X-ray analysis software programme was developed for this particular system which takes into account matrix effects corrections. For sample with U and/or Pu in high concentrations, the hybrid K-edge densitometer is used. A new software programme was also developed. For K-edge densitometry spectra processing, no calibration process is used. Spectra processing is based on theoretical equation and uses XCOM database for mass attenuation coefficients. Measured spectra on K-edge densitometer of Rokkasho Safeguards Analytical Laboratory were processed with this software and a very good agreement was found with IDTIMS results. The new graphical user interface allows to manually correct the defined edge. For the XRF spectra processing, new algorithms are used to define the base line and to find/integrate peaks. With these two analytical devices in laboratory, U and Pu concentrations can be measured from 0.5 mg/l to several hundred of g/l. (author)

  11. Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

    Science.gov (United States)

    Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

    1987-08-01

    Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula.

  12. Multiwalled carbon nanotubes effect on the bioavailability of artemisinin and its cytotoxity to cancerous cells

    Science.gov (United States)

    Rezaei, Behzad; Majidi, Najmeh; Noori, Shokoofe; Hassan, Zuhair M.

    2011-12-01

    Artemisinin regarded as one of the most promising anticancer drugs can bind to DNA with a binding constant of 1.04 × 104 M-1. The electrochemical experiments indicated that for longer incubation time periods, the reduction peak current of artemisinin on carbon nanotube modified electrode increases. Therefore, the uptake of drug molecules from a solution into CNTs will be achieved automatically by adsorption of 88.7% of artemisinin onto carbon nanotubes surface without alteration in drug properties. Hence, capability of carbon nanotubes to have synergistic effect on the bioavailability of artemisinin was investigated. Experimental tests on K562 cancer cell lines growth by MTT assay proved that multi-walled carbon nanotubes can enhance the cytotoxity of artemisinin to the targeted cancer cells with unprecedented accuracy and efficiency. The IC50 values were 65 and 35 μM for artemisinin and artemisinin loaded on multi-walled carbon nanotubes, respectively; demonstrating that artemisinin loaded on multi-walled carbon nanotubes is more effective in inhibition of cancer cell lines growth.

  13. Multiwalled carbon nanotubes effect on the bioavailability of artemisinin and its cytotoxity to cancerous cells

    International Nuclear Information System (INIS)

    Rezaei, Behzad; Majidi, Najmeh; Noori, Shokoofe; Hassan, Zuhair M.

    2011-01-01

    Artemisinin regarded as one of the most promising anticancer drugs can bind to DNA with a binding constant of 1.04 × 10 4 M −1 . The electrochemical experiments indicated that for longer incubation time periods, the reduction peak current of artemisinin on carbon nanotube modified electrode increases. Therefore, the uptake of drug molecules from a solution into CNTs will be achieved automatically by adsorption of 88.7% of artemisinin onto carbon nanotubes surface without alteration in drug properties. Hence, capability of carbon nanotubes to have synergistic effect on the bioavailability of artemisinin was investigated. Experimental tests on K562 cancer cell lines growth by MTT assay proved that multi-walled carbon nanotubes can enhance the cytotoxity of artemisinin to the targeted cancer cells with unprecedented accuracy and efficiency. The IC 50 values were 65 and 35 μM for artemisinin and artemisinin loaded on multi-walled carbon nanotubes, respectively; demonstrating that artemisinin loaded on multi-walled carbon nanotubes is more effective in inhibition of cancer cell lines growth.

  14. 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

  15. K-Line Patterns’ Predictive Power Analysis Using the Methods of Similarity Match and Clustering

    Directory of Open Access Journals (Sweden)

    Lv Tao

    2017-01-01

    Full Text Available Stock price prediction based on K-line patterns is the essence of candlestick technical analysis. However, there are some disputes on whether the K-line patterns have predictive power in academia. To help resolve the debate, this paper uses the data mining methods of pattern recognition, pattern clustering, and pattern knowledge mining to research the predictive power of K-line patterns. The similarity match model and nearest neighbor-clustering algorithm are proposed for solving the problem of similarity match and clustering of K-line series, respectively. The experiment includes testing the predictive power of the Three Inside Up pattern and Three Inside Down pattern with the testing dataset of the K-line series data of Shanghai 180 index component stocks over the latest 10 years. Experimental results show that (1 the predictive power of a pattern varies a great deal for different shapes and (2 each of the existing K-line patterns requires further classification based on the shape feature for improving the prediction performance.

  16. Survey of magnetic fields near BPA 230-kV and 500-kV transmission lines

    International Nuclear Information System (INIS)

    Perrin, N.; Aggarwal, R.P.

    1991-01-01

    The purpose of this study was to characterize typical levels and variability of 60Hz magnetic fields at the centerline and edge of right-of-way of Bonneville Power Administration (BPA) 230-kV and 500-kV transmission lines. This was accomplished by taking magnetic field measurements at over 800 spans in Oregon and Washington. The spans were sampled using a stratified random sampling procedure with region (East vs. West), voltage (230-kV vs 500-kV), and circuit configuration as strata. There were five different circuit configuration groups for each region/voltage category requiring a total of 200 strata. Magnetic field measurements were taken at 13 locations under each span using an EMDEX-C as a survey meter. Additional information recorded for each span included conductor height (at 10 locations), right-of-way width, longitudinal and lateral slope, time of day, vegetation, terrain, weather conditions, temperature, wind speed, span length and presence of other lines in the corridor. 9 refs., 17 figs., 26 tabs

  17. Identifying Neurofibromin Specific Regulatory Nodes for Therapeutic Targeting in NF1

    Science.gov (United States)

    2017-10-01

    suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704...imatinib, an approved BCR-ABL inhibitor. Given the robustness of these results, we believe K562 is an ideal cancer cell line system to interrogate ...fusion gene partner NOL4L greatly increased viability in the NF1 knockout cells, suggesting that these genes normally suppress growth through

  18. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Science.gov (United States)

    Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

    2016-01-01

    MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  19. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    Directory of Open Access Journals (Sweden)

    Ting Lu

    Full Text Available MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye could firmly bind to the surface of adherent cells (Hela and suspended cells (K562 even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  20. Frozen cord blood hematopoietic stem cells differentiate into higher numbers of functional natural killer cells in vitro than mobilized hematopoietic stem cells or freshly isolated cord blood hematopoietic stem cells.

    Directory of Open Access Journals (Sweden)

    Martha Luevano

    Full Text Available Adoptive natural killer (NK cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC has become an alluring option for NK cell therapy, with umbilical cord blood (UCB and mobilized peripheral blood (PBCD34(+ being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34(+ and frozen PBCD34(+ to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34(+ cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34(+ cultures. NK cells generated from CBCD34(+ and PBCD34(+ expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34(+-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34(+-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34(+ for the production of NK cells in vitro results in higher cell numbers than PBCD34(+, without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.

  1. Identification of apoptosis-related PLZF target genes

    International Nuclear Information System (INIS)

    Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes; Campillo, Jose Antonio; Parrado, Antonio

    2007-01-01

    The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localization is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression

  2. Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells

    International Nuclear Information System (INIS)

    Duncan, L; Shelmerdine, H; Hughes, M P; Coley, H M; Huebner, Y; Labeed, F H

    2008-01-01

    Dielectrophoresis (DEP)-the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K + and Ca 2+ levels were extremely similar between sensitive and resistant lines, levels of Cl - were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function. (note)

  3. Room/corner tests of wall linings with 100/300 kW burner

    Science.gov (United States)

    M. A. Dietenberger; O. Grexa; R. H. White; M. S. Sweet; M. Janssens

    1995-01-01

    Six room/comer tests of common wall linings were conducted with gypsum-lined ceiling exposed to propane burning at 100 kW for 10 min followed by 300 kW for 10 min. This test protocol is an option provided by ISO 9705. The flashover event occurred at 1,000 kW rate of heat release within several seconds of observing flames out the doorway. The time to flashover of the...

  4. Localization Study of Co-Phthalocyanines in Cells by Raman Micro(spectro)scopy

    NARCIS (Netherlands)

    Arzhantsev, S.Y.; Arzhantsev, S.Y.; Chikishev, A.Y.; Chikishev, A.Y.; Koroteev, N.I.; Greve, Jan; Otto, Cornelis; Sijtsema, N.M.

    1999-01-01

    An investigation of intracellular localization of Co-phthalocyanines is reported. The Raman images of K562 cells stained with phthalocyanine were acquired. To understand the peculiarities of the Raman images, measurements were performed at different z-axis positions. The intracellular concentration

  5. Localization study of Co-phthalocyanines in cells by Raman micro(spectro)scopy

    NARCIS (Netherlands)

    Arzhantsev, S Y; Chikishev, A Y; Koroteev, N I; Greve, J; Otto, C; Sijtsema, N M

    An investigation of intracellular localization of Co-phthalocyanines is reported. The Raman images of K562 cells stained with phthalocyanine were acquired. To understand the peculiarities of the Raman images, measurements were performed at different z-axis positions. The intracellular concentration

  6. Influence of terbutaline on natural killer cell activity

    International Nuclear Information System (INIS)

    Stenius-Aarniala, B.; Vesterinen, E.; Kiviranta, K.; Timonen, T.

    1988-01-01

    Studies on the effect of cAMO-inducing agents on NK activity have been contradictory. The aim of this study was to investigate the acute effects of beta-agonists on NK activity in vivo in 15 asthmatics and 3 healthy volunteers. Blood samples of NK activity were taken at regular intervals after placebo and after subcutaneous injection of 7 μg/kg of terbutaline. NK activity was measured by the standard 4-h Chromium 51 release assay against the leukemic line K 562 at a 50:1 effector/target cell ratio. Compared with placebo, terbutaline induced within 30-60 min a significant increase in NK activity which lasted less than 2 h. Further studies are necessary to investigate the effect of long-term beta-agonist treatment on NK activity. (author)

  7. Experimental determination of line strengths for selected carbon monoxide and carbon dioxide absorption lines at temperatures between 295 and 1250 K

    International Nuclear Information System (INIS)

    Medvecz, P.J.; Nichols, K.M.

    1994-01-01

    Fourier transform infrared absorption spectroscopy has been used for the determination of the line strengths of 41 CO and CO 2 absorption lines at temperatures between 295 and 1250 K. The CO vibrational-rotational lines were from the P branch of the fundamental absorption band (2150--1950 cm -1 ) while the CO 2 vibrational-rotational lines were from the far wing of the R branch of the ν 3 fundamental band (2395--2380 cm -1 ). The intensities of the lines were measured from absorption spectra recorded in a high-temperature gas cell containing known concentrations of CO/CO 2 /N 2 gas mixtures at atmospheric pressure. Absorption spectra were recorded through the cell with the use of a moderate-resolution Fourier transform infrared spectrometer. The absorption spectra were mathematically corrected for distortions resulting from the finite resolution of the spectrometer and for peak overlap. Line strength measurements were made from the corrected peaks by using the Bouguer-Lambert law and assuming a Lorentzian line profile. The experimentally obtained line strengths were evaluated by statistical calculations, by consideration of the validity of the Bouguer-Lambert assumption for these data, by comparison with existing room-temperature and high-temperature data, and by comparison with theoretical calculations. For CO, the statistical analysis suggests that the reported values have an uncertainty of ±10--12%, which is similar to the observed discrepancies with other reported values at room temperature. At high temperatures, the difference between these data and previously reported data and theoretical predictions is less than 10%. For CO 2 , the statistical uncertainty associated with the line strength calculations is less than 5%, which is also the approximate level of agreement with existing room-temperature data

  8. El coactivador de receptores nucleares RAC3 tiene un rol protector de la Apoptosis inducida por distintos estímulos RAC3 nuclear receptor co-activator has a protective role in the apoptosis induced by different stimuli

    Directory of Open Access Journals (Sweden)

    Georgina P. Coló

    2007-10-01

    Full Text Available RAC3 pertenece a la familia de coactivadores de receptores nucleares p160, y se encuentra sobreexpresado en varios tumores. Demostramos previamente que RAC3 es coactivador del factor de transcripción anti-apoptótico NF-kapa;B. En este trabajo investigamos su rol en la apoptosis inducida por H2O2 en una línea celular no tumoral derivada de riñón embrionario humano (HEK293, y por el ligando inductor de apoptosis relacionado a TNF (TRAIL en una línea de leucemia mieloide crónica humana (K562, naturalmente resistente a la muerte por este estímulo. Observamos que las células tumorales K562 poseen niveles altos de RAC3 comparados con las células no tumorales HEK293. La sobreexpresión normal de coactivador o por transfección, inhibe la apoptosis mediante una disminución de la activación de caspasas, translocación del factor inductor de apoptosis (AIF al núcleo, aumento de la actividad de NF-kapa;B y las quinasas AKT y p38 y disminución de la quinasa ERK. Lo opuesto fue observado por disminución de RAC3 mediante la técnica de ARN interferente (RNAi en K562, aumentando así la apoptosis inducida por TRAIL. Estas evidencias sugieren que una sobreexpresión de RAC3 contribuye al desarrollo de tumores, participando en las cascadas que controlan la muerte celular por mecanismos no estrictamente dependientes de hormonas esteroideas y/o de acetilación, constituyendo esto un posible blanco de ataque para el tratamiento de tumores.RAC3 belongs to the family of p160 nuclear receptors coactivators and it is over-expressed in several tumors. We have previously shown that RAC3 is a NF-kappa;B coactivator. In this paper, we investigated the role of RAC3 in cell-sensitivity to apoptosis, using H2O2 in the human embryonic kidney cell line (HEK293, and tumor necrosis factor-related apoptosis inducing ligand (TRAIL in a human chronic myeloid leukemia cell line (K562 naturally resistant to TRAIL. We observed that the tumoral K562 cells have high levels

  9. Two New Sphingolipids from the Leaves of Piper betle L.

    OpenAIRE

    Chen, Duo-Zhi; Xiong, Hua-Bin; Tian, Kai; Guo, Jun-Ming; Huang, Xiang-Zhong; Jiang, Zhi-Yong

    2013-01-01

    Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

  10. Two new sphingolipids from the leaves of Piper betle L.

    Science.gov (United States)

    Chen, Duo-Zhi; Xiong, Hua-Bin; Tian, Kai; Guo, Jun-Ming; Huang, Xiang-Zhong; Jiang, Zhi-Yong

    2013-09-12

    Two new sphingolipids, pipercerebrosides A (1) and B (2), were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

  11. Modulation of cell polarization by the Na+-K+-ATPase-associated protein FXYD5 (dysadherin).

    Science.gov (United States)

    Lubarski, Irina; Asher, Carol; Garty, Haim

    2014-06-01

    FXYD5 (dysadherin or also called a related to ion channel, RIC) is a transmembrane auxiliary subunit of the Na(+)-K(+)-ATPase shown to increase its maximal velocity (Vmax). FXYD5 has also been identified as a cancer-associated protein whose expression in tumor-derived cell lines impairs cytoskeletal organization and increases cell motility. Previously, we have demonstrated that the expression of FXYD5 in M1 cells derived from mouse kidney collecting duct impairs the formation of tight and adherence junctions. The current study aimed to further explore effects of FXYD5 at a single cell level. It was found that in M1, as well as three other cell lines, FXYD5 inhibits transformation of adhered single cells from the initial radial shape to a flattened, elongated shape in the first stage of monolayer formation. This is also correlated to less ordered actin cables and fewer focal points. Structure-function analysis has demonstrated that the transmembrane domain of FXYD5, and not its unique extracellular segment, mediates the inhibition of change in cell shape. This domain has been shown before to be involved in the association of FXYD5 with the Na(+)-K(+)-ATPase, which leads to the increase in Vmax. Furthermore, specific transmembrane point mutations in FXYD5 that either increase or decrease its effect on cell elongation had a corresponding effect on the coimmunoprecipitation of FXYD5 with α Na(+)-K(+)-ATPase. These findings lend support to the possibility that FXYD5 affects cell polarization through its transmembrane domain interaction with the Na(+)-K(+)-ATPase. Yet interaction of FXYD5 with other proteins cannot be excluded. Copyright © 2014 the American Physiological Society.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    OpenAIRE

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

    1988-01-01

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

  14. K Basin Sandfilter Backwash Line Characterization Project and Analytical Results for Campaign 24

    International Nuclear Information System (INIS)

    STEEN, F.H.

    1999-01-01

    Sample 203KWBMF was taken from the K West Sandfilter Backwash Pit on November 4, 1999 and received by 2224 Laboratory on November 4, 1999. Analyses were performed on sample 203KWBMF in accordance with ''Letter of Instruction for K Basins sandfilter Backwash Line Samples'' (LOI) in support of the K Basin Sandfilter Backwash Line Characterization Project

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

    International Nuclear Information System (INIS)

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

    1993-01-01

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

  16. Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kito, Hiroaki [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Yamazaki, Daiju [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Ohya, Susumu; Yamamura, Hisao [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2011-07-29

    Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

  17. The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

    Science.gov (United States)

    Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

    2012-10-01

    Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

  18. The Identification of Interferon-gamma as a Key Supportive Factor for Retinal Differentiation of Murine Mesenchymal Stem Cells

    Czech Academy of Sciences Publication Activity Database

    Heřmánková, Barbora; Kössl, Jan; Javorková, Eliška; Boháčová, Pavla; Hájková, Michaela; Zajícová, Alena; Krulová, Magdaléna; Holáň, Vladimír

    2017-01-01

    Roč. 26, č. 19 (2017), s. 1399-1408 ISSN 1547-3287 R&D Projects: GA ČR(CZ) GA17-04800S; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : mesenchymal stem cell * differentiation * retina Subject RIV: FF - HEENT, Dentistry OBOR OECD: Cell biology Impact factor: 3.562, year: 2016

  19. Generation and functional analysis of T cell lines and clones specific for schistosomula released products (SRP-A).

    Science.gov (United States)

    Damonneville, M; Velge, F; Verwaerde, C; Pestel, J; Auriault, C; Capron, A

    1987-01-01

    Antigens present in the products released by the larval stage of schistosome (SRP-A) were shown to induce a strong cytotoxic and protective IgE response both in the rat and the monkey. T cell lines and clones specific for SRP-A or 26 kD antigens which are the main target of the cytotoxic IgE have been derived. The passive transfer of SRP-A specific T lymphocytes into infected rats led to an increase of the IgE response, conferring a significant level of protection to the rats. In coculture assays in vitro, these cell lines significantly enhanced the production of IgE by SRP-A sensitized rat spleen cells. This helper effect on the IgE response was confirmed with 26 kD T cell clone supernatants. Moreover, supernatants obtained after stimulation with phorbol myristate acetate were able to enhance the IgE production of a hybridoma B cell line (B48-14) producing a monoclonal IgE antibody, cytotoxic for the schistosomula. PMID:3498590

  20. Two New Sphingolipids from the Leaves of Piper betle L.

    Directory of Open Access Journals (Sweden)

    Zhi-Yong Jiang

    2013-09-01

    Full Text Available Two new sphingolipids, pipercerebrosides A (1 and B (2, were isolated from the leaves of Piper betle L. Their structures, including absolute configurations, were determined by spectroscopic analysis and chemical degradation. These two compounds did not show significant cytotoxic activity against the cancer cell lines K562 and HL-60 in a MTT assay.

  1. Effect of Allium sativum (garlic) methanol extract on viability and ...

    African Journals Online (AJOL)

    Allium sativum extract following 48-h treatment on U-937, Jurkat Clone E6-1 and K-562 cell lines. The mode of ... correlated with down-regulation of Bcl-2, XIAP, and cIAP-1 ... Kuala Lumpur, Malaysia and then dried at room temperature ..... the authors named in this article and all liabilities ... health maintenance: A review.

  2. Antioxidative and in vitro antiproliferative activity of Arctium lappa root extracts

    Science.gov (United States)

    2011-01-01

    Background Arctium lappa, known as burdock, is widely used in popular medicine for hypertension, gout, hepatitis and other inflammatory disorders. Pharmacological studies indicated that burdock roots have hepatoprotective, anti-inflammatory, free radical scavenging and antiproliferative activities. The aim of this study was to evaluate total phenolic content, radical scavenging activity by DPPH and in vitro antiproliferative activity of different A. lappa root extracts. Methods Hot and room temperature dichloromethanic, ethanolic and aqueous extracts; hydroethanolic and total aqueous extract of A. lappa roots were investigated regarding radical scavenging activity by DPPH, total phenolic content by Folin-Ciocalteau method and antiproliferative in vitro activity was evaluated in human cancer cell lines. The hydroethanolic extract analyzed by high-resolution electrospray ionization mass spectroscopy. Results Higher radical scavenging activity was found for the hydroethanolic extract. The higher phenolic contents were found for the dichloromethane, obtained both by Soxhlet and maceration extraction and hydroethanolic extracts. The HRESI-MS demonstrated the presence of arctigenin, quercetin, chlorogenic acid and caffeic acid compounds, which were identified by comparison with previous data. The dichloromethane extracts were the only extracts that exhibited activity against cancer cell lines, especially for K562, MCF-7 and 786-0 cell lines. Conclusions The hydroethanolic extracts exhibited the strongest free radical scavenging activity, while the highest phenolic content was observed in Soxhlet extraction. Moreover, the dichloromethanic extracts showed selective antiproliferative activity against K562, MCF-7 and 786-0 human cancer cell lines. PMID:21429215

  3. Antioxidative and in vitro antiproliferative activity of Arctium lappa root extracts.

    Science.gov (United States)

    Predes, Fabricia S; Ruiz, Ana L T G; Carvalho, João E; Foglio, Mary A; Dolder, Heidi

    2011-03-23

    Arctium lappa, known as burdock, is widely used in popular medicine for hypertension, gout, hepatitis and other inflammatory disorders. Pharmacological studies indicated that burdock roots have hepatoprotective, anti-inflammatory, free radical scavenging and antiproliferative activities. The aim of this study was to evaluate total phenolic content, radical scavenging activity by DPPH and in vitro antiproliferative activity of different A. lappa root extracts. Hot and room temperature dichloromethanic, ethanolic and aqueous extracts; hydroethanolic and total aqueous extract of A. lappa roots were investigated regarding radical scavenging activity by DPPH, total phenolic content by Folin-Ciocalteau method and antiproliferative in vitro activity was evaluated in human cancer cell lines. The hydroethanolic extract analyzed by high-resolution electrospray ionization mass spectroscopy. Higher radical scavenging activity was found for the hydroethanolic extract. The higher phenolic contents were found for the dichloromethane, obtained both by Soxhlet and maceration extraction and hydroethanolic extracts. The HRESI-MS demonstrated the presence of arctigenin, quercetin, chlorogenic acid and caffeic acid compounds, which were identified by comparison with previous data. The dichloromethane extracts were the only extracts that exhibited activity against cancer cell lines, especially for K562, MCF-7 and 786-0 cell lines. The hydroethanolic extracts exhibited the strongest free radical scavenging activity, while the highest phenolic content was observed in Soxhlet extraction. Moreover, the dichloromethanic extracts showed selective antiproliferative activity against K562, MCF-7 and 786-0 human cancer cell lines. © 2011 Predes et al; licensee BioMed Central Ltd.

  4. Antioxidative and in vitro antiproliferative activity of Arctium lappa root extracts

    Directory of Open Access Journals (Sweden)

    Carvalho João E

    2011-03-01

    Full Text Available Abstract Background Arctium lappa, known as burdock, is widely used in popular medicine for hypertension, gout, hepatitis and other inflammatory disorders. Pharmacological studies indicated that burdock roots have hepatoprotective, anti-inflammatory, free radical scavenging and antiproliferative activities. The aim of this study was to evaluate total phenolic content, radical scavenging activity by DPPH and in vitro antiproliferative activity of different A. lappa root extracts. Methods Hot and room temperature dichloromethanic, ethanolic and aqueous extracts; hydroethanolic and total aqueous extract of A. lappa roots were investigated regarding radical scavenging activity by DPPH, total phenolic content by Folin-Ciocalteau method and antiproliferative in vitro activity was evaluated in human cancer cell lines. The hydroethanolic extract analyzed by high-resolution electrospray ionization mass spectroscopy. Results Higher radical scavenging activity was found for the hydroethanolic extract. The higher phenolic contents were found for the dichloromethane, obtained both by Soxhlet and maceration extraction and hydroethanolic extracts. The HRESI-MS demonstrated the presence of arctigenin, quercetin, chlorogenic acid and caffeic acid compounds, which were identified by comparison with previous data. The dichloromethane extracts were the only extracts that exhibited activity against cancer cell lines, especially for K562, MCF-7 and 786-0 cell lines. Conclusions The hydroethanolic extracts exhibited the strongest free radical scavenging activity, while the highest phenolic content was observed in Soxhlet extraction. Moreover, the dichloromethanic extracts showed selective antiproliferative activity against K562, MCF-7 and 786-0 human cancer cell lines.

  5. Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

    Science.gov (United States)

    Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

    2018-01-01

    Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

  6. Microelectrical Impedance Spectroscopy for the Differentiation between Normal and Cancerous Human Urothelial Cell Lines: Real-Time Electrical Impedance Measurement at an Optimal Frequency

    Directory of Open Access Journals (Sweden)

    Yangkyu Park

    2016-01-01

    Full Text Available Purpose. To distinguish between normal (SV-HUC-1 and cancerous (TCCSUP human urothelial cell lines using microelectrical impedance spectroscopy (μEIS. Materials and Methods. Two types of μEIS devices were designed and used in combination to measure the impedance of SV-HUC-1 and TCCSUP cells flowing through the channels of the devices. The first device (μEIS-OF was designed to determine the optimal frequency at which the impedance of two cell lines is most distinguishable. The μEIS-OF trapped the flowing cells and measured their impedance at a frequency ranging from 5 kHz to 1 MHz. The second device (μEIS-RT was designed for real-time impedance measurement of the cells at the optimal frequency. The impedance was measured instantaneously as the cells passed the sensing electrodes of μEIS-RT. Results. The optimal frequency, which maximized the average difference of the amplitude and phase angle between the two cell lines (p<0.001, was determined to be 119 kHz. The real-time impedance of the cell lines was measured at 119 kHz; the two cell lines differed significantly in terms of amplitude and phase angle (p<0.001. Conclusion. The μEIS-RT can discriminate SV-HUC-1 and TCCSUP cells by measuring the impedance at the optimal frequency determined by the μEIS-OF.

  7. ORIGIN OF THE GALACTIC DIFFUSE X-RAY EMISSION: IRON K-SHELL LINE DIAGNOSTICS

    Energy Technology Data Exchange (ETDEWEB)

    Nobukawa, Masayoshi [Department of Teacher Training and School Education, Nara University of Education, Takabatake-cho, Nara, 630-8528 (Japan); Uchiyama, Hideki [Faculty of Education, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529 (Japan); Nobukawa, Kumiko K.; Koyama, Katsuji [Department of Physics, Graduate School of Science, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto, 606-8502 (Japan); Yamauchi, Shigeo, E-mail: nobukawa@nara-edu.ac.jp [Department of Physics, Nara Women’s University, Kitauoyanishimachi, Nara, 630-8506 (Japan)

    2016-12-20

    This paper reports detailed K-shell line profiles of iron (Fe) and nickel (Ni) of the Galactic Center X-ray Emission (GCXE), Galactic Bulge X-ray Emission (GBXE), Galactic Ridge X-ray Emission (GRXE), magnetic Cataclysmic Variables (mCVs), non-magnetic Cataclysmic Variables (non-mCVs), and coronally Active Binaries (ABs). For the study of the origin of the GCXE, GBXE, and GRXE, the spectral analysis is focused on equivalent widths of the Fe i-K α , Fe xxv-He α , and Fe xxvi-Ly α  lines. The global spectrum of the GBXE is reproduced by a combination of the mCVs, non-mCVs, and ABs spectra. On the other hand, the GRXE spectrum shows significant data excesses at the Fe i-K α and Fe xxv-He α  line energies. This means that additional components other than mCVs, non-mCVs, and ABs are required, which have symbiotic phenomena of cold gas and very high-temperature plasma. The GCXE spectrum shows larger excesses than those found in the GRXE spectrum at all the K-shell lines of iron and nickel. Among them the largest ones are the Fe i-K α , Fe xxv-He α , Fe xxvi-Ly α , and Fe xxvi-Ly β  lines. Together with the fact that the scale heights of the Fe i-K α , Fe xxv-He α , and Fe xxvi-Ly α lines are similar to that of the central molecular zone (CMZ), the excess components would be related to high-energy activity in the extreme envelopment of the CMZ.

  8. Increased production of functional recombinant human clotting factor IX by baby hamster kidney cells engineered to overexpress VKORC1, the vitamin K 2,3-epoxide-reducing enzyme of the vitamin K cycle.

    Science.gov (United States)

    Wajih, Nadeem; Hutson, Susan M; Owen, John; Wallin, Reidar

    2005-09-09

    Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent gamma-carboxylation system that converts the proteins to gamma-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully gamma-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the gamma-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of gamma-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin K-dependent gamma-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the rate-limiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.

  9. The Cellosaurus, a Cell-Line Knowledge Resource

    Science.gov (United States)

    Bairoch, Amos

    2018-01-01

    The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences. PMID:29805321

  10. Secondary Metabolites from Two Species of Tolpis and Their Biological Activities

    Directory of Open Access Journals (Sweden)

    Francisco León

    2012-11-01

    Full Text Available Phytochemical research of two Tolpis species, T. proustii and T. lagopoda, led to the isolation of three new compounds: 30-chloro-3β-acetoxy-22α-hydroxyl-20(21-taraxastene (1, 3β,22α-diacetoxy-30-ethoxy-20(21-taraxastene (2 and 3β,28-dihydroxy-11α-hydroperoxy-12-ursene (3. The structures of the new compounds were elucidated by means of extensive IR, NMR, and MS data and by comparison of data reported in the literature. The in vitro antioxidant activities of the extracts were assessed by the DPPH and ABTS scavenging methods. The cytotoxicity of several known compounds and its derivatives was also assessed against human myeloid leukemia K-562 and K-562/ADR cell lines.

  11. Effect of 5-aminolevulinic acid on erythropoiesis: A preclinical in vitro characterization for the treatment of congenital sideroblastic anemia

    International Nuclear Information System (INIS)

    Fujiwara, Tohru; Okamoto, Koji; Niikuni, Ryoyu; Takahashi, Kiwamu; Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi; Ishizawa, Kenichi; Ichinohasama, Ryo; Nakamura, Yukio; Nakajima, Motowo; Tanaka, Tohru; Harigae, Hideo

    2014-01-01

    Highlights: • Treatment with ALA induces erythroid differentiation of K562 cells. • Transportation of ALA into erythroid cells occurs predominantly via SLC36A1. • ALA restores defects in ALAS2 in human iPS cell-derived erythroblasts. • ALA may represent a novel therapeutic option for CSA caused by ALAS2 mutations. - Abstract: Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly

  12. Effect of 5-aminolevulinic acid on erythropoiesis: A preclinical in vitro characterization for the treatment of congenital sideroblastic anemia

    Energy Technology Data Exchange (ETDEWEB)

    Fujiwara, Tohru [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Department of Molecular Hematology/Oncology, Tohoku University Graduate School, Sendai (Japan); Okamoto, Koji; Niikuni, Ryoyu [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Takahashi, Kiwamu [SBI Pharmaceuticals Co., Ltd., Tokyo (Japan); Okitsu, Yoko; Fukuhara, Noriko; Onishi, Yasushi [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Ishizawa, Kenichi [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Clinical Research, Innovation and Education Center, Tohoku University Hospital, Sendai (Japan); Ichinohasama, Ryo [Department of Hematopathology, Tohoku University Graduate School, Sendai (Japan); Nakamura, Yukio [Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki (Japan); Nakajima, Motowo; Tanaka, Tohru [SBI Pharmaceuticals Co., Ltd., Tokyo (Japan); Harigae, Hideo, E-mail: harigae@med.tohoku.ac.jp [Department of Hematology and Rheumatology, Tohoku University Graduate School, Sendai (Japan); Department of Molecular Hematology/Oncology, Tohoku University Graduate School, Sendai (Japan)

    2014-11-07

    Highlights: • Treatment with ALA induces erythroid differentiation of K562 cells. • Transportation of ALA into erythroid cells occurs predominantly via SLC36A1. • ALA restores defects in ALAS2 in human iPS cell-derived erythroblasts. • ALA may represent a novel therapeutic option for CSA caused by ALAS2 mutations. - Abstract: Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly

  13. Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

    International Nuclear Information System (INIS)

    Ristic, Biljana; Bosnjak, Mihajlo; Arsikin, Katarina; Mircic, Aleksandar; Suzin-Zivkovic, Violeta; Bogdanovic, Andrija; Perovic, Vladimir; Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

    2014-01-01

    We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade.

  14. Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

    Energy Technology Data Exchange (ETDEWEB)

    Ristic, Biljana [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Bosnjak, Mihajlo [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Arsikin, Katarina [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Mircic, Aleksandar; Suzin-Zivkovic, Violeta [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Bogdanovic, Andrija [Clinic for Hematology, Clinical Centre of Serbia, School of Medicine, University of Belgrade, Belgrade (Serbia); Perovic, Vladimir [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Trajkovic, Vladimir, E-mail: vtrajkovic@med.bg.ac.rs [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Harhaji-Trajkovic, Ljubica, E-mail: buajk@yahoo.com [Institute for Biological Research, University of Belgrade, Belgrade, Despot Stefan Blvd. 142, 11000 Belgrade (Serbia)

    2014-08-01

    We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade.

  15. Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer

    International Nuclear Information System (INIS)

    Andrade, Sheila Siqueira; Gouvea, Iuri Estrada; Silva, Mariana Cristina C.; Castro, Eloísa Dognani; Paula, Cláudia A. A. de; Okamoto, Debora; Oliveira, Lilian; Peres, Giovani Bravin; Ottaiano, Tatiana; Facina, Gil; Nazário, Afonso Celso Pinto; Campos, Antonio Hugo J. F. M.; Paredes-Gamero, Edgar Julian; Juliano, Maria; Silva, Ismael D. C. G. da; Oliva, Maria Luiza V.; Girão, Manoel J. B. C.

    2016-01-01

    Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and −4 are highly expressed, but PAR-3 shows low expression and unclear functions. Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFβ monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and −4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFβ in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells. The online version of this article (doi:10.1186/s12885-016-2203-7) contains supplementary material, which is available to authorized users

  16. Opposing effects of PI3K/Akt and Smad-dependent signaling pathways in NAG-1-induced glioblastoma cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Zhiguo Zhang

    Full Text Available Nonsteroidal anti-inflammatory drug (NSAID activated gene-1 (NAG-1 is a divergent member of the transforming growth factor-beta (TGF-β superfamily. NAG-1 plays remarkable multifunctional roles in controlling diverse physiological and pathological processes including cancer. Like other TGF-β family members, NAG-1 can play dual roles during cancer development and progression by negatively or positively modulating cancer cell behaviors. In glioblastoma brain tumors, NAG-1 appears to act as a tumor suppressor gene; however, the precise underlying mechanisms have not been well elucidated. In the present study, we discovered that overexpression of NAG-1 induced apoptosis in U87 MG, U118 MG, U251 MG, and T98G cell lines via the intrinsic mitochondrial pathway, but not in A172 and LN-229 cell lines. NAG-1 could induce the phosphorylation of PI3K/Akt and Smad2/3 in all six tested glioblastoma cell lines, except Smad3 phosphorylation in A172 and LN-229 cell lines. In fact, Smad3 expression and its phosphorylation were almost undetectable in A172 and LN-229 cells. The PI3K inhibitors promoted NAG-1-induced glioblastoma cell apoptosis, while siRNAs to Smad2 and Smad3 decreased the apoptosis rate. NAG-1 also stimulated the direct interaction between Akt and Smad3 in glioblastoma cells. Elevating the level of Smad3 restored the sensitivity to NAG-1-induced apoptosis in A172 and LN-229 cells. In conclusion, our results suggest that PI3K/Akt and Smad-dependent signaling pathways display opposing effects in NAG-1-induced glioblastoma cell apoptosis.

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

    Science.gov (United States)

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

    1992-10-01

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

  18. Two-color cytofluorometry and cellular properties of the urokinase receptor associated with a human metastatic carcinomatous cell line

    International Nuclear Information System (INIS)

    Takahashi, K.; Gojobori, T.; Tanifuji, M.

    1991-01-01

    Purified human urokinase was labeled with either fluorescein isothiocyanate or iodine-125 and used as a probe for binding to the human metastatic carcinomatous cell line, Detroit 562. Cytofluorometry showed that the ligand bound preferentially to cells that had been exposed to acidic pH. The binding was competitive and decreased after mild tryptic digestion. The bound ligand could be removed by restoration of the cells to a low pH. Therefore, the cells had specific binding sites. The bound urokinase was involved in the breakdown of fibrin. Two-color cytofluorometric maps were constructed by counterstaining with propidium iodide. Results suggested that there were different cell populations that had different numbers of receptors and amounts of DNA. We cloned cells and found that single clones had homogeneous levels of receptors with different dissociation constants (from 10(-13) to 10(-11) mol/mg protein) for different clones. Cells of one clone, C5, which had high levels of receptor production, moved characteristically on a glass substratum coated with gold particles and reacted with wheat germ agglutinin, but not with concanavalin A. The receptors were found together with adhesion proteins at the sites where the cells adhered to the substrate. These results and the data obtained by zymography of the cellular proteins suggested that the urokinase-type plasminogen activators were bound to the receptors. The membrane-associated activator may stimulate local proteolysis, facilitating the migration of the tumor cell across the substrate

  19. Two-color cytofluorometry and cellular properties of the urokinase receptor associated with a human metastatic carcinomatous cell line

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, K.; Gojobori, T.; Tanifuji, M. (Shimane Medical Univ., Izumo (Japan))

    1991-02-01

    Purified human urokinase was labeled with either fluorescein isothiocyanate or iodine-125 and used as a probe for binding to the human metastatic carcinomatous cell line, Detroit 562. Cytofluorometry showed that the ligand bound preferentially to cells that had been exposed to acidic pH. The binding was competitive and decreased after mild tryptic digestion. The bound ligand could be removed by restoration of the cells to a low pH. Therefore, the cells had specific binding sites. The bound urokinase was involved in the breakdown of fibrin. Two-color cytofluorometric maps were constructed by counterstaining with propidium iodide. Results suggested that there were different cell populations that had different numbers of receptors and amounts of DNA. We cloned cells and found that single clones had homogeneous levels of receptors with different dissociation constants (from 10(-13) to 10(-11) mol/mg protein) for different clones. Cells of one clone, C5, which had high levels of receptor production, moved characteristically on a glass substratum coated with gold particles and reacted with wheat germ agglutinin, but not with concanavalin A. The receptors were found together with adhesion proteins at the sites where the cells adhered to the substrate. These results and the data obtained by zymography of the cellular proteins suggested that the urokinase-type plasminogen activators were bound to the receptors. The membrane-associated activator may stimulate local proteolysis, facilitating the migration of the tumor cell across the substrate.

  20. Effect of saw palmetto extract on PI3K cell signaling transduction in human glioma.

    Science.gov (United States)

    Yang, Yang; Hui, Lv; Yuqin, Che; Jie, Li; Shuai, Hou; Tiezhu, Zhou; Wei, Wang

    2014-08-01

    Saw palmetto extract can induce the apoptosis of prostate cancer cells. The aim of the present study was to investigate the effect of saw palmetto extract on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling transduction pathway in human glioma U87 and U251 cell lines. Suspensions of U87 and U251 cells in a logarithmic growth phase were seeded into six-well plates at a density of 10 4 cells/well. In the experimental group, 1 μl/ml saw palmetto extract was added, while the control group was cultured without a drug for 24 h. The expression levels of PI3K, B-cell lymphoma-extra large (Bcl-xL) and p53 were evaluated through western blot analysis. In the experimental group, the U87 and U251 cells exhibited a lower expression level of PI3K protein as compared with the control group (t=6.849; Psaw palmetto extract induces glioma cell growth arrest and apoptosis via decreasing PI3K/Akt signal transduction.

  1. Deficient natural killer cell function in preeclampsia

    International Nuclear Information System (INIS)

    Alanen, A.; Lassila, O.

    1982-01-01

    Natural killer cell activity of peripheral blood lymphocytes was measured against K-562 target cells with a 4-hour 51 Cr release assay in 15 primigravid women with preeclamptic symptoms. Nineteen primigravid women with an uncomplicated pregnancy and 18 nonpregnant women served as controls. The natural killer cell activity of preeclamptic women was observed to be significantly lower than that of both control groups. Natural killer cells in preeclamptic women responded normally to augmentation caused by interferon. These findings give further evidence for the participation of the maternal immune system in this pregnancy disorder

  2. Deficient natural killer cell function in preeclampsia

    Energy Technology Data Exchange (ETDEWEB)

    Alanen, A.; Lassila, O.

    1982-11-01

    Natural killer cell activity of peripheral blood lymphocytes was measured against K-562 target cells with a 4-hour /sup 51/Cr release assay in 15 primigravid women with preeclamptic symptoms. Nineteen primigravid women with an uncomplicated pregnancy and 18 nonpregnant women served as controls. The natural killer cell activity of preeclamptic women was observed to be significantly lower than that of both control groups. Natural killer cells in preeclamptic women responded normally to augmentation caused by interferon. These findings give further evidence for the participation of the maternal immune system in this pregnancy disorder.

  3. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

    Science.gov (United States)

    Arabski, Michał; Węgierek-Ciuk, Aneta; Czerwonka, Grzegorz; Lankoff, Anna; Kaca, Wiesław

    2012-01-01

    Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed. PMID:22500084

  4. Effects of Saponins against Clinical E. coli Strains and Eukaryotic Cell Line

    Directory of Open Access Journals (Sweden)

    Michał Arabski

    2012-01-01

    Full Text Available Saponins are detergent-like substances showing antibacterial as well as anticancer potential. In this study, the effects of saponins from Quillaja saponaria were analyzed against prokaryotic and eukaryotic cells. Multidrug-resistant clinical E. coli strains were isolated from human urine. As eukaryotic cells, the CHO-K1 cell lines were applied. Antibacterial effect of ampicillin, streptomycin, and ciprofloxacin in the presence of saponins was measured by cultivation methods. Properties of saponins against CHO-K1 cells were measured by the MTT test, hemolysis assay and flow cytometry. Saponin from Quillaja saponaria has a cytotoxic effect at concentrations higher than 25 μg/mL and in the range of 12–50 μg/mL significantly increases the level of early apoptotic cells. Saponin at dose of 12 μg/mL enhances the six E. coli strains growth. We postulate that saponins increase the influx of nutrients from the medium into E. coli cells. Saponins do not have synergetic effects on antibacterial action of tested antibiotics. In contrary, in the presence of saponins and antibiotics, more CFU/mL E. coli cells were observed. This effect was similar to saponins action alone towards E. coli cells. In conclusion, saponins was cytotoxic against CHO-K1 cells, whereas against E. coli cells this effect was not observed.

  5. Nepheliosyne B, a New Polyacetylenic Acid from the New Caledonian Marine Sponge Niphates sp.

    Directory of Open Access Journals (Sweden)

    Patrick Auberger

    2013-06-01

    Full Text Available A new C47 polyoxygenated acetylenic acid, nepheliosyne B (2, along with the previously described nepheliosyne A (1, have been isolated from the New Caledonian marine sponge Niphates sp. Their structures have been elucidated on the basis of extensive spectroscopic analyses. These metabolites exhibited a moderate cytotoxicity against K562, U266, SKM1, and Kasumi cancer cell lines.

  6. Cost-effective master cell bank validation of multiple clinical-grade human pluripotent stem cell lines from a single donor.

    Science.gov (United States)

    Devito, Liani; Petrova, Anastasia; Miere, Cristian; Codognotto, Stefano; Blakely, Nicola; Lovatt, Archie; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

    2014-10-01

    Standardization guidelines for human pluripotent stem cells are still very broadly defined, despite ongoing clinical trials in the U.S., U.K., and Japan. The requirements for validation of human embryonic (hESCs) and induced pluripotent stem cells (iPSCs) in general follow the regulations for other clinically compliant biologics already in place but without addressing key differences between cell types or final products. In order to realize the full potential of stem cell therapy, validation criteria, methodology, and, most importantly, strategy, should address the shortfalls and efficiency of current approaches; without this, hESC- and, especially, iPSC-based therapy will not be able to compete with other technologies in a cost-efficient way. We addressed the protocols for testing cell lines for human viral pathogens and propose a novel strategy that would significantly reduce costs. It is highly unlikely that the multiple cell lines derived in parallel from a tissue sample taken from one donor would have different profiles of endogenous viral pathogens; we therefore argue that samples from the Master Cell Banks of sibling lines could be safely pooled for validation. We illustrate this approach with tiered validation of two sibling clinical-grade hESC lines, KCL033 and KCL034 (stage 1, sterility; stage 2, specific human pathogens; and stage 3, nonspecific human pathogens). The results of all tests were negative. This cost-effective strategy could also be applied for validation of Master Cell Banks of multiple clinical-grade iPSC lines derived from a single donor. ©AlphaMed Press.

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

    Directory of Open Access Journals (Sweden)

    Raquel Sarafian

    2018-04-01

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

  8. Plasma membrane organization and dynamics is probe and cell line dependent.

    Science.gov (United States)

    Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

    2017-09-01

    The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue

  9. Stellar and interstellar K lines - Gamma Pegasi and iota Herculis.

    Science.gov (United States)

    Hobbs, L. M.

    1973-01-01

    High-resolution scans show that the relatively strong (about 90 mA) K lines of Ca II in the early B stars gamma-Peg and iota-Her are almost entirely stellar in origin, although the latter case includes a small interstellar contribution. Such stellar lines can be of great importance in augmenting the interstellar absorption, up through the earliest of the B stars.

  10. 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

  11. Ethanol extracts of black pepper or turmeric down-regulated SIRT1 protein expression in Daudi culture cells.

    Science.gov (United States)

    Nishimura, Yuri; Kitagishi, Yasuko; Yoshida, Hitomi; Okumura, Naoko; Matsuda, Satoru

    2011-01-01

    SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

  12. Emission lines of [K V] in the optical spectra of gaseous nebulae.

    Science.gov (United States)

    Keenan, Francis P; Aller, Lawrence H; Espey, Brian R; Exter, Katrina M; Hyung, Siek; Keenan, Michael T C; Pollacco, Don L; Ryans, Robert S I

    2002-04-02

    Recent R-matrix calculations of electron impact excitation rates in K v are used to derive the nebular emission line ratio R = I(4122.6 A)/I(4163.3 A) as a function of electron density (N(e)). This ratio is found to be very sensitive to changes in N(e) over the density range 10(3) to 10(6) cm(-3), but does not vary significantly with electron temperature, and hence in principle should provide an excellent optical N(e) diagnostic for the high-excitation zones of nebulae. The observed value of R for the planetary nebula NGC 7027, measured from a spectrum obtained with the Hamilton Echelle spectrograph on the 3-m Shane Telescope, implies a density in excellent agreement with that derived from [Ne iv], formed in the same region of the nebula as [K v]. This observation provides observational support for the accuracy of the theoretical [K v] line ratios, and hence the atomic data on which they are based. However, the analysis of a high-resolution spectrum of the symbiotic star RR Telescopii, obtained with the University College London Echelle Spectrograph on the 3.9-m Anglo-Australian Telescope, reveals that the [K v] 4122.6 A line in this object is badly blended with Fe ii 4122.6 A. Hence, the [K v] diagnostic may not be used for astrophysical sources that show a strong Fe ii emission line spectrum.

  13. A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

    Science.gov (United States)

    Abraham, S; Solomon, W B

    2000-09-19

    We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin

  14. Signaling via class IA Phosphoinositide 3-kinases (PI3K in human, breast-derived cell lines.

    Directory of Open Access Journals (Sweden)

    Veronique Juvin

    Full Text Available We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K in human breast-derived MCF10a (and iso-genetic derivatives and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5-trisphosphate (PtdIns(3,4,5P3 that can activate effectors, eg protein kinase B (PKB, and responses, eg migration. The PtdIns(3,4,5P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K, basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/- cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN(-/- MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely

  15. Plumbagin induces cell cycle arrest and autophagy and suppresses epithelial to mesenchymal transition involving PI3K/Akt/mTOR-mediated pathway in human pancreatic cancer cells

    Science.gov (United States)

    Wang, Feng; Wang, Qi; Zhou, Zhi-Wei; Yu, Song-Ning; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Yin-Xue; Yang, Tianxing; Sun, Tao; Li, Min; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5′-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of

  16. Phosphorylation events implicating p38 and PI3K mediate tungstate-effects in MIN6 beta cells

    International Nuclear Information System (INIS)

    Piquer, Sandra; Barcelo-Batllori, Silvia; Julia, Marta; Marzo, Nuria; Nadal, Belen; Guinovart, Joan J.; Gomis, Ramon

    2007-01-01

    Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. Several lines of evidence indicate the pancreatic beta cell as one of the targets of tungstate action. Here, we examined the molecular mechanism by which this compound exerts its effects on the beta cell line MIN6. Tungstate treatment induced phosphorylation and subsequent activation of p38 and PI3K which in turn are implicated in tungstate PDX-1 nuclear localization and activation. Although no effect was observed in glucose-induced insulin secretion we found that tungstate activates basal insulin release, a process driven, at least in part, by activation of p38. These results show a direct involvement of p38 and PI3K phosphorylation in the mechanism of action of tungstate in the beta cell

  17. 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,

  18. K lines in the sulphur isonuclear sequence

    International Nuclear Information System (INIS)

    Palmeri, P.; Mendoza, C.; Bautista, M.A.; Garcia, J.; Kallman, T.R.

    2006-01-01

    Accurate and complete sets of atomic data are being computed for the radiative and Auger processes giving rise to K lines in ions of sulphur. AUTOSTRUCTURE, HFR and BPRM codes are used, taking into account configuration interaction, core relaxation and relativistic effects so as to generate data following an approach used in our previous studies on iron and oxygen. Level energies, wavelengths, A-values and Auger rates are compared with available measurements and previous calculations

  19. 64 kDa protein is a candidate for a thyrotropin-releasing hormone receptor in prolactin-producing rat pituitary tumor cells (GH4C1 cells)

    International Nuclear Information System (INIS)

    Wright, M.; Hogset, A.; Alestrom, P.; Gautvik, K.M.

    1988-01-01

    A thyrotropin-releasing hormone (TRH) binding protein of 64 kDa has been identified by covalently crosslinking [ 3 H]TRH to GH4C1 cells by ultraviolet illumination. The crosslinkage of [ 3 H]TRH is UV-dose dependent and is inhibited by an excess of unlabeled TRH. A 64 kDa protein is also detected on immunoblots using an antiserum raised against GH4C1 cell surface epitopes. In a closely related cell line (GH12C1) which does not bind [ 3 H]TRH, the 64 kDa protein cannot be demonstrated by [ 3 H]TRH crosslinking nor by immunoblotting. These findings indicate that the 64 kDa protein is a candidate for a TRH-receptor protein in GH4C1 cells

  20. Wild Horse 69-kV transmission line environmental assessment

    International Nuclear Information System (INIS)

    1996-12-01

    Hill County Electric Cooperative Inc. (Hill County) proposes to construct and operate a 69-kV transmission line from its North Gildford Substation in Montana north to the Canadian border. A vicinity project area map is enclosed as a figure. TransCanada Power Corporation (TCP), a Canadian power-marketing company, will own and construct the connecting 69-kV line from the international border to Express Pipeline's pump station at Wild Horse, Alberta. This Environmental Assessment is prepared for the Department of Energy (DOE) as lead federal agency to comply with the requirements of the National Environmental Policy Act (NEPA), as part of DOE's review and approval process of the applications filed by Hill County for a DOE Presidential Permit and License to Export Electricity to a foreign country. The purpose of the proposed line is to supply electric energy to a crude oil pump station in Canada, owned by Express Pipeline Ltd. (Express). The pipeline would transport Canadian-produced oil from Hardisty, Alberta, Canada, to Caster, Wyoming. The Express Pipeline is scheduled to be constructed in 1996--97 and will supply crude oil to refineries in Wyoming and the midwest

  1. Wild Horse 69-kV transmission line environmental assessment

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-12-01

    Hill County Electric Cooperative Inc. (Hill County) proposes to construct and operate a 69-kV transmission line from its North Gildford Substation in Montana north to the Canadian border. A vicinity project area map is enclosed as a figure. TransCanada Power Corporation (TCP), a Canadian power-marketing company, will own and construct the connecting 69-kV line from the international border to Express Pipeline`s pump station at Wild Horse, Alberta. This Environmental Assessment is prepared for the Department of Energy (DOE) as lead federal agency to comply with the requirements of the National Environmental Policy Act (NEPA), as part of DOE`s review and approval process of the applications filed by Hill County for a DOE Presidential Permit and License to Export Electricity to a foreign country. The purpose of the proposed line is to supply electric energy to a crude oil pump station in Canada, owned by Express Pipeline Ltd. (Express). The pipeline would transport Canadian-produced oil from Hardisty, Alberta, Canada, to Caster, Wyoming. The Express Pipeline is scheduled to be constructed in 1996--97 and will supply crude oil to refineries in Wyoming and the midwest.

  2. Hydrogen peroxide removal with magnetically responsive Saccharomyces cerevisiae cells

    Czech Academy of Sciences Publication Activity Database

    Šafařík, Ivo; Maděrová, Zdeňka; Šafaříková, Miroslava

    2008-01-01

    Roč. 56, - (2008), s. 7925-7928 ISSN 0021-8561 R&D Projects: GA MPO 2A-1TP1/094; GA MŠk OC 157 Institutional research plan: CEZ:AV0Z60870520 Keywords : magnetic alginate beads * catalase * magnetic separation * Saccharomyces cerevisiae cells * hydrogen peroxide Subject RIV: GM - Food Processing Impact factor: 2.562, year: 2008

  3. Comparison of steroid receptors from the androgen responsive DDT1 cell line and the nonresponsive HVP cell line.

    Science.gov (United States)

    Norris, J S; Kohler, P O

    1978-01-01

    Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.

  4. Visualization of proteolytic activity associated with the apoptotic response in cancer cells

    Science.gov (United States)

    Tice, Brian George

    Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation

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

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

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

  6. Expression of p89c-Mybex9b, an alternatively spliced form of c-Myb, is required for proliferation and survival of p210BCR/ABL-expressing cells

    International Nuclear Information System (INIS)

    Manzotti, G; Mariani, S A; Corradini, F; Bussolari, R; Cesi, V; Vergalli, J; Ferrari-Amorotti, G; Fragliasso, V; Soliera, A R; Cattelani, S; Raschellà, G; Holyoake, T L; Calabretta, B

    2012-01-01

    The c-Myb gene encodes the p75 c-Myb isoform and less-abundant proteins generated by alternatively spliced transcripts. Among these, the best known is p c-Mybex9b , which contains 121 additional amino acids between exon 9 and 10, in a domain involved in protein–protein interactions and negative regulation. In hematopoietic cells, expression of p c-Mybex9b accounts for 10–15% of total c-Myb; these levels may be biologically relevant because modest changes in c-Myb expression affects proliferation and survival of leukemic cells and lineage choice and frequency of normal hematopoietic progenitors. In this study, we assessed biochemical activities of p c-Mybex9b and the consequences of perturbing its expression in K562 and primary chronic myeloid leukemia (CML) progenitor cells. Compared with p75 c-Myb , p c-Mybex9b is more stable and more effective in transactivating Myb-regulated promoters. Ectopic expression of p c-Mybex9b enhanced proliferation and colony formation and reduced imatinib (IM) sensitivity of K562 cells; conversely, specific downregulation of p c-Mybex9b reduced proliferation and colony formation, enhanced IM sensitivity of K562 cells and markedly suppressed colony formation of CML CD34 + cells, without affecting the levels of p75 c-Myb . Together, these studies indicate that expression of the low-abundance p c-Mybex9b isoform has an important role for the overall biological effects of c-Myb in BCR/ABL-transformed cells

  7. Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV-3) and possible latency of CyHV-3 by temperature shift in the cells.

    Science.gov (United States)

    Imajoh, M; Fujioka, H; Furusawa, K; Tamura, K; Yamasaki, K; Kurihara, S; Yamane, J; Kawai, K; Oshima, S

    2015-06-01

    A new cell line named CCF-K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV-3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV-3-infected CCF-K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV-3 was produced stably in CCF-K104 cells over 30 viral passages. Our findings showed that CCF-K104 is a useful cell line for isolation and productive replication of CyHV-3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real-time PCR showed that CyHV-3 was present with low viral DNA loads, suggesting that CyHV-3 may establish latent infection in CCF-K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV-3 genome arranged in a head-to-tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV-3. © 2014 John Wiley & Sons Ltd.

  8. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels

    International Nuclear Information System (INIS)

    Cotten, M.; Laengle-Rouault, F.; Kirlappos, H.; Wagner, E.; Mechtler, K.; Zenke, M.; Beug, H.; Birnstiel, M.L.

    1990-01-01

    The authors have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, they have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle. They demonstrate here that this procedure is useful for a human leukemic cell line. They enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, they show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks transferrinfection, as does incubation of the cells at 18 degree C

  9. Chemosensitivity and radiosensitivity of small cell lung cancer cell lines studied by a newly developed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) hybrid assay

    International Nuclear Information System (INIS)

    Hida, T.; Ueda, R.; Takahashi, T.; Watanabe, H.; Kato, T.; Suyama, M.; Sugiura, T.; Ariyoshi, Y.

    1989-01-01

    The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) hybrid assay was developed by technically combining the human tumor clonogenic assay and the MTT assay to make the most of both assays. This assay was able to estimate the in vitro growth of cultured cell lines and of tumor cells in pleural effusion, suggesting the possibility of its use for assessment of chemosensitivity and radiosensitivity of fresh tumor samples. Multiple cell lines [including morphological and/or phenotypical in vitro converters and cisplatin (CDDP)-resistant lines] were established from three patients with small cell lung cancer at different stages of the disease. Chemosensitivity of these cell lines to four commonly used chemotherapeutic drugs was tested by the MTT hybrid assay. SK1 and SK3 lines were established from Patient S. K. before and after chemotherapy and radiotherapy, respectively. SK3/CDDP, a CDDP-resistant line derived from the SK3 line, was 30-fold more resistant to CDDP [50% inhibiting dose (IC50), 21.5 micrograms/ml] than the SK1 line. In Patient M. O., MOA2/CDDP, a CDDP-resistant line derived from MOA2 (an in vitro converter from the MO line), was 41-fold more resistant to CDDP (IC50, 37 micrograms/ml) than the parent MO line. From Patient T. M., TM1 and TM2 lines were established before and after chemotherapy, respectively. The latter showed 6-fold more resistance to CDDP than the former. Chemosensitivity of these lines to three other drugs, 4-hydroperoxycyclophosphamide, Adriamycin, and etoposide, suggested cross-resistance between CDDP and 4-hydroperoxycyclophosphamide. Radiosensitivity study was also carried out with the MTT hybrid assay. The MOA2 line was more resistant [Do, 3.0 Gy; extrapolation number (n), 4.0] than the parental MO line (Do, 1.6 Gy; n, 2.1). There was no clear difference in radiosensitivity between the cell lines established before and after radiation therapy in Patient S. K

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

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2006-11-01

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

  11. Characterization of stem-like cells in a new astroblastoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Coban, Esra Aydemir; Kasikci, Ezgi [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Karatas, Omer Faruk [Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum (Turkey); Suakar, Oznur; Kuskucu, Aysegul [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey); Altunbek, Mine [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Türe, Uğur [Department of Neurosurgery, Yeditepe University School of Medicine, Istanbul (Turkey); Sahin, Fikrettin [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Bayrak, Omer Faruk, E-mail: ofbayrak@yeditepe.edu.tr [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey)

    2017-03-15

    Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. - Highlights: • An establishment of a novel astroblastoma cell line was proposed. • The presence of astroblastic cells and cancer stem-like cells was investigated. • The molecular pathways underlying astroblastomas may be investigated. • New therapeutic strategies for patients with astroblastoma may be developed.

  12. Comparison of micronucleus frequencies and proliferation kinetics in three X-irradiated cell lines

    International Nuclear Information System (INIS)

    Kaffenberger, W.; Becker, K.; Beuningen, D. van

    1990-01-01

    The kinetics of the occurrence of micronuclei was correlated with the survival of three mammalian cell lines of human, monkey, and mouse origin after irradiation with 240 kV X-rays. Particular attention was paid to the evaluation of the individual proliferation kinetics of the cell lines as well as to the characterization of micronuclei subpopulation with respect to size and possible biological importance using DNA and BUdR labelling techniques, fluorescence microscopy, and image analysis. The results demonstrate very characteristic size distributions of micronuclei for the three cell lines independent of radiation dose and time after irradiation. A close correlation between cell death and the occurrence of micronuclei (expressed as a calculated 'MN index') after irradiation could be established only when the kinetics of progression of cells through the cell cycle (e.g. the doubling time) and the biological characteristics of micronuclei (e.g. BUdR positivity, the micronucleus frequencies, and the number of micronuclei per main nucleus) were taken into account. Therefore, the micronucleus assay might not be useful as a quantitative perdictive assay in vivo but may allow qualitative estimations of radiation damage only because the necessary proliferation parameters of the cells might not be possible to establish in vivo. (orig.) [de

  13. Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kalle, Arunasree M., E-mail: arunasreemk@ilsresearch.org [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Mallika, A. [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Badiger, Jayasree [HKE' s Smt. V.G. College for Women, Aiwan-E-Shahi Area, Gulbarga, KA 585 102 (India); Alinakhi [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Talukdar, Pinaki [Department of Chemistry, Indian Institute of Science Education and Research, First Floor, Central Tower, Sai Trinity Building Garware Circle, Sutarwadi, PashanPune, Maharashtra 411 021 (India); Sachchidanand [Lupin Research Park, 46/47, A, Village Nande, Taluka Mulshi, Dist. Pune 411 042 (India)

    2010-10-08

    Research highlights: {yields} Novel small molecule SIRT1 inhibitor better than sirtinol. {yields} IC{sub 50} 500 nM. {yields} Specific tumor cytotoxicity towards breast cancer cells. {yields} Restoration of H3K9 acetylation levels to baseline when co-treated with SIRT1 activator (Activator X) and inhibitor (ILS-JGB-1741). -- Abstract: Overexpression of SIRT1, a NAD{sup +}-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC{sub 50} of 1, 10 and 0.5 {mu}M, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.

  14. Glial K(+) Clearance and Cell Swelling

    DEFF Research Database (Denmark)

    Macaulay, Nanna; Zeuthen, Thomas

    2012-01-01

    An important feature of neuronal signalling is the increased concentration of K(+) in the extracellular space. The K(+) concentration is restored to its original basal level primarily by uptake into nearby glial cells. The molecular mechanisms by which K(+) is transferred from the extracellular...... space into the glial cell are debated. Although spatial buffer currents may occur, their quantitative contribution to K(+) clearance is uncertain. The concept of spatial buffering of K(+) precludes intracellular K(+) accumulation and is therefore (i) difficult to reconcile with the K(+) accumulation...

  15. Imidazole Alkaloids from the South China Sea Sponge Pericharax heteroraphis and Their Cytotoxic and Antiviral Activities

    Directory of Open Access Journals (Sweden)

    Kai-Kai Gong

    2016-01-01

    Full Text Available Marine sponges continue to serve as a rich source of alkaloids possessing interesting biological activities and often exhibiting unique structural frameworks. In the current study, chemical investigation on the marine sponge Pericharax heteroraphis collected from the South China Sea yielded one new imidazole alkaloid named naamidine J (1 along with four known ones (2–5. Their structures were established by extensive spectroscopic methods and comparison of their data with those of the related known compounds. All the isolates possessed a central 2-aminoimidazole ring, substituted by one or two functionalized benzyl groups in some combination of the C4 and C5 positions. The cytotoxicities against selected HL-60, HeLa, A549 and K562 tumor cell lines and anti-H1N1 (Influenza a virus (IAV activity for the isolates were evaluated. Compounds 1 and 2 exhibited cytotoxicities against the K562 cell line with IC50 values of 11.3 and 9.4 μM, respectively. Compound 5 exhibited weak anti-H1N1 (influenza a virus, IAV activity with an inhibition ratio of 33%.

  16. DNA-binding, DNA cleavage and cytotoxicity studies of two anthraquinone derivatives.

    Science.gov (United States)

    Gholivand, M B; Kashanian, S; Peyman, H

    2012-02-15

    The interaction of native calf thymus DNA (CT-DNA) with two anthraquinones including quinizarin (1,4-dihydroxy anthraquinone) and danthron (1,8-dihydroxy anthraquinone) in a mixture of 0.04M Brittone-Robinson buffer and 50% of ethanol were studied at physiological pH by spectrofluorometric and cyclic voltammetry techniques. The former technique was used to calculate the binding constants of anthraquinones-DNA complexes at different temperatures. Thermodynamic study indicated that the reactions of both anthraquinone-DNA systems are predominantly entropically driven. Furthermore, the binding mechanisms on the reaction of the two anthraquinones with DNA and the effect of ionic strength on the fluorescence property of the system have also been investigated. The results of the experiments indicated that the binding modes of quinizarin and danthron with DNA were evaluated to be groove binding. Moreover, the cytotoxic activity of both compounds against human chronic myelogenous leukemia K562 cell line and DNA cleavage were investigated. The results indicated that these compounds slightly cleavage pUC18 plasmid DNA and showed minor antitumor activity against K562 (human chronic myeloid leukemia) cell line. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Arginase induction by sodium phenylbutyrate in mouse tissues and human cell lines.

    Science.gov (United States)

    Kern, R M; Yang, Z; Kim, P S; Grody, W W; Iyer, R K; Cederbaum, S D

    2007-01-01

    Hyperargininemia is a urea cycle disorder caused by mutations in the gene for arginase I (AI) resulting in elevated blood arginine and ammonia levels. Sodium phenylacetate and a precursor, sodium phenylbutyrate (NaPB) have been used to lower ammonia, conjugating glutamine to produce phenylacetylglutamine which is excreted in urine. The elevated arginine levels induce the second arginase (AII) in patient kidney and kidney tissue culture. It has been shown that NaPB increases expression of some target genes and we tested its effect on arginase induction. Eight 9-week old male mice fed on chow containing 7.5 g NaPB/kg rodent chow and drank water with 10 g NaPB/L, and four control mice had a normal diet. After one week all mice were sacrificed. The arginase specific activities for control and NaPB mice, respectively, were 38.2 and 59.4 U/mg in liver, 0.33 and 0.42 U/mg in kidney, and 0.29 and 1.19 U/mg in brain. Immunoprecipitation of arginase in each tissue with AI and AII antibodies showed the activity induced by NaPB is mostly AI. AII may also be induced in kidney. AI accounts for the fourfold increased activity in brain. In some cell lines, NaPB increased arginase activity up to fivefold depending on dose (1-5 mM) and exposure time (2-5 days); control and NaPB activities, respectively, are: erythroleukemia, HEL, 0.06 and 0.31 U/mg, and K562, 0.46 and 1.74 U/mg; embryonic kidney, HEK293, 1.98 and 3.58 U/mg; breast adenocarcinoma, MDA-MB-468, 1.11 and 4.06 U/mg; and prostate adenocarcinoma, PC-3, 0.55 and 3.20 U/mg. In MDA-MB-468 and HEK most, but not all, of the induced activity is AI. These studies suggest that NaPB may induce AI when used to treat urea cycle disorders. It is relatively less useful in AI deficiency, although it could have some effect in those patients with missense mutations.

  18. A 1H nuclear magnetic resonance study of structural and organisational changes in the cell

    International Nuclear Information System (INIS)

    Tunnah, Susan K.

    2000-01-01

    Increasing importance is being placed on understanding the role of membrane lipids in many different areas of biochemistry. It is of interest to determine what interactions may occur between membrane lipids and drug species. Furthermore, an increasing body of evidence suggests that membrane lipids are involved in the pathology of numerous diseases such as rheumatoid arthritis, cancer and HIV. Clearly, the more information available on the mechanisms involved in diseases, the greater the potential for identifying a cure or even a prevention. 1 H nuclear magnetic resonance (NMR) spectroscopy was used to study the alterations in membrane lipid organisation and structure in intact, viable cultured cells. Changes in the 1 H NMR spectra and the spin-lattice relaxation measurements of the human K562 and the rat FRTL-5 cell lines were observed on the addition of the fatty acid species: triolein, evening primrose oil, arachidonic acid and ITF 1779. Results indicate that the membrane lipids are reorganised to accommodate the interpolation of these molecules. The spatial arrangement adopted by each of these species appeared to dictate its effect on the lipids. Doxorubicin and menadione, both known to cause oxidative stress, were added to K562 cells. Although both agents are known to act by different mechanisms, the NMR data and scanning electron microscopy suggested that both caused similar alterations in the membrane organisation and lipid fluidity. Protrusions were formed indicating areas of weakness in the membrane. Spin-echo NMR was employed to investigate the action of the thiol-containing compounds, penicillamine, captopril and N-acetylcysteine in erythrocytes under conditions of oxidative stress. Results indicate that while captopril acts as a free radical scavenger, penicillamine may act as either oxidant or reductant. N-acetylcysteine was observed to act as a reducing agent. (author)

  19. Wnt/β-catenin pathway regulates ABCB1 transcription in chronic myeloid leukemia

    International Nuclear Information System (INIS)

    Corrêa, Stephany; Binato, Renata; Du Rocher, Bárbara; Castelo-Branco, Morgana TL; Pizzatti, Luciana; Abdelhay, Eliana

    2012-01-01

    The advanced phases of chronic myeloid leukemia (CML) are known to be more resistant to therapy. This resistance has been associated with the overexpression of ABCB1, which gives rise to the multidrug resistance (MDR) phenomenon. MDR is characterized by resistance to nonrelated drugs, and P-glycoprotein (encoded by ABCB1) has been implicated as the major cause of its emergence. Wnt signaling has been demonstrated to be important in several aspects of CML. Recently, Wnt signaling was linked to ABCB1 regulation through its canonical pathway, which is mediated by β-catenin, in other types of cancer. In this study, we investigated the involvement of the Wnt/β-catenin pathway in the regulation of ABCB1 transcription in CML, as the basal promoter of ABCB1 has several β-catenin binding sites. β-catenin is the mediator of canonical Wnt signaling, which is important for CML progression. In this work we used the K562 cell line and its derived MDR-resistant cell line Lucena (K562/VCR) as CML study models. Real time PCR (RT-qPCR), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), flow cytometry (FACS), western blot, immunofluorescence, RNA knockdown (siRNA) and Luciferase reporter approaches were used. β-catenin was present in the protein complex on the basal promoter of ABCB1 in both cell lines in vitro, but its binding was more pronounced in the resistant cell line in vivo. Lucena cells also exhibited higher β-catenin levels compared to its parental cell line. Wnt1 and β-catenin depletion and overexpression of nuclear β-catenin, together with TCF binding sites activation demonstrated that ABCB1 is positively regulated by the canonical pathway of Wnt signaling. These results suggest, for the first time, that the Wnt/β-catenin pathway regulates ABCB1 in CML

  20. Effect of Thai saraphi flower extracts on WT1 and BCR/ABL protein ...

    African Journals Online (AJOL)

    In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. Materials and Methods: The ...

  1. Closure Report for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

    Energy Technology Data Exchange (ETDEWEB)

    NSTec Environmental Restoration

    2012-08-15

    This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 562, Waste Systems, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 562 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 as amended). CAU 562 consists of the following 13 Corrective Action Sites (CASs), located in Areas 2, 23, and 25 of the Nevada National Security Site: · CAS 02-26-11, Lead Shot · CAS 02-44-02, Paint Spills and French Drain · CAS 02-59-01, Septic System · CAS 02-60-01, Concrete Drain · CAS 02-60-02, French Drain · CAS 02-60-03, Steam Cleaning Drain · CAS 02-60-04, French Drain · CAS 02-60-05, French Drain · CAS 02-60-06, French Drain · CAS 02-60-07, French Drain · CAS 23-60-01, Mud Trap Drain and Outfall · CAS 23-99-06, Grease Trap · CAS 25-60-04, Building 3123 Outfalls Closure activities began in October 2011 and were completed in April 2012. Activities were conducted according to the Corrective Action Plan for CAU 562 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste and hazardous waste. Some wastes exceeded land disposal limits and required offsite treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite or offsite landfills. NNSA/NSO requests the following: · A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for closure of CAU 562 · The transfer of CAU 562 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO

  2. X-ray K-edge analysis of drain lines in Wilhelm Hall, Ames Laboratory

    International Nuclear Information System (INIS)

    Jensen, T.; Whitmore, C.; Iowa State Univ., Ames, IA

    1999-01-01

    From August 12--27, 1998 X-ray K-edge measurements were made on drain lines in seven rooms in Wilhelm Hall, Ames Laboratory. The purpose of these measurements was to determine the extent of thorium (and other heavy metal) contamination inside these pipes. The K-edge method is a noninvasive inspection technique that can provide accurate quantification of heavy metal contamination interior to an object. Of the seven drain lines inspected, one was found to have no significant contamination, three showed significant thorium deposits, two showed mercury contamination, and one line was found to contain mercury, thorium and uranium. The K-edge measurements were found to be consistent with readings from hand-held survey meters, and provided much greater detail on the location and amount of heavy metal contamination

  3. Cell fusion induced by ionizing radiation in various cell lines

    International Nuclear Information System (INIS)

    Khair, M.B.

    1994-07-01

    Cell fusion induced by ionizing radiation has been studied in rat's hepatocytes in vivo and in different cell lines in vitro. These cell lines were: Hela cells, V-79 fibroblasts, human and rat lymphocytes. For irradiation, 0.85 MeV fission neutrons and 14 MeV fast neutrons were used. Cell analyses were performed by fluorescent dyes using immunofluorescent microscope and flow cytometre. Our results in vivo showed that, regardless the dose-rate, a dose of 1 Gy approximately was enough to induce a significant level of cell fusion depending on neutron energy and the age of rats. The level of cell fusion was also significant in Hela cells at a dose of 0.5 Gy. Similar effect, but to a lesser extent, was observed in V-79 cells. Whereas, in lymphocytes insignificant cell fusion was noticed. The varying levels of cell-fusion in different cell lines could be attributed to the type of cells and mutual contact between cells. Furthermore irradiation did not show any influence on cell division ability in both hepatocytes and Hela cells and that fused cells were also able to divide forming a new generation of cells. (author). 36 refs., 8 figs., 10 tabs

  4. 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.

  5. Biological and Molecular Effects of Small Molecule Kinase Inhibitors on Low-Passage Human Colorectal Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Falko Lange

    2014-01-01

    Full Text Available Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal cancer (CRC patients, to evaluate effects of the small molecule kinase inhibitors (SMI vemurafenib, trametinib, perifosine, and regorafenib in an in vitro setting. The mutant BRAF (V600E/V600K inhibitor vemurafenib, but also the MEK1/2 inhibitor trametinib efficiently inhibited DNA synthesis, signaling through ERK1/2 and expression of genes downstream of ERK1/2 in BRAF mutant cells only. In case of the AKT inhibitor perifosine, three cell lines showed a high or intermediate responsiveness to the drug while one cell line was resistant. The multikinase inhibitor regorafenib inhibited proliferation of all CRC lines with similar efficiency and independent of the presence or absence of KRAS, BRAF, PIK3CA, and TP53 mutations. Regorafenib action was associated with broad-range inhibitory effects at the level of gene expression but not with a general inhibition of AKT or MEK/ERK signaling. In vemurafenib-sensitive cells, the antiproliferative effect of vemurafenib was enhanced by the other SMI. Together, our results provide insights into the determinants of SMI efficiencies in CRC cells and encourage the further use of low-passage CRC cell lines as preclinical models.

  6. xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies.

    Science.gov (United States)

    Martinez-Serra, Jordi; Gutierrez, Antonio; Muñoz-Capó, Saúl; Navarro-Palou, María; Ros, Teresa; Amat, Juan Carlos; Lopez, Bernardo; Marcus, Toni F; Fueyo, Laura; Suquia, Angela G; Gines, Jordi; Rubio, Francisco; Ramos, Rafael; Besalduch, Joan

    2014-01-01

    The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

  7. Cathepsin K in Lymphangioleiomyomatosis: LAM Cell-Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification.

    Science.gov (United States)

    Dongre, Arundhati; Clements, Debbie; Fisher, Andrew J; Johnson, Simon R

    2017-08-01

    Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Effect of dabrafenib on melanoma cell lines harbouring the BRAFV600D/R mutations

    Directory of Open Access Journals (Sweden)

    Gentilcore Giusy

    2013-01-01

    Full Text Available Abstract Background Conventional therapeutic agents are largely unsatisfactory into the treatment of malignant melanoma. Recently, an innovative approach based on inhibitors of the mutated BRAF gene (which represents the most prevalent alteration in melanoma patients appears very promising from the clinical point of view. On this regard, a new compound, dabrafenib (GSK2118436, has been demonstrated to be effective in patients carrying the BRAFV600E/K mutations. We here tested dabrafenib for its capability to inhibit cell growth on primary melanoma cell lines, established from patients' tumour tissues and carrying the BRAFV600D/R mutations. Methods Three melanoma cell lines were tested: M257 wild-type BRAF, LCP BRAFV600R and WM266 BRAFV600D. The MTT assays were performed using standardized approaches. To evaluate the inhibition of MAPK pathway and the consequent inhibition of cellular proliferation, the phosphorylation of ERK was examined by Western Blot analysis performed on total protein extracts from cell lines after treatment with dabrafenib. Results Our experiments demonstrated an effective action of Dabrafenib (GSK2118436 and the inhibition of MAPK pathway in melanoma cell lines carrying BRAFV600D/R mutations. Conclusion These results could be helpful to enlarge the number of melanoma patients who may benefit of a more effective targeted treatment.

  9. Calcium K-line network in coronal holes

    Energy Technology Data Exchange (ETDEWEB)

    Marsh, K A [Hale Observatories, Pasadena, Calif. (USA)

    1977-05-01

    Microphotometry of calcium K-line photographs in the regions of polar coronal holes shows that the chromospheric network exterior to a hole has a slightly broader intensity distribution than that inside the hole itself, a fact which can be attributed to a greater number of bright network elements outside the hole. These bright elements presumably represent the enhanced network resulting from the dispersal of magnetic flux from old active regions, a hypothesis which is consistent with current ideas of coronal hole formation.

  10. California-Oregon 500-kV transmission line development of design criteria

    International Nuclear Information System (INIS)

    Simpson, K.D.

    1990-01-01

    The California-Oregon Transmission Project (COTP) encompassed the design and construction of a third 500-kV ac intertie between California and the Pacific Northwest Transmission system. Sargent ampersand Lundy's (S ampersand L) scope of work in the COTP includes the design of approximately 150 miles of new single-circuit, 500-kV transmission line from southern Oregon to the vicinity of Redding, California. This paper presents the development of the design criteria for this segment of the project, which crosses diverse topographic and climatic regions. This project is an example of the increasing utilization of computers in transmission line engineering. Almost all aspects of design involved the use of the computer. Also, the development of the design criteria for this project coincided with an early release of the TLWorkstation software package by EPRI. TLWorkstation is an engineering workstation containing a family of programs for various aspects of transmission line design. This engineering software allows for increasing refinement in the design and economic optimization of transmission lines and is becoming an important design tool for transmission engineers

  11. Resistance of some leukemic blasts to lysis by lymphokine activated killer (LAK) cells

    Energy Technology Data Exchange (ETDEWEB)

    Panayotides, P; Sjoegren, A -M; Reizenstein, P; Porwit, A. Immunopathology Lab., Dept. of Pathology, Karolinska Hospital, Stockholm; Wasserman, J

    1988-01-01

    Peripheral blood mononuclear cells (PBMC) from healthy donors and AML patients in remission were stimulated with phytohemagglutinin (PHA) and recombinant interleukin-2 (IL-2). These stimulated cells (lymphokine activated killer (LAK) cells) showed increased DNA synthesis as measured by /sup 3/H-Thymidine uptake. A synergistic effect of PHA and IL-2 was found. LAK cells' ability to kill acute myeloid leukemia (AML) blasts was investigated by the /sup 51/Cr release assay. LAK cells showed a cytotoxicity (over 10% specific /sup 51/Cr release) against 9/12 leukemic blasts, even at effector/target (E/T) ratios as low as 5:1. However, on average only 22.2% (SD 11.8) and 36.5% (SD 12.5) /sup 51/Cr release were obtained in 4- and 18-hour cytotoxicity assays, respectively, at an E/T ratio of 20:1. Leukemic blasts in 3/12 AML cases and normal PBMC were entirely resistant to lysis, even at an E/T ratio of 80:1. Susceptibility to lysis was not correlated to peanut-agglutinin receptor expression. LAK cells were more cytotoxic towards the K-562 cell line (natural killer activity) than unstimulated PBMC.

  12. Putative contribution of CD56 positive cells in cetuximab treatment efficacy in first-line metastatic colorectal cancer patients

    International Nuclear Information System (INIS)

    Maréchal, Raphaël; De Schutter, Jef; Nagy, Nathalie; Demetter, Pieter; Lemmers, Arnaud; Devière, Jacques; Salmon, Isabelle; Tejpar, Sabine; Van Laethem, Jean-Luc

    2010-01-01

    Activity of cetuximab, a chimeric monoclonal antibody targeting the epidermal growth factor receptor, is largely attributed to its direct antiproliferative and proapoptotic effects. Antibody-dependent cell-mediated cytotoxicity (ADCC) could be another possible mechanism of cetuximab antitumor effects and its specific contribution on the clinical activity of cetuximab is unknown. We assessed immune cells infiltrate (CD56, CD68, CD3, CD4, CD8, Foxp3) in the primary tumor of metastatic colorectal cancer (mCRC) patients treated with a first-line cetuximab-based chemotherapy in the framework of prospective trials (treatment group) and in a matched group of mCRC patients who received the same chemotherapy regimen without cetuximab (control group). The relationship between intra-tumoral immune effector cells, the K-ras status and the efficacy of the treatment were investigated. We also evaluated in vitro, the ADCC activity in healthy donors and chemonaive mCRC patients and the specific contribution of CD56 + cells. ADCC activity against DLD1 CRC cell line is maintained in cancer patients and significantly declined after CD56 + cells depletion. In multivariate analysis, K-ras wild-type (HR: 4.7 (95% CI 1.8-12.3), p = 0.001) and tumor infiltrating CD56 + cells (HR: 2.6, (95%CI:1.14-6.0), p = 0.019) were independent favourable prognostic factors for PFS and response only in the cetuximab treatment group. By contrast CD56 + cells failed to predict PFS and response in the control group. CD56 + cells, mainly NK cells, may be the major effector of ADCC related-cetuximab activity. Assessment of CD56 + cells infiltrate in primary colorectal adenocarcinoma may provide additional information to K-ras status in predicting response and PFS in mCRC patients treated with first-line cetuximab-based chemotherapy

  13. The methane absorption spectrum near 1.73 μm (5695-5850 cm-1): Empirical line lists at 80 K and 296 K and rovibrational assignments

    Science.gov (United States)

    Ghysels, M.; Mondelain, D.; Kassi, S.; Nikitin, A. V.; Rey, M.; Campargue, A.

    2018-07-01

    The methane absorption spectrum is studied at 297 K and 80 K in the center of the Tetradecad between 5695 and 5850 cm-1. The spectra are recorded by differential absorption spectroscopy (DAS) with a noise equivalent absorption of about αmin≈ 1.5 × 10-7 cm-1. Two empirical line lists are constructed including about 4000 and 2300 lines at 297 K and 80 K, respectively. Lines due to 13CH4 present in natural abundance were identified by comparison with a spectrum of pure 13CH4 recorded in the same temperature conditions. About 1700 empirical values of the lower state energy level, Eemp, were derived from the ratios of the line intensities at 80 K and 296 K. They provide accurate temperature dependence for most of the absorption in the region (93% and 82% at 80 K and 296 K, respectively). The quality of the derived empirical values is illustrated by the clear propensity of the corresponding lower state rotational quantum number, Jemp, to be close to integer values. Using an effective Hamiltonian model derived from a previously published ab initio potential energy surface, about 2060 lines are rovibrationnally assigned, adding about 1660 new assignments to those provided in the HITRAN database for 12CH4 in the region.

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

    Science.gov (United States)

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

    2018-05-01

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

  15. A {sup 1}H nuclear magnetic resonance study of structural and organisational changes in the cell

    Energy Technology Data Exchange (ETDEWEB)

    Tunnah, Susan K

    2000-07-01

    Increasing importance is being placed on understanding the role of membrane lipids in many different areas of biochemistry. It is of interest to determine what interactions may occur between membrane lipids and drug species. Furthermore, an increasing body of evidence suggests that membrane lipids are involved in the pathology of numerous diseases such as rheumatoid arthritis, cancer and HIV. Clearly, the more information available on the mechanisms involved in diseases, the greater the potential for identifying a cure or even a prevention. {sup 1}H nuclear magnetic resonance (NMR) spectroscopy was used to study the alterations in membrane lipid organisation and structure in intact, viable cultured cells. Changes in the {sup 1}H NMR spectra and the spin-lattice relaxation measurements of the human K562 and the rat FRTL-5 cell lines were observed on the addition of the fatty acid species: triolein, evening primrose oil, arachidonic acid and ITF 1779. Results indicate that the membrane lipids are reorganised to accommodate the interpolation of these molecules. The spatial arrangement adopted by each of these species appeared to dictate its effect on the lipids. Doxorubicin and menadione, both known to cause oxidative stress, were added to K562 cells. Although both agents are known to act by different mechanisms, the NMR data and scanning electron microscopy suggested that both caused similar alterations in the membrane organisation and lipid fluidity. Protrusions were formed indicating areas of weakness in the membrane. Spin-echo NMR was employed to investigate the action of the thiol-containing compounds, penicillamine, captopril and N-acetylcysteine in erythrocytes under conditions of oxidative stress. Results indicate that while captopril acts as a free radical scavenger, penicillamine may act as either oxidant or reductant. N-acetylcysteine was observed to act as a reducing agent. (author)

  16. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    International Nuclear Information System (INIS)

    Okabe, Seiichi; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-01-01

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance

  17. Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko; Ohyashiki, Kazuma

    2013-06-07

    Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzed the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.

  18. Vitamin K metabolism in Chinese Hamster Ovary cells

    International Nuclear Information System (INIS)

    Hoffman, H.S.

    1986-01-01

    Recent investigations suggest that vitamin K may have functions other than in blood coagulation and calcification. The present study was undertaken to investigate this hypothesis using cells in culture. Chinese Hamster Ovary (CHO) cells were chosen due to their active metabolism and growth and lack of similarity to liver and bone cells, in which vitamin K metabolism is well known. Cells were adapted to serum-free media, incubated in media containing the appropriate concentrations of vitamin K for specified times, scraped from plates, pelleted, extensively washed to remove adhering vitamin K, extracted with chloroform:methanol (2:1, v/v) and analyzed on C18 HPLC columns. Uptake of vitamin K by CHO cells follows saturation kinetics at vitamin K concentrations up to 25 μ M and is transported into cells at the rate of 10 pmol/min. 10 6 cells. After 24 hours, 3 H vitamin K is metabolized by CHO cells to several compounds, the major of which was isolated and identified as vitamin K epoxide. In 3 experiments, after 24 hours, the average cellular uptake of vitamin K was 8% with approximately half being metabolized to vitamin K epoxide. These results demonstrate that vitamin K is metabolized in cells with widely different functions and suggest a generalized function for vitamin K which has yet to be elucidated

  19. Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

    Science.gov (United States)

    The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

  20. Isolation and characterization of a neoplastic epithelial cell line derived from irradiated human submaxillary gland

    International Nuclear Information System (INIS)

    Shirasuna, Kanemitsu; Sato, Mitsunobu; Yura, Yoshiaki; Yanagawa, Tetuo; Kubo, Kazuko

    1979-01-01

    Submaxillary tissues taken from a patient whose oral base was irradiated for squamous cell carcinoma were cultured in order to isolate transformed epithelial cells in vitro. The cells showed a fine structure similar to an intermediate duct cell. When they were transplanted in nude mice, salivary tumors developed. It is epidemiologically known that irradiation induces salivary tumors. In this study, the risk of inducement was revealed and a salivary epithelial cell line was used as a model for the analysis of salivary tumors. (Ichikawa, K.)

  1. Huaier Extract Induces Autophagic Cell Death by Inhibiting the mTOR/S6K Pathway in Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Huaier extract is attracting increased attention due to its biological activities, including antitumor, anti-parasite and immunomodulatory effects. Here, we investigated the role of autophagy in Huaier-induced cytotoxicity in MDA-MB-231, MDA-MB-468 and MCF7 breast cancer cells. Huaier treatment inhibited cell viability in all three cell lines and induced various large membranous vacuoles in the cytoplasm. In addition, electron microscopy, MDC staining, accumulated expression of autophagy markers and flow cytometry revealed that Huaier extract triggered autophagy. Inhibition of autophagy attenuated Huaier-induced cell death. Furthermore, Huaier extract inhibited the mammalian target of the rapamycin (mTOR/S6K pathway in breast cancer cells. After implanting MDA-MB-231 cells subcutaneously into the right flank of BALB/c nu/nu mice, Huaier extract induced autophagy and effectively inhibited xenograft tumor growth. This study is the first to show that Huaier-induced cytotoxicity is partially mediated through autophagic cell death in breast cancer cells through suppression of the mTOR/S6K pathway.

  2. Application of DNA fingerprints for cell-line individualization.

    Science.gov (United States)

    Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

    1990-09-01

    DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.

  3. Comparative analysis of mitochondrial genomes between a wheat K-type cytoplasmic male sterility (CMS) line and its maintainer line.

    Science.gov (United States)

    Liu, Huitao; Cui, Peng; Zhan, Kehui; Lin, Qiang; Zhuo, Guoyin; Guo, Xiaoli; Ding, Feng; Yang, Wenlong; Liu, Dongcheng; Hu, Songnian; Yu, Jun; Zhang, Aimin

    2011-03-29

    Plant mitochondria, semiautonomous organelles that function as manufacturers of cellular ATP, have their own genome that has a slow rate of evolution and rapid rearrangement. Cytoplasmic male sterility (CMS), a common phenotype in higher plants, is closely associated with rearrangements in mitochondrial DNA (mtDNA), and is widely used to produce F1 hybrid seeds in a variety of valuable crop species. Novel chimeric genes deduced from mtDNA rearrangements causing CMS have been identified in several plants, such as rice, sunflower, pepper, and rapeseed, but there are very few reports about mtDNA rearrangements in wheat. In the present work, we describe the mitochondrial genome of a wheat K-type CMS line and compare it with its maintainer line. The complete mtDNA sequence of a wheat K-type (with cytoplasm of Aegilops kotschyi) CMS line, Ks3, was assembled into a master circle (MC) molecule of 647,559 bp and found to harbor 34 known protein-coding genes, three rRNAs (18 S, 26 S, and 5 S rRNAs), and 16 different tRNAs. Compared to our previously published sequence of a K-type maintainer line, Km3, we detected Ks3-specific mtDNA (> 100 bp, 11.38%) and repeats (> 100 bp, 29 units) as well as genes that are unique to each line: rpl5 was missing in Ks3 and trnH was absent from Km3. We also defined 32 single nucleotide polymorphisms (SNPs) in 13 protein-coding, albeit functionally irrelevant, genes, and predicted 22 unique ORFs in Ks3, representing potential candidates for K-type CMS. All these sequence variations are candidates for involvement in CMS. A comparative analysis of the mtDNA of several angiosperms, including those from Ks3, Km3, rice, maize, Arabidopsis thaliana, and rapeseed, showed that non-coding sequences of higher plants had mostly divergent multiple reorganizations during the mtDNA evolution of higher plants. The complete mitochondrial genome of the wheat K-type CMS line Ks3 is very different from that of its maintainer line Km3, especially in non

  4. Comparative analysis of mitochondrial genomes between a wheat K-type cytoplasmic male sterility (CMS line and its maintainer line

    Directory of Open Access Journals (Sweden)

    Liu Dongcheng

    2011-03-01

    Full Text Available Abstract Background Plant mitochondria, semiautonomous organelles that function as manufacturers of cellular ATP, have their own genome that has a slow rate of evolution and rapid rearrangement. Cytoplasmic male sterility (CMS, a common phenotype in higher plants, is closely associated with rearrangements in mitochondrial DNA (mtDNA, and is widely used to produce F1 hybrid seeds in a variety of valuable crop species. Novel chimeric genes deduced from mtDNA rearrangements causing CMS have been identified in several plants, such as rice, sunflower, pepper, and rapeseed, but there are very few reports about mtDNA rearrangements in wheat. In the present work, we describe the mitochondrial genome of a wheat K-type CMS line and compare it with its maintainer line. Results The complete mtDNA sequence of a wheat K-type (with cytoplasm of Aegilops kotschyi CMS line, Ks3, was assembled into a master circle (MC molecule of 647,559 bp and found to harbor 34 known protein-coding genes, three rRNAs (18 S, 26 S, and 5 S rRNAs, and 16 different tRNAs. Compared to our previously published sequence of a K-type maintainer line, Km3, we detected Ks3-specific mtDNA (> 100 bp, 11.38% and repeats (> 100 bp, 29 units as well as genes that are unique to each line: rpl5 was missing in Ks3 and trnH was absent from Km3. We also defined 32 single nucleotide polymorphisms (SNPs in 13 protein-coding, albeit functionally irrelevant, genes, and predicted 22 unique ORFs in Ks3, representing potential candidates for K-type CMS. All these sequence variations are candidates for involvement in CMS. A comparative analysis of the mtDNA of several angiosperms, including those from Ks3, Km3, rice, maize, Arabidopsis thaliana, and rapeseed, showed that non-coding sequences of higher plants had mostly divergent multiple reorganizations during the mtDNA evolution of higher plants. Conclusion The complete mitochondrial genome of the wheat K-type CMS line Ks3 is very different from that of

  5. Heme-dependent up-regulation of the α-globin gene expression by transcriptional repressor Bach1 in erythroid cells

    International Nuclear Information System (INIS)

    Tahara, Tsuyoshi; Sun Jiying; Igarashi, Kazuhiko; Taketani, Shigeru

    2004-01-01

    The transcriptional factor Bach1 forms a heterodimer with small Maf family, and functions as a repressor of the Maf recognition element (MARE) in vivo. To investigate the involvement of Bach1 in the heme-dependent regulation of the expression of the α-globin gene, human erythroleukemia K562 cells were cultured with succinylacetone (SA), a heme biosynthetic inhibitor, and the level of α-globin mRNA was examined. A decrease of α-globin mRNA was observed in SA-treated cells, which was restored by the addition of hemin. The heme-dependent expression of α-globin occurred at the transcriptional level since the expression of human α-globin gene promoter-reporter gene containing hypersensitive site-40 (HS-40) was decreased when K562 cells were cultured with SA. Hemin treatment restored the decrease of the promoter activity by SA. The regulation of the HS-40 activity by heme was dependent on the NF-E2/AP-1 (NA) site, which is similar to MARE. The NA site-binding activity of Bach1 in K562 increased upon SA-treatment, and the increase was diminished by the addition of hemin. The transient expression of Bach1 and mutated Bach1 lacking CP motifs suppressed the HS-40 activity, and cancellation of the repressor activity by hemin was observed when wild-type Bach1 was expressed. The expression of NF-E2 strengthened the restoration of the Bach1-effect by hemin. Interestingly, nuclear localization of Bach1 increased when cells were treated with SA, while hemin induced the nuclear export of Bach1. These results indicated that heme plays an important role in the induction of α-globin gene expression through disrupting the interaction of Bach1 and the NA site in HS-40 enhancer in erythroid cells

  6. Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R. (Brigham and Women' s Hospital, Boston, MA (USA))

    1989-12-01

    Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 nmol.min-1.mg protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake.

  7. Endothelin, a peptide inhibitor of Na(+)-K(+)-ATPase in intact renaltubular epithelial cells

    International Nuclear Information System (INIS)

    Zeidel, M.L.; Brady, H.R.; Kone, B.C.; Gullans, S.R.

    1989-01-01

    Endothelin, a potent vasoconstrictor released by vascular endothelial cells, can induce natriuresis in vivo. These studies examined the regulation of Na+ transport by endothelin in suspensions of rabbit proximal tubule (PT) and inner medullary collecting duct (IMCD) cells. Endothelin reduced oxygen consumption (QO2) by 18 +/- 1% in IMCD cells but did not alter QO2 in PT cells. In IMCD cells, endothelin inhibited QO2 half maximally at approximately 5 x 10(-12) M. Several lines of evidence indicate that endothelin reduces QO2 by inhibiting the Na(+)-K(+)-ATPase. (1) Endothelin gave no further inhibition of QO2 after ouabain and blunted the stimulatory effect of amphotericin B on QO2 (+29 +/- 4% in absence of endothelin, 0 +/- 5% in presence of endothelin; n = 6 preparations, P less than 0.001). (2) Endothelin inhibited ouabain-sensitive 86Rb+ uptake by 46.6 +/- 8.6% at 10 s and by 35.4 +/- 5.3% at 30 s without altering uptake at (60 min. 3) Addition of endothelin to IMCD cells induced a net K+ efflux with an initial rate of 32.2 +/- 4.8 nmol.min-1.mg protein-1, consistent with inhibition of the Na(+)-K(+)-ATPase. In contrast to the response observed in intact cells, in permeabilized IMCD cells endothelin did not inhibit ouabain-sensitive ATPase. Several observations indicated that prostaglandin E2 (PGE2) mediates endothelin inhibition of Na(+)-K(+)-ATPase activity. (1) The response to endothelin was blocked by ibuprofen in assays of QO2, net K+ flux, and 86Rb+ uptake. (2) Endothelin and PGE2 gave equivalent, nonadditive inhibition of ouabain-sensitive 86Rb+ uptake

  8. Radiation Response in Two HPV-Infected Head-and-Neck Cancer Cell Lines in Comparison to a Non-HPV-Infected Cell Line and Relationship to Signaling Through AKT

    International Nuclear Information System (INIS)

    Gupta, Anjali K.; Lee, John H.; Wilke, Werner W.; Quon, Harry; Smith, Gareth; Maity, Amit; Buatti, John M.; Spitz, Douglas R.

    2009-01-01

    Purpose: Human papilloma virus (HPV)-associated cancers of the head and neck (H and N) are increasing in frequency and are often treated with radiation. There are conflicting data in the literature regarding the radiation response in the presence of HPV infection, with some data suggesting they may be more sensitive to radiation. There are few studies looking at in vitro effects of HPV and further sensitization by inhibitors of specific signaling pathways. We are in the process of starting a clinical trial in H and N cancer patients using nelfinavir (NFV) (which inhibits Akt) and it would be important to know the effect of HPV on radiation response ± NFV. Methods and Materials: Two naturally infected HPV-16 cell lines (UPCI-SCC90 and UMSCC47) and the HPV-negative SQ20B H and N squamous carcinoma cells were used. Western blots with or without 10 uM NFV were done to evaluate signaling from the PI3K-Akt pathway. Clonogenic assays were done in the three cell lines with or without NFV. Results: Both UPCI-SCC90 and UMSCC47 cells were sensitive to radiation as compared with SQ20B and the degree corresponded to Akt activation. The SQ20B cell line has an activating mutation in EGFR resulting in phosphorylation (P) of Akt; UMSCC47 has decreased P-phosphatase and TENsin (PTEN), resulting in increased P-Akt; UPCI-SCC90 had overexpression of P-PTEN and decreased P-Akt. NFV resulted in downregulation of Akt in all three cell lines, resulting in sensitization to radiation. Conclusions: HPV-infected H and N cancers are sensitive to radiation. The degree of sensitivity correlates to Akt activation and they can be further sensitized by NFV.

  9. Mitochondrial modulation of oxygen-dependent radiosensitivity in some human tumour cell lines.

    LENUS (Irish Health Repository)

    Anoopkumar-Dukie, S

    2009-10-01

    Oxygen-dependent radiosensitivity of tumour cells reflects direct oxidative damage to DNA, but non-nuclear mechanisms including signalling pathways may also contribute. Mitochondria are likely candidates because not only do they integrate signals from each of the main kinase pathways but mitochondrial kinases responsive to oxidative stress communicate to the rest of the cell. Using pharmacological and immunochemical methods, we tested the role of mitochondrial permeability transition (MPT) and the Bcl-2 proteins in oxygen-dependent radiosensitivity. Drug-treated or untreated cervical cancer HeLa, breast cancer MCF-7 and melanoma MeWo cell lines were irradiated at 6.2 Gy under normoxic and hypoxic conditions then allowed to proliferate for 7 days. The MPT blocker cyclosporin A (2 microM) strongly protected HeLa but not the other two lines against oxygen-dependent radiosensitivity. By contrast, bongkrekic acid (50 microM), which blocks MPT by targeting the adenine nucleotide transporter, had only marginal effect and calcineurin inhibitor FK-506 (0.1 microM) had none. Nor was evidence found for the modulation of oxygen-dependent radiosensitivity by Bax\\/Bcl-2 signalling, mitochondrial ATP-dependent potassium (mitoK(ATP)) channels or mitochondrial Ca(2+) uptake. In conclusion, calcineurin-independent protection by cyclosporin A suggests that MPT but not mitoK(ATP) or the mitochondrial apoptosis pathway plays a causal role in oxygen-dependent radiosensitivity of HeLa cells. Targeting MPT may therefore improve the effectiveness of radiotherapy in some solid tumours.

  10. Natural killer cells for immunotherapy – Advantages of cell lines over blood NK cells

    Directory of Open Access Journals (Sweden)

    Hans eKlingemann

    2016-03-01

    Full Text Available Natural killer cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells form a patient’s blood since they represent only 10% of the lymphocytes. Especially, cancer patients are known to have dysfunctional NK cells. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T-cells. Establishing cell lines from donor blood NK cells have not been successful, in contrast to blood NK cells obtained from patients with a clonal NK cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. However, except for the NK-92 cell line none of the other six known cell lines has consistent and reproducibly high anti-tumor cytotoxicity, nor can they be easily genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through ADCC. NK-92 is also the only cell line product that has been widely given to patients with advanced cancer with demonstrated efficiency and minimal side effects.

  11. Susceptibility testing of fish cell lines for virus isolation

    DEFF Research Database (Denmark)

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

    2009-01-01

    and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...... sensitivity for surveillance purposes within a cell line and between laboratories.In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination...

  12. A novel derivative of doxorubicin, AD198, inhibits canine transitional cell carcinoma and osteosarcoma cells in vitro.

    Science.gov (United States)

    Rathore, Kusum; Cekanova, Maria

    2015-01-01

    Doxorubicin (DOX) is one of the most commonly used chemotherapeutic treatments for a wide range of cancers. N-benzyladriamycin-14-valerate (AD198) is a lipophilic anthracycline that has been shown to target conventional and novel isoforms of protein kinase C (PKC) in cytoplasm of cells. Because of the adverse effects of DOX, including hair loss, nausea, vomiting, liver dysfunction, and cardiotoxicity, novel derivatives of DOX have been synthesized and validated. In this study, we evaluated the effects of DOX and its derivative, AD198, on cell viability of three canine transitional cell carcinoma (K9TCC) (K9TCC#1-Lillie, K9TCC#2-Dakota, K9TCC#4-Molly) and three canine osteosarcoma (K9OSA) (K9OSA#1-Zoe, K9OSA#2-Nashville, K9OSA#3-JJ) primary cancer cell lines. DOX and AD198 significantly inhibited cell proliferation in all tested K9TCC and K9OSA cell lines in a dose-dependent manner. AD198 inhibited cell viability of tested K9TCC and K9OSA cell lines more efficiently as compared to DOX at the same concentration using MTS (3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium) assay. AD198 had lower IC50 values as compared to DOX for all tested K9TCC and K9OSA cell lines. In addition, AD198 increased apoptosis in all tested K9TCC and K9OSA cell lines. AD198 increased the caspase activity in tested K9TCC and K9OSA cell lines, which was confirmed by caspase-3/7 assay, and cleavage of poly (ADP-ribose) polymerase (PARP) was confirmed by Western blotting analysis. In addition, AD198 cleaved PKC-δ, which subsequently activated the p38 signaling pathway, resulting in the apoptosis of tested K9TCC and K9OSA cell lines. Inhibition of the p38 signaling pathway by SB203580 rescued DOX- and AD198-induced apoptosis in tested K9TCC and K9OSA cell lines. Our in vitro results suggest that AD198 might be considered as a new treatment option for K9TCC and K9OSA cell lines cancers in vivo.

  13. 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.

  14. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome

    DEFF Research Database (Denmark)

    Lewis, Nathan E; Liu, Xin; Li, Yuxiang

    2013-01-01

    stymied by the lack of a unifying genomic resource for CHO cells. Here we report a 2.4-Gb draft genome sequence of a female Chinese hamster, Cricetulus griseus, harboring 24,044 genes. We also resequenced and analyzed the genomes of six CHO cell lines from the CHO-K1, DG44 and CHO-S lineages...

  15. The 12th 735-kV transmission line project: Hydro-Quebec network

    International Nuclear Information System (INIS)

    1992-01-01

    Hydro-Quebec is proposing to construct a 735-kV transmission line from the Chissibi station near the LG3 hydroelectric power plant to the Jacques-Cartier station near Quebec City. The width of the right-of-way will generally be 90 m and it is intended to install the line in two stages. The Chissibi-Chibougamau section should enter service in 1993 and the Chibougamau-Cartier section in 1994. The cost of the line is estimated at $1.21 billion, of which $900 million is for line construction and $310 million for station equipment. Prospective corridors for the line route have been selected, portions of which enable twinning with Hydro-Quebec's existing 9th 735-kV line. A series of hearings on the environmental and other impacts of the power line project was held by the Bureau d'audiences publiques sur l'environnement and results are summarized along with the views of the Bureau. The summary describes the justification for the project, the choice of the transmission line route, the evaluation of the impacts of the project on the natural and human environments, health effects due to magnetic fields, and effects on the economic development of northern Quebec. Recommendations are given for mitigating the impacts of the project. 13 refs., 8 figs., 2 tabs

  16. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-05-28

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

  17. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

    Science.gov (United States)

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

    2010-05-28

    Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  18. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  19. Lyn kinase is activated following thrombopoietin stimulation of the megakaryocytic cell line B1647

    DEFF Research Database (Denmark)

    Santini, Valeria; Scappini, Barbara; Gozzini, Antonella

    2002-01-01

    BACKGROUND AND OBJECTIVES: B1647 is a cell line derived from bone marrow cells of a patient with acute myeloid leukemia (M2) with a complete erythro-megakaryocytic phenotype and bears both k and p isoforms of c-mpl. Interestingly, spontaneous B1647 cell proliferation is significantly potentiated...... by thrombopoietin (TPO). DESIGN AND METHODS: We aimed to evaluate the proliferative signal transduction events following the activation of c-mpl and we stimulated B1647 cells with TPO 40 ng/mL for 3, 7, 15 and 30 minutes; cells were then lysed and whole lysates were immunoprecipitated with anti...

  20. Phenotype-gene: 562 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 562 http://metadb.riken.jp/db/SciNetS_ria224i/cria224u3ria224u1559i non-functional response to gravity...ken.jp/db/SciNetS_ria224i/cria224u4ria224u16690816i non-functional response to gravity http://metadb.riken.jp/db/SciNetS_ria224i/cria224u1ria224u947i AT1G54990

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

    Science.gov (United States)

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

    1988-09-01

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

  2. Different G2/M accumulation in M059J and M059K cells after exposure to DNA double-strand break-inducing agents

    International Nuclear Information System (INIS)

    Holgersson, Asa; Heiden, Thomas; Castro, Juan; Edgren, Margareta R.; Lewensohn, Rolf; Meijer, Annelie E.

    2005-01-01

    Purpose: To investigate and compare the cell cycle progression in relation to cell death in the human glioma cell lines, M059J and M059K, after exposure to DNA double-strand break-inducing agents. Methods and materials: The M059J and M059K cells, deficient and proficient in the catalytic subunit of the DNA-dependent protein kinase, respectively, were exposed to 1 and 4 Gy of photons or accelerated nitrogen ions. In addition, M059J and M059K cells were treated with 10 and 40 μg/mL of bleomycin for 30 min, respectively. Cell cycle progression, monitored by DNA flow cytometry, was measured up to 72 h after treatment. Results: M059J, but not M059K, cells displayed G 2 /M accumulation after low linear energy transfer irradiation. High linear energy transfer radiation exposure however, resulted in a substantial increase of M059K cells in the G 2 /M phase detected at 48 h. At 72 h, the number of cells in the G 2 /M phase was equivalent to its control. M059J cells accumulated mainly in S phase after high linear energy transfer irradiation. In contrast to M059K, M059J cells were still blocked at 72 h. Bleomycin induced G 2 /M accumulation for both M059J and M059K cells detected 24 h after treatment. At 48 h, the percentage of bleomycin-treated M059J cells in G 2 /M phase remained high, and the number of M059K cells had decreased to control levels. Neither cell line showed cell cycle arrest (≤10 h) after exposure to these agents. Conclusion: Distinct cell cycle block and release is dependent on the complexity of the induced DNA damage and the presence of the DNA-dependent protein kinase catalytic subunit

  3. Solar polarimetry in the K I D2 line : A novel possibility for a stratospheric balloon

    Science.gov (United States)

    Quintero Noda, C.; Villanueva, G. L.; Katsukawa, Y.; Solanki, S. K.; Orozco Suárez, D.; Ruiz Cobo, B.; Shimizu, T.; Oba, T.; Kubo, M.; Anan, T.; Ichimoto, K.; Suematsu, Y.

    2018-03-01

    Of the two solar lines, K I D1 and D2, almost all attention so far has been devoted to the D1 line, as D2 is severely affected by an O2 atmospheric band. This, however, makes the latter appealing for balloon and space observations from above (most of) the Earth's atmosphere. We estimate the residual effect of the O2 band on the K I D2 line at altitudes typical for stratospheric balloons. Our aim is to study the feasibility of observing the 770 nm window. Specifically, this paper serves as a preparation for the third flight of the Sunrise balloon-borne observatory. The results indicate that the absorption by O2 is still present, albeit much weaker, at the expected balloon altitude. We applied the obtained O2 transmittance to K I D2 synthetic polarimetric spectra and found that in the absence of line-of-sight motions, the residual O2 has a negligible effect on the K I D2 line. On the other hand, for Doppler-shifted K I D2 data, the residual O2 might alter the shape of the Stokes profiles. However, the residual O2 absorption is sufficiently weak at stratospheric levels that it can be divided out if appropriate measurements are made, something that is impossible at ground level. Therefore, for the first time with Sunrise III, we will be able to perform polarimetric observations of the K I D2 line and, consequently, we will have improved access to the thermodynamics and magnetic properties of the upper photosphere from observations of the K I lines.

  4. Masses and K-line absolute magnitudes of γ Leonis and 35 Comae

    International Nuclear Information System (INIS)

    Deming, D.; Dykton, M.

    1979-01-01

    The visual binary system γ Leonis consists of two K giants whose total mass was found by Wilson to be less than 0.6M/sub sun/ when a distance was obtained from K-line absolute magnitudes of both components. Since the orbital period of the system is of order 600 years, the orbital solution has usually been regarded as the most likely explanation of the anomalous mass. We have investigated the uniqueness of the orbital solution. We find that the individual orbital elements are only poorly determined, but that a 3 /P 2 is known to within +- 10%. The anomalous mass of this binary system implies either extensive mass-loss or the need for revisions in the K-line absolute magnitudes. We also investigate the binary system 35 Comae. Wilson has noted that in this system the K-line absolute magnitude of the G8 III primary component implies a total mass of 8 M/sub sun/. However the A component of 35 Comae is not sufficiently luminous to justify such a large total mass. The system has a period of order 700 years, and the uniqueness of the orbital solution is therefore suspect. However, in this case also we have investigated the uniqueness of the orbital solution and find that individual orbital elements are poorly determined, but that a 3 /P 2 is known to within +- 17%. We point out that 35 Comae is known to be very metal rich, in contrast to γ Leonis which is metal poor. We conclude that a composition-dependent correction to the K-line absolute magnitudes provides the simplest explanation of these mass anomalies. Such a correction would also give better agreement with the trigonometric parallaxes of these binary systems

  5. Molecular analysis of urothelial cancer cell lines for modeling tumor biology and drug response.

    Science.gov (United States)

    Nickerson, M L; Witte, N; Im, K M; Turan, S; Owens, C; Misner, K; Tsang, S X; Cai, Z; Wu, S; Dean, M; Costello, J C; Theodorescu, D

    2017-01-05

    The utility of tumor-derived cell lines is dependent on their ability to recapitulate underlying genomic aberrations and primary tumor biology. Here, we sequenced the exomes of 25 bladder cancer (BCa) cell lines and compared mutations, copy number alterations (CNAs), gene expression and drug response to BCa patient profiles in The Cancer Genome Atlas (TCGA). We observed a mutation pattern associated with altered CpGs and APOBEC-family cytosine deaminases similar to mutation signatures derived from somatic alterations in muscle-invasive (MI) primary tumors, highlighting a major mechanism(s) contributing to cancer-associated alterations in the BCa cell line exomes. Non-silent sequence alterations were confirmed in 76 cancer-associated genes, including mutations that likely activate oncogenes TERT and PIK3CA, and alter chromatin-associated proteins (MLL3, ARID1A, CHD6 and KDM6A) and established BCa genes (TP53, RB1, CDKN2A and TSC1). We identified alterations in signaling pathways and proteins with related functions, including the PI3K/mTOR pathway, altered in 60% of lines; BRCA DNA repair, 44%; and SYNE1-SYNE2, 60%. Homozygous deletions of chromosome 9p21 are known to target the cell cycle regulators CDKN2A and CDKN2B. This loci was commonly lost in BCa cell lines and we show the deletions extended to the polyamine enzyme methylthioadenosine (MTA) phosphorylase (MTAP) in 36% of lines, transcription factor DMRTA1 (27%) and antiviral interferon epsilon (IFNE, 19%). Overall, the BCa cell line genomic aberrations were concordant with those found in BCa patient tumors. We used gene expression and copy number data to infer pathway activities for cell lines, then used the inferred pathway activities to build a predictive model of cisplatin response. When applied to platinum-treated patients gathered from TCGA, the model predicted treatment-specific response. Together, these data and analysis represent a valuable community resource to model basic tumor biology and to study

  6. A Fe K Line in GRB 970508

    Science.gov (United States)

    Protassov, R.; van Dyk, D.; Connors, A.; Kashyap, V.; Siemiginowska, A.

    2000-12-01

    We examine the x-ray spectrum of the afterglow of GRB 970508, analyzed for Fe line emission by Piro et al (1999, ApJL, 514, L73). This is a difficult and extremely important measurement: the detection of x-ray afterglows from γ -ray bursts is at best a tricky business, relying on near-real satellite time response to unpredictable events; and a great deal of luck in catching a burst bright enough for a useful spectral analysis. Detecting a clear atomic (or cyclotron) line in the generally smooth and featureless afterglow (or burst) emission not only gives one of the few very specific keys to the physics local to the emission region, but also provides clues or confirmation of its distance (via redshift). Unfortunately, neither the likelihood ratio test or the related F-statistic commonly used to detect spectral lines adhere to their nominal Chi square and F-distributions. Thus we begin by calibrating the F-statistic used in Piro et al (1999, ApJL, 514, L73) via a simulation study. The simulation study relies on a completely specified source model, i.e. we do Monte Carlo simulations with all model parameters fixed (so--called ``parametric bootstrapping''). Second, we employ the method of posterior predictive p-values to calibrate a LRT statistic while accounting for the uncertainty in the parameters of the source model. Our analysis reveals evidence for the Fe K line.

  7. Corona performance of a compact 230-kV line

    International Nuclear Information System (INIS)

    Chartier, V.L.; Blair, D.E.

    1994-01-01

    Permitting requirements and the acquisition of new rights-of-way for transmission facilities has in recent years become increasingly difficult for most utilities, including Puget Sound Power and Light Company. In order to maintain a high degree of reliability of service while being responsive to public concerns regarding the siting of high voltage (HV) transmission facilities, Puget Power has found it necessary to more heavily rely upon the use of compact lines in franchise corridors. Compaction does, however, precipitant increased levels of audible noise (AN) and radio and TV interference (RI and TVI) due to corona on the conductors and insulator assemblies. Puget Power relies upon the Bonneville Power Administration (BPA) Corona and Field Effects computer program to calculate AN and RI for new lines. Since there was some question of the program's ability to accurately represent quiet 230-kV compact designs, a joint project was undertaken with BPA to verify the program's algorithms. Long-term measurements made on an operating Puget Power 230-kV compact line confirmed the accuracy of BPA's AN model; however, the RI measurements were much lower than predicted by the BPA computer and other programs. This paper also describes how the BPA computer program can be used to calculate the voltage needed to expose insulator assemblies to the correct electric field in single test setups in HV laboratories

  8. KHYG-1 and NK-92 represent different subtypes of LFA-1-mediated NK cell adhesiveness.

    Science.gov (United States)

    Suck, Garnet; Tan, Suet-Mien; Chu, Sixian; Niam, Madelaine; Vararattanavech, Ardcharaporn; Lim, Tsyr Jong; Koh, Mickey B C

    2011-01-01

    Novel cancer cellular therapy approaches involving long-term ex vivo IL-2 stimulated highly cytotoxic natural killer (NK) cells are emerging. However, adhesion properties of such NK cells are not very well understood. Herein, we describe the novel observation of permanently activated alphaLbeta2 integrin leukocyte function-associated antigen (LFA)-1 adhesion receptor in long-term IL-2 activated NK cells and the permanent NK cell lines KHYG-1 and NK-92. We show that such cytokine activated NK effectors constitutively adhered to the LFA-1-ligand ICAM-1, whereas binding to the lower affinity ligand ICAM-3 required additional exogenous activating conditions. The results demonstrate an extended conformation and an intermediate affinity state for the LFA-1 population expressed by the NK cells. Interestingly, adhesion to ICAM-1 or K562 induced pronounced cell spreading in KHYG-1, but not in NK-92, and partially in long-term IL-2 stimulated primary NK cells. It is conceivable that such differential adhesion characteristics may impact motility potential of such NK effectors with relevance to clinical tumor targeting. KHYG-1 could be a useful model in planning future targeted therapeutic approaches involving NK effectors with augmented functions.

  9. Chromatin status of apoptosis genes correlates with sensitivity to chemo-, immune- and radiation therapy in colorectal cancer cell lines.

    Science.gov (United States)

    Benard, Anne; Janssen, Connie M; van den Elsen, Peter J; van Eggermond, Marja C J A; Hoon, Dave S B; van de Velde, Cornelis J H; Kuppen, Peter J K

    2014-12-01

    The apoptosis pathway of programmed cell death is frequently deregulated in cancer. An intact apoptosis pathway is required for proper response to anti-cancer treatment. We investigated the chromatin status of key apoptosis genes in the apoptosis pathway in colorectal cancer cell lines in relation to apoptosis induced by chemo-, immune- or radiation therapy. Using chromatin immunoprecipitation (ChIP), we measured the presence of transcription-activating histone modifications H3Ac and H3K4me3 and silencing modifications H3K9me3 and H3K27me3 at the gene promoter regions of key apoptosis genes Bax, Bcl2, Caspase-9, Fas (CD95) and p53. Cell lines DLD1, SW620, Colo320, Caco2, Lovo and HT29 were treated with cisplatin, anti-Fas or radiation. The apoptotic response was measured by flow cytometry using propidium iodide and annexin V-FITC. The chromatin status of the apoptosis genes reflected the activation status of the intrinsic (Bax, Bcl2, Caspase-9 and p53) and extrinsic (Fas) pathways. An active intrinsic apoptotic pathway corresponded to sensitivity to cisplatin and radiation treatment of cell lines DLD1, SW620 and Colo320. An active Fas promoter corresponded to an active extrinsic apoptotic pathway in cell line DLD1. mRNA expression data correlated with the chromatin status of the apoptosis genes as measured by ChIP. In conclusion, the results presented in this study indicate that the balance between activating and silencing histone modifications, reflecting the chromatin status of apoptosis genes, can be used to predict the response of tumor cells to different anti-cancer therapies and could provide a novel target to sensitize tumors to obtain adequate treatment responses.

  10. In vitro generation of Epstein-Barr virus-specific cytotoxic T cells in patients receiving haplo-identical allogeneic stem cell transplantation.

    Science.gov (United States)

    Musk, P; Szmania, S; Galloway, A T; Johnson, K; Scott, A; Guttman, S; Bridges, K; Bruorton, M; Gatlin, J; Garcia, J V; Lamb, L; Chiang, K Y; Spencer, T; Henslee-Downey, J; van Rhee, F

    2001-01-01

    Use of a partially mismatched related donor (PMRD) is an option for patients who require allogeneic transplantation but do not have a matched sibling or unrelated donor. Epstein-Barr virus (EBV)-induced lymphoma is a major cause of mortality after PMRD transplantation. In this study, we present a clinical grade culture system for donor-derived EBV-specific cytotoxic T cells (CTLs) that do not recognize haplo-identical recipient cells. The EBV-specific CTLs were tested for cytolytic specificity and other functional properties, including ability to transgress into tissues, propensity for apoptosis, degree of clonality, stability of dominant T-cell clones, and Tc and Th phenotypes. The EBV-specific CTLs were routinely expanded to greater than 80 x 10(6) over a period of 5 weeks, which is sufficient for clinical application. A CD8+ phenotype predominated, and the CTLs were highly specific for donor lymphoblastoid cell lines (LCLs) without killing of recipient targets or K562. Vbeta spectratyping showed an oligoclonal population that was stable on prolonged culture. The EBV-specific CTLs were activated (D-related human leukocyte antigen [HLA-DR+], L-selectin+/-) and of memory phenotype (CD45RO+). Expression of the integrin VLA-4 suggested that these CTLs could adhere to endothelium and migrate into tissues. The Bcl-2 message was upregulated, which may protect the CTLs from the apoptosis. The first demonstration of overexpression of bcl-2 in human memory CTLs. In addition, we show that lymphoblastoid cell lines used to generate CTLs are readily genetically modified with recombinant lentivirus, indicating that genetically engineered antigen presentation is feasible.

  11. Effects of Vitamin K3 and K5 on Daunorubicin-resistant Human T Lymphoblastoid Leukemia Cells.

    Science.gov (United States)

    Nakaoka, Eri; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Hirano, Toshihiko

    2015-11-01

    Anticancer efficacy of vitamin K derivatives on multidrug-resistant cancer cells has been scarcely investigated. The effects of vitamins K3 and K5 on proliferation of human leukemia MOLT-4 cells and on daunorubicin-resistant MOLT-4/DNR cells were estimated by a WST assay. Apoptotic cells were detected by Annexin V and propidium iodide staining, followed by flow cytometry. Vitamins K3 and K5 significantly inhibited proliferation of leukemic cells at 10 and 100 μM (pVitamin K3 induced cell apoptosis at 10 and 100 μM in both MOLT-4 and MOLT-4/DNR cells (pVitamin K5 also increased apoptotic cells, while rather inducing necrotic cell death. Vitamins K3 and K5 suppress MOLT-4 and MOLT-4/DNR cell-proliferation partially through induction of apoptosis, and these vitamin derivatives can overcome drug resistance due to P-glycoprotein expression. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  12. Xylitol induces cell death in lung cancer A549 cells by autophagy.

    Science.gov (United States)

    Park, Eunjoo; Park, Mi Hee; Na, Hee Sam; Chung, Jin

    2015-05-01

    Xylitol is a widely used anti-caries agent that has anti-inflammatory effects. We have evaluated the potential of xylitol in cancer treatment. It's effects on cell proliferation and cytotoxicity were measured by MTT assay and LDH assay. Cell morphology and autophagy were examined by immunostaining and immunoblotting. Xylitol inhibited cell proliferation in a dose-dependent manner in these cancer cells: A549, Caki, NCI-H23, HCT-15, HL-60, K562, and SK MEL-2. The IC50 of xylitol in human gingival fibroblast cells was higher than in cancer cells, indicating that it is more specific for cancer cells. Moreover, xylitol induced autophagy in A549 cells that was inhibited by 3-methyladenine, an autophagy inhibitor. These results indicate that xylitol has potential in therapy against lung cancer by inhibiting cell proliferation and inducing autophagy of A549 cells.

  13. 562,3 MWP installed in european union

    International Nuclear Information System (INIS)

    Anon.

    2004-01-01

    In the end, the growth problems that were announced for the European photovoltaic market did not happen. With 170 MWp of additional capacity installed in the European Union 2003 has marked a new record high in installations bringing European installed capacity up to the neighbourhood of 562 MWp. While this sector's expansion has been remarkable (+ 43.4 % with respect to 2002), a lot of questions still remain with respect to the true will of certain countries to develop this form of energy. (author)

  14. NON-LTE INVERSIONS OF THE Mg ii h and k AND UV TRIPLET LINES

    Energy Technology Data Exchange (ETDEWEB)

    De la Cruz Rodríguez, Jaime; Leenaarts, Jorrit [Institute for Solar Physics, Dept. of Astronomy, Stockholm University, AlbaNova University Centre, SE-106 91 Stockholm Sweden (Sweden); Ramos, Andrés Asensio [Instituto de Astrofísica de Canarias, E-38205, La Laguna, Tenerife (Spain)

    2016-10-20

    The Mg ii h and k lines are powerful diagnostics for studying the solar chromosphere. They have become particularly popular with the launch of the Interface Region Imaging Spectrograph ( IRIS ) satellite, and a number of studies that include these lines have lead to great progress in understanding chromospheric heating, in many cases thanks to the support from 3D MHD simulations. In this study, we utilize another approach to analyze observations: non-LTE inversions of the Mg ii h and k and UV triplet lines including the effects of partial redistribution. Our inversion code attempts to construct a model atmosphere that is compatible with the observed spectra. We have assessed the capabilities and limitations of the inversions using the FALC atmosphere and a snapshot from a 3D radiation-MHD simulation. We find that Mg ii h and k allow reconstructing a model atmosphere from the middle photosphere to the transition region. We have also explored the capabilities of a multi-line/multi-atom setup, including the Mg ii h and k, the Ca ii 854.2 nm, and the Fe i 630.25 lines to recover the full stratification of physical parameters, including the magnetic field vector, from the photosphere to the chromosphere. Finally, we present the first inversions of observed IRIS spectra from quiet-Sun, plage, and sunspot, with very promising results.

  15. Effect of gamma-ray irradiated natural herbal extracts on NF-kB activation in HMC-1 cells

    International Nuclear Information System (INIS)

    Kim, Yong Soo; Lim, Youn Mook; Gwon, Hui Jeong; Choi, Bo Ram; Nho, Young Chang

    2009-01-01

    Recently, studies have documented various health benefits of some natural herbal extracts (NHE) such as Houttuynia cordata (H), Centella asiatica (C), Plantago asiatica (P), Morus alba L. (M), and Ulmus davidiana (U). The aim of the present study was to demonstrate the radiation effect on NF-kB activation of the NHE in the human mast cell line (HMC-1). The HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus A23187. Both non-and irradiated NHE also significantly inhibited the PMA plus A23187-induced nuclear factor NF-kB activation and also suppressed the expression of activation of NF-kB. These results indicated that the NHE exerted a regulatory effect on inflammatory reactions mediated by mast cells

  16. Effect of gamma-ray irradiated natural herbal extracts on NF-kB activation in HMC-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yong Soo; Lim, Youn Mook; Gwon, Hui Jeong; Choi, Bo Ram; Nho, Young Chang [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-12-15

    Recently, studies have documented various health benefits of some natural herbal extracts (NHE) such as Houttuynia cordata (H), Centella asiatica (C), Plantago asiatica (P), Morus alba L. (M), and Ulmus davidiana (U). The aim of the present study was to demonstrate the radiation effect on NF-kB activation of the NHE in the human mast cell line (HMC-1). The HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus A23187. Both non-and irradiated NHE also significantly inhibited the PMA plus A23187-induced nuclear factor NF-kB activation and also suppressed the expression of activation of NF-kB. These results indicated that the NHE exerted a regulatory effect on inflammatory reactions mediated by mast cells.

  17. Investigation of Cytocidal Activity of Bacillus Thuringiensis Parasporal Toxin on CCRF-CEM Cell Line

    Directory of Open Access Journals (Sweden)

    Elham Moazamian

    2013-03-01

    Full Text Available Background & Objective: Parasporin is a parasporal protein of Bacillus thuringiensis and exhibits special cytocidal activity against human cancer cells. Similar to other insecticidal Bacillus thuringiensis crystal toxins, parasporin shows target specificity and damages the cellular membrane. In this study, different strains of Bacillus thuringiensis isolated from various regions of Iran and their cytocidal activity against CCRF-CEM cell line and human erythrocyte were investigated.   Materials & Methods: Fifty soil samples were collected from different Iranian provinces, and characterization was performed based on protein crystal morphology by phase-contrast microscope and variations of Cry protein toxin using SDS-PAGE. After parasporin was processed with proteinase K, the active form was produced and protein activity on the cell line was evaluated. Results: Parasporal inclusion proteins showed different cytotoxicity against acute lymphoblastic leukemia cells (ALL, but not against normal lymphocyte. Isolated parasporin demonstrated no hemolytic activity against human erythrocyte. It appears that these proteins have the ability to differentiate between normal lymphocytes and leukemia cells and have specific receptors on specific cancer cell lines. Conclusion: Our results provide evidence that the parasporin-producing organism is a common member in Bacillus thuringiensis populations occurring in the natural environments of Iran.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-12-01

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

  19. Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary

    Science.gov (United States)

    Low, Donald W.; Hill, Michael G.; Carrasco, Michael R.; Kent, Stephen B. H.; Botti, Paolo

    2001-01-01

    We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet7]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the α-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet7]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by ≈45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments. PMID:11390992

  20. Phosphorylation of ribosomal protein S6 kinase 1 at Thr421/Ser424 and dephosphorylation at Thr389 regulates SP600125-induced polyploidization of megakaryocytic cell lines.

    Science.gov (United States)

    Li, Chang-Ling; Yang, Jin-Gang; Lin, Di; Zhao, Yong-Shan; Liu, Shuo; Xing, Si-Ning; Zhao, Song; Chen, Cong-Qin; Jiang, Zhi-Ming; Pu, Fei-Fei; Cao, Jian-Ping; Ma, Dong-Chu

    2014-01-01

    Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125