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Sample records for jurkat nso hl-60

  1. [Preparation of cytoplasts from HL-60 cells].

    Science.gov (United States)

    Wang, Lili; Yu, Huangfei; Fang, Ning; Chen, Daixiong

    2013-06-01

    This experimental research was aimed to establish an optimum system of enucleation, purification and identification for preparing the cytoplasts of suspension culture cells in order to undertake cell recombination. Human leukemia HL-60 cells in suspension culture were purified by 42% Percoll density gradient centrifugation and low-speed centrifugation at 1 500r/min, respectively. The purified HL-60 cells were treated with cytochalasin B (CB) alone or combined with colcchicine and enucleated by isopycnic gradient centrifugation on 50% Percoll at 25 degrees C and 34 degrees C, respectively. Cytoplasts made from HL-60 cells were purified through gradient centrifugation by 37%, 38% and 40% Percoll, respectively. The final cytoplasts were identified by Wright-Giemsa staining and 4,6-diamidino-2-phenylidole dihydrochloride (DAPI)/5, 6-carboxyflu-orescein diacetate succinimidyl ester (CFSE) double-staining. The phenotype and mitochondrial membrane potential of HL-60 cytoplasts were analyzed by flow cytometry. The results indicated that the enucleation ratio of HL-60 cells induced by CB combined with colcchicine was up to 91. 98% +/-4. 29%, which was significantly higher than that in CB alone group (74. 95% +/- 3. 02%)(PHL-60 cytoplasts had no significant change, and the activity of the cytoplasts was above 80% within 12h. It is concluded that enucleation throuth density gradient centrifugation on 50% Percoll mediated by CB combined with colcchicine, 38%Percoll of purification followed by DAPI/CFSE double labeling and MMP detection is an optimum scheme for preparation and identification of cytoplast from suspension culture cells.

  2. Proliferation inhibition, cell cycle arrest and apoptosis induced in HL-60 HL-60HL-60 HL-60 cells by a natural diterpene ester from Daphne mucronata

    Directory of Open Access Journals (Sweden)

    R Yazdanparast

    2011-05-01

    Full Text Available "n  "nBackground and the purpose of the study: Gnidilatimonoein (Gn, a new diterpene ester from Daphne mucronata, possesses strong anti-metastasis and anti-tumor activities. In this study, its apoptosis and differentiation capabilities were evaluated by using the leukemia HL-60 cell line. "nMaterial and methods: Cell prolifaration inhibition was estimated by MTT assay. The occurrence of apoptosis was evaluated by EtBr/AO double staining technique, cell cycle analyses and detection of apoptotic cells by Annexin V-FITC and propodium iodide (PI. Differentiation of the cells was determined by NBT reduction assay and the expression of specific cell surface markers such as CD14 and CD11b, were analyzed by flow cytometry.   "nResults: The drug decreased the growth of the cells dose- and time- dependently and the IC50 was found to be 1.3 µM. Our data suggested that Gn induced both monocytic differentiation  and apoptosis among HL-60 cells. In addition, cell cycle analyses showed an increase in G1 phase population by 24 hrs, which was gradually replaced by Sub-G1 cell population (apoptotic cells by 72 hrs. "nConclusion: Based on these data, the Gn-treated HL-60 cells displayed differentiation-dependent apoptosis. Thus, Gn might be a good candidate for differentiation therapy of leukemia, pending full biological evaluation of the compound among the wide array of leukemia cells. "nevaluation of the compound among the wide array of leukemia cells.

  3. Microarray analysis of responsible genes in increased growth rate in the subline of HL60 (HL60RG) cells.

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    Luan, Yang; Kogi, Mieko; Rajaguru, Palanisamy; Ren, Jin; Yamaguchi, Teruhide; Suzuki, Kazuhiro; Suzuki, Takayoshi

    2012-03-01

    HL60RG, a subline of human promyelocytic leukemia HL60 cells, has a increased growth rate than their parental cells. To gain information of the mechanisms involved in the increased growth rate of HL60RG, we performed a multiplex fluorescence in situ hybridization (M-FISH), standard cytogenetics analysis (G-banding) and genome scan using 10K SNP mapping array on both cell types. Characteristic genomic alterations in HL60RG cells were identified including uniparental disomy (UPD) of chromosome 1, and hemizygous deletion in 10p and 11p. However, no such defects were observed in HL60 cells. Changes in gene expression in HL60RG cells were determined using expression arrays (Affymetrix GeneChip, HU133A). Candidate genes associated with the rapid growth of HL60RG cells were identified. Two tumor necrosis factor receptors, TNFRSF1B (type II tumor necrosis factor-α receptor) and TNFRSF8 (also known as a tumor marker CD30), which are adjacently located on chromosome 1 showed opposing changes in gene expression in HL60RG cells-over-expression of TNFRSF8 and repression of TNFRSF1B. Differences in the DNA methylation status in the transcriptional regulatory regions of both genes between HL60 and HL60RG was detected by a methylation-specific PCR assay. In conclusion, alterations in chromosome and gene expression in HL60RG may be associated with increased growth rate.

  4. Hematopoietic Pyk2 regulates migration of differentiated HL-60 cells

    Directory of Open Access Journals (Sweden)

    Duan Yingli

    2010-05-01

    Full Text Available Abstract Background Pyk2 is a non-receptor cytoplasmic tyrosine kinase that belongs to the focal adhesion kinase family and has been implicated in neutrophil spreading and respiratory burst activity caused by TNF-α. However, the role of Pyk2 in neutrophil migration is incompletely defined. In this study, we tested the hypothesis that Pyk2 regulates the migration of neutrophil-like differentiated HL-60 cells subsequent to β2-integrin mediated cell adhesion. Methods HL-60 cells were induced to differentiate into neutrophil-like cells (dHL60 by incubation in medium containing 1.25% DMSO for up to 4 days. Pyk2 expression and tyrosine phosphorylation was measured by Western blot analysis. Adhesion of dHL60 cells to plated fibrinogen was measured by residual myeloperoxidase activity. dHL60 cell migration was evaluated using a 96-well chemoTx chamber. Results Western blot analysis demonstrated that hematopoietic Pyk2 was predominantly expressed after HL60 cell differentiation. Pyk2 was tyrosine phosphorylated upon adhesion of dHL60 cells to plated fibrinogen in the presence of fMLP. By contrast, tyrosine phosphorylation of Pyk2 was insignificant in dHL60 cells treated in suspension with fMLP. Antibodies against CD18 blocked both phosphorylation of Pyk2 and adhesion of dHL60 cells to fibrinogen, demonstrating that phosphorylation of Pyk2 was β2-integrin dependent. TAT-Pyk2-CT, a dominant negative fusion protein in which the TAT protein transduction domain was fused to the c-terminal Pyk2, attenuated fMLP-stimulated spreading, migration and phosphorylation of endogenous Pyk2 without blocking adhesion of dHL-60 cells to fibrinogen. Similarly, silencing of Pyk2 expression by siRNA in dHL60 cells also attenuated dHL60 cell migration caused by fMLP. Phospho-Pyk2 was evenly distributed around cell membrane circumferentially in unstimulated dHL-60 cells adherent to plated fibrinogen. In dHL60 cells treated with fMLP to cause cell spreading and polarization

  5. [Transfection of HL-60 cells by Venus lentiviral vector].

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    Li, Zheng; Hu, Shao-Yan; Cen, Jian-Nong; Chen, Zi-Xing

    2013-06-01

    In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  7. Dystroglycan depletion inhibits the functions of differentiated HL-60 cells.

    Science.gov (United States)

    Martínez-Zárate, Alma Delia; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Cisneros, Bulmaro; Winder, Steve J; Cerecedo, Doris

    2014-06-01

    Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

  8. [Inhibitory effect of 14-3-3ζ on the proliferation of HL-60 cells and HL-60/VCR cells].

    Science.gov (United States)

    Liang, Rong; Chen, Xie-Qun; Wang, Zhe; Xiong, Hua; Bai, Qing-Xian; Gao, Guang-Xun; Dong, Bao-Xia; Zhu, Hua-Feng

    2013-08-01

    This study was aimed to investigate the expression and role of 14-3-3ζ in the AML cell lines: sensitive HL-60 and drug-resistant HL-60/VCR cells. Semi-quantitative RT-PCR and Western blot were respectively used to examine the expression of mdr1 mRNA and Pgp in AML cell lines to validate the results of microarray. Western blot was performed to investigate the expression of Pgp, 14-3-3ζ, and anti-apoptosis protein BCL-2, MCL-1 proteins. Immunofluorescence assay was used to detect the subcellular location of 14-3-3ζ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy. Transduction with siRNA was used to silence 14-3-3ζ in AML cell lines. Cell count method and flow cytometry of cell cycle were used to analyze the changes of growth of AML cells. The results found that mdr1 mRNA and Pgp did not expressed in HL-60 cells, but significantly overexpressed in HL-60/VCR cells. Except 14-3-3σ, the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells, especially 14-3-3ζ. The higher expression of 14-3-3ζ, BCL-2, MCL-1 protein was observed in HL-60/VCR cells than that in HL-60 cells. These results were same results from gene chip. It was also noticed that 14-3-3ζ was located in the cytoplasma and nuclei of AML cell lines, especially over-expressed in HL-60/VCR cells. Furthermore, suppression of 14-3-3ζ by RNA interference resulted in inhibition of the proliferation of AML cells with decreased protein expression of BCL-2 and MCL-1, especially in HL-60/VCR cells. It is concluded that 14-3-3ζ plays an important role in proliferation of AML cells and associates with BCL-2 and MCL-1 expression. These results suggested that development of therapy targeting 14-3-3ζ may provide novel, effective strategies for refractory and relapsed AML.

  9. Dimethylsulfoxide exposure modulates HL-60 cell rolling interactions.

    Science.gov (United States)

    Gee, David J; Wright, L Kate; Zimmermann, Jonathan; Cole, Kayla; Soule, Karen; Ubowski, Michelle

    2012-08-01

    Human leukaemic HL-60 cells are widely used for studying interactions involving adhesion molecules [e.g. P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1)] since their rolling behaviour has been shown to mimic the dynamics of leucocyte rolling in vitro. HL-60 cells are neutrophilic promyelocytes that can undergo granulocytic differentiation upon exposure to compounds such as DMSO (dimethylsulfoxide). Using a parallel plate flow chamber functionalized with recombinant P-selectin-Fc chimaera, undifferentiated and DMSO-induced (48, 72 and 96 h) HL-60 cells were assayed for rolling behaviour. We found that depending on P-selectin incubation concentration, undifferentiated cells incurred up to a 6-fold increase in rolling velocity while subjected to an approximately 10-fold increase in biologically relevant shear stress. HL-60 cells exposed to DMSO for up to 72 h incurred up to a 3-fold increase in rolling velocity over the same shear stress range. Significantly, cells exposed for up to 96 h incurred up to a 9-fold decrease in rolling velocity, compared with undifferentiated HL-60 cells. Although cell surface and nuclear morphological changes were evident upon exposure to DMSO, flow cytometric analysis revealed that PSGL-1 expression was unchanged, irrespective of treatment duration. The results suggest that DMSO-treated HL-60 cells may be problematic as a substitute for neutrophils for trafficking studies during advanced stages of the LAC (leucocyte adhesion cascade). We suggest that remodelling of the cell surface during differentiation may affect rolling behaviour and that DMSO-treated HL-60 cells would behave differently from the normal leucocytes during inflammatory response in vivo.

  10. ARSENIC TRIOXIDE DOWNREGULATES TELOMERASE ACTIVITY IN HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    何冬梅; 张洹

    2002-01-01

    Objective: To evaluate whether arsenic trioxide (AS2O3) could downregulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity during induction of apoptosis of HL-60 cells. Methods: Apoptosis was detected by morphological observation and flow cytomertric cell cycle analysis. The expression of hTERT at mRNA and protein levels was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence using fluoresce isothiocyanate (FITC) label, respectively. Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Results: Treatment of 2 μmol/L at As2O3 could induce apoptosis of HL-60 cells. hTERT was decreased at both mRNA and protein levels during apoptosis of HL-60 cells. Telomerase activity of HL-60 cells was significantly inhibited. Conclusion:It is suggested that telomerase activity of HL-60 cells might be specifically inhibited by AS2O3 through the downregulation of hTERT gene expression.

  11. Identification of genes responsive to apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Wei JIN; Le-feng QU; Ping MIN; Shan CHEN; Hong LI; He LU; Yong-tai HOU

    2004-01-01

    AIM: To identify genes responsive to apoptosis in HL-60 cells treated by homoharringtonine. METHODS: cDNA microarray technology was used to detect gene expression and the result of microarrays for genes (TIEG and VDUP1) was confirmed by Northern analysis. RESULTS: Seventy-five individual mRNAs whose mass changed significantly were identified. Among these genes (25 were up-regulated and 50 were down-regulated), most are known related to oncogenes and tumor suppressor. Some genes were involved in apoptosis signaling pathways.CONCLUSION: TGFβ and TNF apoptosis signaling pathways were initiated during apoptosis in HL-60 cells.TIEG and VDUP1 play important roles in mediating apoptosis.

  12. Tetramethylpyrazine potentiates arsenic trioxide activity against HL-60 cell lines

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    Wu, Yuni; Xu, Youhua; Shen, Yali; Wang, Cuicui; Guo, Gaili; Hu, Tiantian [Key Laboratory of Developmental Diseases in Childhood, Chongqing (China); Key Laboratory of Pediatrics in Chongqing, Chongqing (China); Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing (China)

    2012-02-17

    The objective of this study was to evaluate the effects of tetramethylpyrazine (TMP) in combination with arsenic trioxide (As{sub 2}O{sub 3}) on the proliferation and differentiation of HL-60 cells. The HL-60 cells were treated with 300 µg/mL TMP, 0.5 µM As{sub 2}O{sub 3}, and 300 µg/mL TMP combined with 0.5 µM As{sub 2}O{sub 3}, respectively. The proliferative inhibition rates were determined with MTT. Differentiation was detected by the nitroblue tetrazolium (NBT) reduction test, Wright's staining and the distribution of CD11b and CD14. Flow cytometry was used to analyze cell cycle distribution. RT-PCR and Western blot assays were employed to detect the expressions of c-myc, p27, CDK2, and cyclin E1. Combination treatment had synergistic effects on the proliferative inhibition rates. The rates were increased gradually after the combination treatment, much higher than those treated with the corresponding concentration of As{sub 2}O{sub 3} alone. The cells exhibited characteristics of mature granulocytes and a higher NBT-reducing ability, being a 2.6-fold increase in the rate of NBT-positive ratio of HL-60 cells within the As{sub 2}O{sub 3} treatment versus almost a 13-fold increase in the TMP + As{sub 2}O{sub 3} group. Cells treated with both TMP and As{sub 2}O{sub 3} expressed far more CD11b antigens, almost 2-fold compared with the control group. Small doses of TMP potentiate As{sub 2}O{sub 3}-induced differentiation of HL-60 cells, possibly by regulating the expression and activity of G0/G1 phase-arresting molecules. Combination treatment of TMP with As{sub 2}O{sub 3} has significant synergistic effects on the proliferative inhibition of HL-60 cells.

  13. Importance of inducible multidrug resistance 1 expression in HL-60 cells resistant to gemtuzumab ozogamicin.

    Science.gov (United States)

    Matsumoto, Taichi; Jimi, Shiro; Hara, Shuuji; Takamatsu, Yasushi; Suzumiya, Junji; Tamura, Kazuo

    2012-07-01

    Resistance to gemtuzumab ozogamicin (GO) hampers the effective treatment of refractory acute myeloid leukemia (AML). To clarify the mechanism of resistance to GO, HL-60 cells were persistently exposed to GO in order to establish GO-resistant HL-60 (HL-60/GOR) cells. Multidrug resistance 1 (MDR-1) was strongly expressed in HL-60/GOR cells, but not in HL-60 cells. Although withdrawal of GO after the chronic exposure of HL-60/GOR cells to this compound gradually decreased MDR-1 expression to trace levels, reintroducing GO restored high MDR-1 expression in HL-60/GOR cells, but not in HL-60 cells. These results indicate that HL-60/GOR cells acquired the ability to induce MDR-1 expression in response to GO. U0126, a MEK1/2 inhibitor, prevented GO-inducible MDR-1 expression and abrogated GO resistance in HL-60/GOR cells. These results suggest that in the clinical use of GO, inducible MDR-1 expression in tumor cells should be investigated before treatment with GO. If the cells are positive then MEK1/2 inhibitors may be effective in overcoming resistance to GO.

  14. Ascorbic acid modulates cell migration in differentiated HL-60 cells and peripheral blood leukocytes.

    Science.gov (United States)

    Schwager, Joseph; Bompard, Albine; Weber, Peter; Raederstorff, Daniel

    2015-08-01

    The impact of L-ascorbic acid (L-AA) on the chemokinesis (CK) and chemotaxis (CT) of HL-60 cells and polymorphonuclear cells (PMN) was investigated. HL-60 cells were differentiated with DMSO, retinoic acid (RA), vitamin D, or L-AA. Chemokinesis and chemotaxis of differentiated HL-cells were assayed. Vitamin D3-treated HL-60 cells (dHL-60vitD3 cells) and RA-treated cells (dHL-60RA cells) acquired monocyte/macrophage-like and neutrophil-like phenotypes, respectively. DMSO induced the differentiation of an intermediate phenotype (dHL-60DMSO cells), whereas L-AA downregulated neutrophil markers (dHL-60L-AA cells). dHL-60DMSO cells had increased CK and potent CT in gradients of IL-8 and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). dHL-60RA cells and dHL-60L-AA cells migrated less toward IL-8 and fMLP; dHL-60vitD3 cells preferably responded to fMLP. L-AA enhanced CK of dHL-60DMSO cells and was a weak chemo-attractant. In human leukocytes, IL-8 and fMLP triggered receptor-mediated chemotaxis. CXCR2 and fMLPR were downregulated by IL-8 and fMLP, respectively. L-AA stimulated chemotaxis although significantly less than IL-8 and fMLP. IL-8 targeted chemotaxis was enhanced both in HL-60 cells and leukocytes when cells were incubated with L-AA. L-AA modulated chemokinesis and had significant chemo-attractant properties, which were independent on fMLP or IL-8 receptors. The results suggest that L-AA improves leukocyte function in innate immune responses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Efflux of potassium ion is an important reason of HL-60 cells apoptosis induced by tachyplesin

    Institute of Scientific and Technical Information of China (English)

    Hai-tao ZHANG; Jun WU; Hai-feng ZHANG; Qi-feng ZHU

    2006-01-01

    Aim: To investigate the role of intercellular potassium in tachyplesin-induced HL-60 cells apoptosis. Methods: The concentration of intercellular potassium, cell volume and mitochondrial membrane potential were examined by flow cytometry. Results: The concentration of intercellular potassium reduced in a time-dependent manner in tachyplesin-treated HL-60 cells. In addition, the loss of mitochondrial membrane potential was tightly coupled with the shrinkage of cell volume. Different caspase inhibitors protected against DNA degradation but did not prevent the loss of HL-60 cell viability induced by tachyplesin. Ba2+, which was a kind of blocker of volume-regulatory K+ channels, increased the viability of tachyplesin-treated HL-60 cells and maintained mitochondrial membrane potential and cell volume. Conclusion: Efflux of K+ was an important reason for apoptosis in tachyplesin-treated HL-60 cells. Efflux of K+ affected the viability of tachyplesin-treated HL-60 cells independent of the process of caspase activation.

  16. [Mechanism of HL-60 cells apoptosis induced by proteasome inhibitor MG132].

    Science.gov (United States)

    Zhou, Yong-Ming; Yu, Mei-Xia; Qiu, Yu-Zhen; Xing, Xiao-Lei; Yao, Chun-Hong; Bai, Ru-Jun

    2013-08-01

    The purpose of this study was to elucidate the apoptosis, apoptotic pathway of HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Apoptosis of HL-60 cells was detected by flow cytometry, the expression of P21, P27 and P53 proteins in HL-60 cells treated with MG132 was assayed by Western blot. The HL-60 cells were treated with 1 µmol/L MG132 for 48 h, and irradiated by 75 Gy of (60)Co γ-ray, but their antigenicity was preserved. The effect of irradiated HL-60 cells treated with MG132 on proliferation of peripheral blood mononuclear cells (PBMNC) was measured by CCK-8 method. The results showed that the apoptotic rate of MG132-treated HL-60 cells increased in dose-and time-dependent manner. No significant changes in MG132-induced apoptosis were observed after inhibiting caspase-8 and caspase-9 pathway. The expression of P21 and P27 protein increased after treatment of HL-60 cells with MG132. CCK-8 test showed that HL-60 cells induced with low-dose of MG132 displayed the enhancing effect on proliferation of PBMNC. It is concluded that high dose of MG132 can induce the apoptosis of HL-60 cells, and has direct killing effect on HL-60 cells, but this inducing apoptotic effect on HL-60 cells can not be realized through caspase-8 and caspase-9 pathway. The P21 and P27 protein may be involved in MG132 induced HL-60 cell apoptosis. Low dose of MG132 promotes the proliferation of PBMNC in healthy individuals and enhance the immunity of organism.

  17. NSO News January 2014

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    Beck, James B. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2014-02-04

    National Security Office (NSO) newsletter's main highlight is on the annual Strategic Weapons in the 21st Century that the Los Alamos and Lawrence Livermore National Laboratories host in Washington, DC.

  18. [Effect of 5-aza-2'-deoxycytidine on DAPK gene expression in human HL-60 cells].

    Science.gov (United States)

    Wang, Chun-Yan; Liu, Wen-Jun

    2014-06-01

    This study was aimed to investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2dC) of different concentrations on the apoptosis of human acute myeloid leukemia (AML) cell line HL-60 and the expression of DAPK gene in HL-60 cells, as well as to explore the possible anti-AML mechanism of 5-aza-2dC. HL-60 cells were treated by 5-aza-2dC of different concentrations. The effect of 5-aza-2dC on the HL-60 cell morphology was observed by Wright's staining. The effect of 5-aza-2dC on HL-60 cell apoptosis and DAPK mRNA expression was detected by flow cytometry and reverse transcription-polymerize chain reaction (RT-PCR) respectively. The results showed that the 5-aza-2dC induced the apoptosis of HL-60 cells in a concentration-dependent manner; the 5-aza-2dC increased the expression levels of DAPK mRNA in HL-60 cells in a concentration-dependent manner. It is concluded that the apoptosis rate of HL-60 cells and DAPK mRNA expression level displayed a rising trend with 5-aza-2dC concentration increasing. Therefore, DAPK gene may participate in HL-60 cell apoptosis induced by 5-aza-2dC.

  19. [Drug resistance reversal of HL-60/ADR cells by simultaneous suppression of XIAP and MRP].

    Science.gov (United States)

    Wang, Xiao-Fang; Wang, Chun; Qin, You-Wen; Yan, Shi-Ke; Gao, Yan-Rong

    2006-12-01

    This study was purposed to explore the mechanisms of drug resistance of HL-60/ADR cells and to compare the reversal drug-resistance effects of antisense oligonucleotides (AS ODN) of XIAP (X-linked inhibitor of apoptosis protein) and AS ODNs of MRP (multidrug resistance-associated protein) by use alone or in combination. Reverse transcription-PCR and Western blot were applied to detect the expression of XIAP, BCL-2, MRP and MDR1 in mRNA and protein levels of HL-60 cells and HL-60/ADR cells, respectively. Fully phosphorothioated AS ODN of XIAP and MRP was delivered into HL-60/ADR cells with Lipofectamine 2000 in the form of liposome-ODN complexes alone or in combination. CCK-8 cell viability assay was used to determine the effect of AS ODN of XIAP and MRP used alone or in combination on the chemotherapy sensitivity of HL-60/ADR cells to daunorubicin (DNR). Reverse transcription-PCR and Western blot were applied to examine the changes of XIAP, MRP in mRNA and protein levels respectively. The results showed that MRP and XIAP were both significantly higher in HL-60/ADR cells than those in HL-60 cells. AS ODN of XIAP and MRP down-regulated the expression of XIAP and MRP in HL-60/ADR cells and increased the sensitivity of HL-60/ADR cells to DNR, respectively. AS ODN of XIAP + MRP did not enhance the inhibition expression of XIAP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly as compared with AS ODN of XIAP (P MRP did not increase the concentration of DNR nor enhanced the inhibition expression of MRP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly (P MRP. It is concluded that both XIAP and MRP may be involved in the drug resistance mechanisms of HL-60/ADR cells. Drug-resistance of HL-60/ADR cells can be reversed significantly when antisense oligonucleotides of XIAP and MRP were used in combination.

  20. MG132 Induced Apoptosis Pathway in HL-60 Cells and Impact of Allogeneic Mixed Lymphocyte Reaction

    Institute of Scientific and Technical Information of China (English)

    Yong-ming Zhou; Wei Guo; Hao Zhou; Jin-hua Zhang; Zhi-ping Liu; Mei-xia Yu

    2009-01-01

    Objective: To investigate the proteasome inhibitor MG132-induced apoptosis pathway in HL-60 cells and the role of allogeneic mixed lymphocyte reaction.Methods: Cell apoptosis was analyzed by flow cytometry. The expressions of p21 protein, p27 protein and p53 protein in HL-60 cells treated with MG132 were measured by Western blot. The proliferation of, peripheral blood mononuclear cells (PBMNCs) after treatment with 75 Gy irradiated HL-60 cells treated with MG132 was measured with CCK-8.Results: High-dose MG132 induced apoptosis in HL-60 cells. No significant change was observed in MG132-induced apoptosis after inhibiting caspase-8 and caspase-9 pathway. The expressions of p21 protein and p27 protein increased in MG132-induced apoptosis. HL-60 cells treated with low-dose MG132 improved the proliferation of PBMNCs from healthy volunteers.Conclusion: High-dose MG132 induced apoptosis and directly killed HL-60 cells. MG132 induced apoptosis in a caspase-8- and caspase-9-independent pathway. p21 protein and p27 protein were involved in MG132-induced apoptosis in HL-60 cells. HL-60 cells treated with Low-dose MG132 improved the effect of promoting the proliferation of PBMNCs from healthy volunteers.

  1. [Influence of TIEG1 on apoptosis of HL-60 cells and expression of Bcl-2/Bax].

    Science.gov (United States)

    Yao, Kun; Yang, Ying; Hu, Rong; Miao, Miao; Liao, Ai-Jun; Yang, Wei; Liu, Zhuo-Gang

    2013-06-01

    This study was aimed to investigate the influence of TIEG1 on apoptosis of HL-60 cells and the expression of Bcl-2/Bax. Different concentration of TIEG1 were used to treat HL-60 cells, the cell growth inhibition rate was detected by MTT method. After treating HL-60 cells with 12.03 ng/ml TIEG1, cell apoptosis was detected with flow cytometry. Bcl-2 and Bax was detected with RT-PCR. The results showed that TIEG1 had inhibitory effect on HL-60 cell proliferation, and in time-and dose-dependent manners. The more obvious inhibitory effect was observed in HL-60 cells treated with TIEG1 of 12.03 ng/ml. During the course of cell apoptosis, Bax expression increased, but Bcl-2 expression decreased (P HL-60 cell proliferation and induces apoptosis in time and dose-dependent manners. During the course of HL-60 cells apoptosis induced by TIEG1, Bcl-2/Bax are associated with HL-60 cell apoptosis induced by TIEG1.

  2. [Human umbilical cord mesenchymal stem cells reduce the sensitivity of HL-60 cells to cytarabine].

    Science.gov (United States)

    Cui, Jun-Jie; Chi, Ying; Du, Wen-Jing; Yang, Shao-Guang; Li, Xue; Chen, Fang; Ma, Feng-Xia; Lu, Shi-Hong; Han, Zhong-Chao

    2013-06-01

    This study was purposed to investigate the impact of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) on the sensitivity of HL-60 cells to therapeutic drugs so as to provide more information for exploring the regulatory effect of hUC-MSC on leukemia cells. Transwell and direct co-culture systems of HL-60 and hUC-MSC were established. The apoptosis and cell cycle of HL-60 cells were detected by flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein levels of Caspase 3, respectively. The results showed that the apoptosis of HL-60 induced by cytarabine (Ara-C) decreased significantly after direct co-cultured with hUC-MSC cycle mRNA (P HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P HL-60 cells were reduced (P HL-60 from Arc-C induced apoptosis through regulating the cell cycle and down-regulating expression of Caspase 3 in HL-60 cells. In addition, this effect is caused by the soluble factors from hUC-MSC.

  3. Regulation of Histone Acetylation and Apoptosis by Trichostatin in HL-60 Cells

    Institute of Scientific and Technical Information of China (English)

    李新刚; 陈维凯; 谷俊侠; 崔国惠; 陈燕

    2004-01-01

    In order to examine the strong anticancer action and low toxicity of Trichostatin A(TSA), the effect of TSA was examined on the growth inhibition, acetylation of histone H3 and apoptosis in HL-60 cells by employing MTT, immunocytochemical techniques, and Annexin-V-FITC/PI assay. Our results showed that TSA could inhibit proliferation of HL- 60 cells in a time- and dose-dependent manner, and the IC50 at the 36th h was 100 ng/ml. The apoptosis-inducing effect of TSA on HL-60 cells was also time- and dose-dependent. But it didn't demonstrate apparent apoptosis induction in NPBMNCs within specific dose and time range. Both of the acetylation of histone H3 in HL-60 cells and NPBMNCs increased significantly (P<0.05) after treated with 100 ng/ml TSA for 4 h. However, there was no significant differences between the two groups (P>0.05). It is concluded that TSA can inhibit growth and induce apoptosis of HL-60 cells in a time- and dosedependent manner, and is able to selectively induce apoptosis in HL-60 cells but does not respond in NPBMNCs under the same conditions. The difference of TSA between HL-60 cells and NPBMNCs can't be explained by the regulation of histone acetylation.

  4. Study on human promyelocytic leukemia HL-60 cells apoptosis induced by fucosterol.

    Science.gov (United States)

    Ji, Yu-Bin; Ji, Chen-Feng; Yue, Lei

    2014-01-01

    In this study, we investigated the effect of fucosterol on HL-60 and the molecular mechanism. HL-60 Cells were treated with fucosterol, and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method was used to study fucosterol anti-tumor activity. Morphology of HL-60 cells was observed. Flow cytometry (FCM) was employed to detect the cell cycle. Laser scanning confocal microscope (LSCM) was used to analyze mitochondrial membrane potential (MMP) and the expressions of Fas, FasL, Fadd and Caspase-8. Western blot was performed to analyze the expressions of Cyt-C, Pro-Caspase-9 and Pro-Caspase-3. Caspase activity kits were used to determine the activity of Caspase-9, Caspase-8 and Caspase-3. The results showed fucosterol could inhibit the growth of HL-60 cells, and the cell cycle was arrested at G2/M phase. HL-60 cells showed obvious apoptosis morphology. After being treated with fucosterol for 24 h, HL-60 cells decreased MMP, induced Cyt-C release and Caspase-9, Caspase-3 activation. Fucosterol also increased the protein expression of Fas, FasL, Fadd and Caspase-8. Moreover, the activity of Caspase-9, Caspase-8 and Caspase-3 was increased significantly. In conclusion, Fucosterol can induce HL-60 cells apoptosis, suggesting that it may be a potent agent for cancer prevention and treatment.

  5. Isolation of furocoumarins from bergamot fruits as HL-60 differentiation-inducing compounds.

    Science.gov (United States)

    Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

    1999-10-01

    The HL-60 differentiation-inducing compounds in bergamot fruits were isolated with column chromatography and identified as bergamottin, bergapten, and citropten by (1)H and (13)C NMR. Their HL-60 differentiation-inducing activity was measured by examining nitro blue tetrazolium (NBT) reducing, nonspecific acid esterase (NSE), specific esterase (SE), and phagocytic activities, and bergamottin showed the strongest activity among the coumarins isolated from bergamot fruits. The structure-activity relationship obtained from HL-60 differentiation assay suggests that hydrophobicity of furocoumarins is correlated with their activity.

  6. PML(NLS(-)) inhibits cell apoptosis and promotes proliferation in HL-60 cells.

    Science.gov (United States)

    Gao, Yuan-Mei; Zhong, Liang; Zhang, Xi; Hu, Xiu-Xiu; Liu, Bei-Zhong

    2013-01-01

    Promyelocytic leukemia (PML) is a cell-growth suppressor, and PML-retinoic acid receptor α (PML-RARα) is known as a fusion gene of acute promyelocytic leukemia (APL). Studies have reported that neutrophil elastase(NE) cleaved bcr-1-derived PML-RARα in early myeloid cells leading to the removal of nuclear localization signal (NLS) from PML. The resultant PML without NLS named PML(NLS(-)). PML(NLS(-)) mainly locates and functions in the cytoplasm. PML(NLS(-)) may act in different ways from PML, but its biological characteristics have not been reported. In this study, the PML (NLS(-)) was silenced with shRNA [HL-60/pPML(NLS(-))-shRNA] and over-expressed by preparation of recombinant adenovirus [HL-60/pAd-PML(NLS(-))]. The mRNA and protein expression of PML(NLS(-)) were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect apoptotic cells. The transcription of BCL-2, BAX and C-MYC was detected in HL-60/pAd-PML(NLS(-)) cells. Our results showed that compared to the control group, the expression of PML(NLS(-)) was significantly reduced in the HL-60/pPML(NLS(-))-shRNA cells, and increased significantly in the HL-60/pAd-PML(NLS(-)) cells. The proliferation was significantly inhibited in the HL-60/pPML(NLS(-))-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-PML(NLS(-)) cells treated with 60 μmol/L emodin. FCM revealed the apoptosis increased in HL-60/pPML(NLS(-))-shRNA cells, and decreased in the HL-60/pAd-PML(NLS(-)) cells. The expression of BAX decreased significantly, while that of BCL-2 and C-MYC increased significantly in HL-60/ pAd-PML(NLS(-)) cells. Down-regulation of PML(NLS(-)) expression inhibits the proliferation and induces the apoptosis of HL-60 cells. On the contrary, over-expression of PML(NLS(-)) promotes the proliferation and reduce the emodin-induced apoptosis of HL-60 cells.

  7. [Effect of astragalus polysaccharide on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism].

    Science.gov (United States)

    Zeng, Peng-Yun; Deng, Li-Li; Yue, Ling-Ling; Zhang, Lian-Sheng

    2012-08-01

    The objective of this study was to explore the effect of astragalus polysaccharide (APS) on sensitivity of leukemic cell line HL-60 to NK cell cytotoxicity and its mechanism. The cytotoxicities of NK cells against HL-60 cells were analyzed by LDH releasing assay at different effect-to-target cell ratios (E:T) before and after treated with APS. The gene expression of MHC class I chain-related (MICA) in HL-60 cells before and after APS treatment was assayed with RT-PCR. Protein expression of MICA in HL-60 cells was assayed by flow cytometry before and after treated by APS. The results showed that after treated with APS 15 mg/ml for 48 h, the cytotoxicities of NK cells against HL-60 cells enhanced at different effect-to-target (P HL-60 cells were up-regulated (P HL-60 cells, thus enhance sensitivity of HL-60 cells to cytotoxicity of NK cells.

  8. 14-3-3ξ对HL-60HL-60/VCR细胞增殖的抑制作用%Inhibitory Effect of 14-3-3ξ on the Proliferation of HL-60 Cells and HL-60/VCR Cells

    Institute of Scientific and Technical Information of China (English)

    梁蓉; 陈协群; 王哲; 熊华; 白庆咸; 高广勋; 董宝侠; 朱华锋

    2013-01-01

    This study was aimed to investigate the expression and role of 14-3-3ξ in the AML cell lines:sensitive HL-60 and drug-resistant HL-60/VCR cells.Semi-quantitative RT-PCR and Western blot were respectively used to examine the expression of mdr1 mRNA and Pgp in AML cell lines to validate the results of microarray.Western blot was performed to investigate the expression of Pgp,14-3-3ξ,and anti-apoptosic protein BCL-2,MCL-1 proteins.Immunofluorescence assay was used to detect the subcellular location of 14-3-3ξ protein in HL-60 and HL-60/VCR cells by laser scanning confocal microscopy.Transduction with siRNA was used to silence 14-3-3ξ in AML cell lines.Cell count method and flow cytometry of cell cycle were used to analyze the changes of growth of AML cells.The results found that mdr1 mRNA and Pgp did not expressed in HL-60 cells,but significantly overexpressed in HL-60/VCR cells.Except 14-3-3(o),the expression of other subtypes of 14-3-3 was higher in HL-60/VCR cells than that in HL-60 cells,especially 14-3-3ξ.The higher expression of 14-3-3ξ,BCL-2,MCL-1 protein was observed in HL-60/VCR cells than that in HL-60 cells.These results were same results from gene chip.It was also noticed that 14-3-3ξ was located in the cytoplasma and nuclei of AML cell lines,especially over-expressed in HL-60/VCR cells.Furthermore,suppression of 14-3-3ξ by RNA interference resulted in inhibition of the proliferation of AML cells with decreased protein expression of BCL-2 and MCL-1,especially in HL-60/VCR cells.It is concluded that 14-3-3ξ plays an important role in proliferation of AML cells and associates with BCL-2 and MCL-1 expression.These results suggested that development of therapy targeting 14-3-3ξ may provide novel,effective strategies for refractory and relapsed AML.%本研究旨在进一步探讨14-3-3ξ在急性髓系白血病(acute myeloid leukemia,AML)敏感细胞HL-60和耐药细胞HL-60/VCR中的表达和作用.用半定量RT-PCR和Western blot分

  9. EFFECTS OF THE HYPERTHERMIA AND HARRINGTONINE ON TELOMERASE ACTIVITY OF HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    王宝燕; 张海涛; 李静

    2002-01-01

    Objective To understand the mechanism of the hype rthermia and harringtonine in purging of leukemia cells In Vitro. telomerase activity of HL-60 cells treated by hyperthermia (42℃ for one hour) and di fferent concentrations of Harringtonine were investigated.Methods Using Telomeric Repeats Amplification Protocol (TRAP) a nd ELISA techniques to analyze the telomerase activity of HL-60 cells. Results Our results showed that harringtonine inhibited the tel omerase activity of HL-60 cells in a dosage related manner. Moreover, the telom erase activity of HL-60 cells was significantly decreased after the hyperthermi a treatment as compared with untreated cells.Conclusion The effect of the hyperthermia and Harringtonine on purging leukemia cells In Vitro may be mediated by down regulation of telome rase activity of tumor cells.

  10. CLONING AND EXPRESSION OF A GENE MEDIATINGγ-RADIATION-INDUCED APOPTOSIS IN HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To identify the member of the caspase family proteases involved in γ-radiation-induced apoptosis in HL-60 cells and to study the expression of the caspase gene in normal, apoptotic cells and in immortal tu mor cells. Methods By using degenerate oligonucleotide primers encoding the highly conserved peptides that were pre sent in all known caspases, we performed RT-PCR on poly(A)RNA from γ-radiation-induced apoptotic HL-60 cells. Caspase-3 mRNA in apoptotic HL-60 cells and in human tumor cell lines was analyzed by Northern blot. Results The amplified DNA fragment was identified with caspase-3 cDNA by cloning and sequencing. The Northern blot analysis of caspase-3 mRNA of different human tumor cell lines showed that the caspase-3 gene transcript was more highly ex pressed in leukemia cell lines and the SH-SY5Y cell line than in HeLa and MCF-7 cells. It was more highly expressed in the radiation-induced apoptotic HL-60 cells than in control HL-60 cells. Conclusion These results indicated that caspase-3 was involved in γ-radiation-induced apoptosis in HL-60 cells. The high level of expression of caspase-3 may aid efforts to understand the insensitivity of some tumor cells to radiation, their inherent ability to survive, and apop tosis.

  11. Ethanolic extract of propolis induces apoptosis of HL-60 cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Yan Shi; Yana Li; Co-first author Naie Li; Min Yu; Dong Wang; Lijun Kong

    2016-01-01

    Objective The aim of the study was to investigate whether ethanolic extract of propolis inhibits the growth and induces apoptosis of HL-60 cel s. Methods HL-60 cel s were treated for 24, 48, 72 h with various concentrations ethanolic extracts of prop-olis (0, 50, 100, and 200 μg/mL). The proliferation of HL-60 cels was determined using the 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Subsequently, Hochest 33258 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to test the apoptosis of HL-60 cel s. We observed the expression levels of Bax and Bcl-2 in HL-60 cel s by immunohistochemistry. Results MTT assay showed that various concentrations of ethanolic extract of propolis had significant inhibitory efect on HL-60 cel proliferation (P Conclusion Ethanolic extract of propolis inhibits leukemia cel proliferation and induces apoptosis in vitro. Its mechanism may be related to the regulation of Bax and Bcl-2 expression and up-regulation of Bcl-2/Bax ratio.

  12. Differential impact of bortezomib on HL-60 and K562 cells.

    Science.gov (United States)

    Kliková, Katarína; Štefaniková, Andrea; Pilchová, Ivana; Hatok, Jozef; Chudý, Peter; Chudej, Juraj; Dobrota, Dušan; Račay, Peter

    2015-01-01

    Bortezomib (PS-341, or Velcade), reversible inhibitor of 20S proteasome approved for the treatment of multiple myeloma and mantle cell lymphoma, exhibited a cytotoxic effect toward other malignancies including leukaemia. In this study, we have documented that incubation of both HL-60 and K562 leukaemia cells with nanomolar concentrations of bortezomib is associated with the death of HL-60 cells observed within 24 hours of incubation with bortezomib and the death of K562 cells that were observed after 72 hours of incubation with bortezomib. The relative resistance of K562 cells to bortezomib correlated well with significantly higher expression of HSP27, HSP70, HSP90α, HSP90β and GRP75 in these cells. Incubation of both HL-60 and K562 cells with bortezomib induced a cleavage of HSP90β as well as expression of HSP70 and HSP90β but bortezomib did not affect levels of HSP27, HSP90α, GRP75 and GRP78. The death of both types of cells was accompanied with proteolytic activation of caspase 3 that was observed in HL-60 cells and proteolytic degradation of procaspase 3 in K562 cells. Our study has also pointed to essential role of caspase 8 in bortezomib-induced cleavage of HSP90β in both HL-60 and K562 cells. Finally, we have shown that bortezomib induced activation of caspase 9/caspase 3 axis in HL-60 cells, while the mechanism of death of K562 cells remains unknown.

  13. [Effect of honokiol on proliferation and apoptosis in HL-60 cells and its potential mechanism].

    Science.gov (United States)

    Fan, Jia-Xin; Zeng, Ying-Jian; Weng, Guang-Yang; Wu, Jian-Wei; Li, Zhang-Qiu; Li, Yuan-Ming; Zheng, Rong; Guo, Kun-Yuan

    2014-12-01

    This study was aimed to investigate the effect of Honokiol (HNK) on proliferation and apoptosis of acute myeloid leukemia HL-60 cells and its potential mechanism. Inhibitory effect of HNK on the HL-60 cell proliferation was detected by MTT assay. Flow cytometry was used to detect the change of cell cycle and AnnexinV/PI staining was used to detect apoptosis. Western blot was applied to analyze the cell cycle protein (cyclins), cyclin-dependent kinase (CDK), P53, P21, P27, BCL-2, BCL-XL, Bax, caspase-3/9 and proteins for MAPK signal pathway. The results showed that HNK could inhibit the proliferation of HL-60 cells in time- and dose dependent ways. HNK arrested HL-60 cells in G0/G1 phase, and S phase cells decreased significantly (P HL-60 cell apoptosis increased significantly with the upregulation of activated caspase-3, -9, BAX expression and the downregulation of BCL-2, BCL-XL expression. The MAPK subfamily, P38 and JNK were not significantly changed, but the expression of MEK1/2-ERK1/2 was significantly downregulated (P HL-60 cell apoptosis through the intervention of MEK1/2-ERK1/2 signaling pathway.

  14. [Effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of HL-60 cells and its mechanism].

    Science.gov (United States)

    Xie, Xia; Li, Jie; Wang, Rui-Cang; Geng, Rui-Li; Wang, Su-Yun; Wang, Chao; Zhao, Xiao-Yun; Hao, Hong-Ling

    2014-06-01

    This study was aimed to investigate the effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of human acute myeloid leukemia cell line HL-60 and its mechanism. HL-60 cells were cultured with different concentrations of celecoxib for 24 h. Cell proliferation was analyzed by CCK-8 assay, cell apoptosis and cell cycle distribution were detected by flow cytometry. Cyclin D1, cyclin E1 and COX-2 mRNA expressions were determined by RT-PCR. The results showed that after the HL-60 cells were treated with different concentrations of celecoxib for 24 h, the cell growth was significantly inhibited in a dose-dependent manner(r = 0.955), IC50 was 63.037 µmol/L of celecoxib. Celecoxib could effectively induce apoptosis in HL-60 cells also in dose-dependent manner(r = 0.988), blocked the HL-60 cells in the G0/G1 phase. The expression of cyclin D1, cyclin E1 and COX-2 mRNA were downregulated. It is concluded that celecoxib can inhibit the proliferation of HL-60 cells in dose-dependent manner, celecoxib causes cell G0/G1 arrest and induces cell apoptosis possibly through down-regulation of the cyclin D1 and cyclin E1 expression, and down-regulation of COX-2 expression respectively.

  15. Raman spectrum reveals Mesenchymal stem cells inhibiting HL60 cells growth

    Science.gov (United States)

    Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; Lu, Xiaoxu; Tian, Jindong; Fan, Jinping; Zhong, Liyun

    2017-04-01

    Though some research results reveals that Mesenchymal stem cells (MSCs) have the ability of inhibiting tumor cells proliferation, it remains controversial about the precise interaction mechanism during MSCs and tumor cells co-culture. In this study, combing Raman spectroscopic data and principle component analysis (PCA), the biochemical changes of MSCs or Human promyelocytic leukemia (HL60) cells during their co-culture were presented. The obtained results showed that some main Raman peaks of HL60 assigned to nucleic acids or proteins were greatly higher in intensity in the late stage of co-culture than those in the early stage of co-culture while they were still lower relative to the control group, implicating that the effect of MSCs inhibiting HL60 proliferation appeared in the early stage but gradually lost the inhibiting ability in the late stage of co-culture. Moreover, some other peaks of HL60 assigned to proteins were decreased in intensity in the early stage of co-culture relative to the control group but rebounded to the level similar to the control group in the late stage, showing that the content and structure changes of these proteins might be generated in the early stage but returned to the original state in the late stage of co-culture. As a result, in the early stage of MSCs-HL60 co-culture, along with the level of Akt phosphorylation of HL60 was lowered relative to its control group, the proliferation rate of HL60 cells was decreased. And in the late stage of co-culture, along with the level of Akt phosphorylation was rebounded, the reverse transfer of Raman peaks within 875-880 cm- 1 appeared, thus MSCs lost the ability to inhibit HL60 growth and HL60 proliferation was increased. In addition, it was observed that the peak at 811 cm- 1, which is a marker of RNA, was higher in intensity in the late stage than that in the control group, indicating that MSCs might be differentiated into myofibroblast-like MSCs. In addition, PCA results also exhibited

  16. [Differentiation of human promyelocytic leukemia HL-60 cells induced by proanthocyanidin and its mechanism].

    Science.gov (United States)

    Xie, Zhao-Yang; Wu, Bin-Hua; Yang, Zhi-Gang; Chen, Xiao-Fang; Chen, Qiu-Shen

    2013-08-01

    This study was purposed to investigate the proliferation, differentiation and apoptosis of human promyelocytic leukemia HL-60 cells induced by proanthocyanidin (PAC). HL-60 cells were incubated with 20 mg/L PAC for 24 h, the cell growth was evaluated by CCK-8 assay. the effect of PAC on HL-60 cells was evaluated and the cells morphology was observed by optical microscopy. Expression of CD14 and CD11b, and cell cycle were analyzed by flow cytometry. The results showed that the growth of HL-60 cells was inhibited after treatment with PAC of different concentration in a dose-dependent manner (P HL-60 cells with inhibition ratio (72.3 ± 1.8)% for 24 h. Microscopy displayed that some cells differentiated to relative mature cells after treating for 48 h. Expression of CD14 increased and the expression of CD11b increased a little after treating with 20 mg/L PAC for 24 h, the ratio of cells in G0/G1 phase increased, but the ratio of cells in S phase decreased. The mRNA and protein expression of P21 gene increased, but the protein expression of CDK4 and Cyclin D1 decreased. It is concluded that PAC may inhibit the proliferation of HL-60 cells in vitro, induces the differentiation of HL-60 cells, and arrests the cells in G0/G1 phase. The possible mechanism may be related to up-regulation of P21 gene expression and down-regulation of the protein expression of CDK4 and Cyclin D1.

  17. Knockdown of nucleophosmin by RNA interference reverses multidrug resistance in resistant leukemic HL-60 cells.

    Science.gov (United States)

    Lin, Minhui; Hu, Jianda; Liu, Tingbo; Li, Jing; Chen, Buyuan; Chen, Xinji

    2013-09-01

    Nucleophosmin, a multifunctional nucleolar phosphoprotein, is involved in many cellular activities. However, the role of NPM in drug-resistance of leukemia has not yet been explored. We designed and selected one shRNA targeting on NPM gene transduction into HL-60 and HL-60/ADR cell lines (an adriamycin resistant cell line) by lentivirus. Cell proliferation, apoptosis and differentiation were assessed. The expressions of the related genes and proteins were detected by real-time quantitative RT-PCR and Western blotting. The results showed obvious down-regulation of NPM mRNA and protein levels after NPM RNAi. NPM-targeted RNAi also resulted in many cellular changes, such as, suppressing cell proliferation and inducing cell differentiation. Down-regulation of NPM gene could arrest the cell cycle progression, an increase in the proportion of G0/G1 phase in knockdown groups. NPM gene silencing could also induce pro-apoptotic genes and proteins expression, and inhibit anti-apoptotic genes/proteins expression. Furthermore, IC50 of two chemotherapeutic agents (adriamycin and ADR; daunorubicin and DNR) to HL-60 and HL-60/ADR cells decreased, especially more remarkable on HL-60/ADR cells. IC50 of ADR on HL-60/ADR cells was reduced from 12.544 ± 0.851 μmol/L (before NPM RNAi) to 6.331 ± 0.522 μmol/L (after NPM RNAi), IC50 of DNR was reduced from 2.152 ± 0.143 μmol/L (before NPM RNAi) to 1.116 ± 0.093 μmol/L (after NPM RNAi). The relative reversal rate of HL-60/ADR cells on ADR was 50.2%, and on DNR was 48.9%. In conclusion, our results demonstrated that shRNA expression vectors could effectively reduce NPM expression and restore the drug sensitivity of resistant leukemic cells to conventional chemotherapeutic agents.

  18. [Reversing effects of emodin on multidrug resistance in resistant HL-60/ADR cells].

    Science.gov (United States)

    Chen, Ying-Yu; Li, Jing; Hu, Jian-Da; Zheng, Jing; Zheng, Zhi-Hong; Zhu, Liang-Fang; Chen, Xin-Ji; Lin, Zhen-Xing

    2013-12-01

    This study was aimed to investigate the reversing effects of emodin on multidrug resistance (MDR) in resistant HL-60/ADR cells, and to explore the underlying mechanisms. The MTT assay was used to assess the chemoresistance of HL-60/ADR cells to emodin and 8 chemotherapeutic agents commonly used in clinic. The reversal effects of emodin on MDR of HL-60/ADR cells were also evaluated by MTT method. DNA ploidy analysis and DNA Ladder assay were used to detect apoptosis-induced effects on HL-60/ADR cells via the adriamycin (ADR) and emodin combination. The expression changes of the drug resistance-associated genes and proteins were detected by RT-PCR and Western Blot respectively. The intracellular accumulation and subcellular distribution of ADR and DNR were measured by flow cytometry and confocal laser scanning microscopy. The results showed that emodin inhibited HL-60/ADR cell proliferation with an average IC50 value of 24.09 ± 1.72 µmol/L, which was similar to that of the parental HL-60 cells (average IC50 = 23.18 ± 0.87 µmol/L). HL-60/ADR cells were resistant to a variety of chemotherapeutic agents, such as ADR, DNR, VP16, VCR,Ara-C, HHT, MTZ and THP. The reversal multiple were between 1.58 and 4.12 after the treatment with low concentration of emodin combined with the above mentioned different agents. The combination of ADR with emodin showed the best reversal effects, and the typical hypodiploid peak (apoptotic peak) and DNA ladder could be detected after the co-treatment.In addition, emodin down-regulated the mRNA and protein expression levels of MRP1, TOPOIIβ, GST π and BCL-2. Furthermore, the addition of emodin enhanced ADR and DNR intracellular accumulation and subcellular distribution in HL-60/ADR cells in dose-dependent manner. It is concluded that the emodin shows reversing effects on the multidrug resistant HL-60/ADR cells, possibly via decreasing the expression levels of drug resistance-associated genes, increasing the intracellular accumulation of

  19. ANTI-PROLIFERATION EFFECT OF ORIDONIN ON HL-60 CELLS AND ITS MECHANISM

    Institute of Scientific and Technical Information of China (English)

    Jia-jun Liu; Xin-yao Wu; Hui-ling Lu; Xiang-lin Pan; Jun Peng; Ren-wei Huang

    2004-01-01

    Objective To investigate the anti-proliferation effect of oridonin on leukemic HL-60 cells and its mechanism.Methods HL-60 cells in vitro in culture medium were given different concentrations oforidonin. The inhibitory rate of cells were measured by microculture tetrazolium (MTT) assay, cell apoptotic rate was detected by flow cytometry (FCM),morphology of cell apoptosis was observed by hoechst 33258 fluorescence staining, and the activity of telomerase was detected using telomere repeat amplification protocol (TRAP) PCR-ELISA before and after apoptosis occurred.Results Oridonin could decrease telomerase activity, inhibit growth of HL-60 cells, and cause apoptosis significantly.The suppression was both in time- and dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by hoechst 33258 fluorescence staining especially after cells were treated 48-60 hours by oridonin.Conclusions Oridonin has apparent anti-proliferation and apoptotic effects on HL-60 cells in vitro, decreasing telomerase activity of HL-60 cells may be one of its most important mechanisms. These results will provide strong laboratory evidence of oridonin for clinical treatment of acute leukemia.

  20. Rare Coumarins Induce Apoptosis, G1 Cell Block and Reduce RNA Content in HL60 Cells

    Directory of Open Access Journals (Sweden)

    Widelski Jarosław

    2017-02-01

    Full Text Available The rare coumarins stenocarpin, stenocarpin isobutyrate, oficinalin, oficinalin isobutyrate, 8-methoxypeucedanin and the known xanthotoxin, isoimperatorin, bergapten, peucedanin and 8–methoxyisoimperatorin were isolated from Peucedanum luxurians Tamamsch. (Apiaceae and identified by means of spectral data (1D and 2D NMR. Their immunomodulating activity was evaluated by flow cytometry and their influence on HL60 cells as well as on PHA-stimulated PBLs was tested. All tested coumarins induce apoptosis (maximal in the 48 h culture and decrease cell proliferation in a time- and dose-dependent manner, especially in HL60 cells. They also induce partial G1 block, but only in HL60 cells (at 100 µM concentrations. Dose-dependent reduction of RNA content was also found in G1 cells treated by the coumarins. All of the tested coumarins also possessed immunomodulatory activities. Bergapten and xanthotoxin were found to be the best candidates for further evaluation as anti-cancer drugs.

  1. EFFECTS OF CURCUMIN ON PROLIFERATION AND APOPTOSIS IN ACUTE MYELOID LEUKEMIA CELLS HL-60

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To investigate the curcumin killing leukemia cells in vitro,. Methods: The myeloid leukemic cell line HL-60 was studied by using cell culture, flow cytometrydetermining DNA content and TUNEL method measuring apoptotic cell percentage. Results: The data showed that curcumin selectively inhibited proliferation of acute myeloid leukemia (AML) HL-60 cell lines in a dose- and time-dependent manner. The growth inhibition rate was gradually increased and reached the peak at concentration of 25 m mol/L curcumin at 24h. The sub-G1 peak appeared after 12h treatment and was increased to 34.4% at 24h. The TUNEL method further certified that apoptotic cells reached 41% at the same phase. Conclusion: curcumin possesses obvious potent of anti-leukemia cell proliferation, which is contributed to the induction of HL-60 cells apoptosis. The concentration and action time of curcumin in vitro provide some reference for clinical use.

  2. OPTIMIZATIONS FOR 5-AMINOLEVULINIC ACID BASED PHOTODYNAMIC THERAPY IN PURGING LEUKEMIA CELL HL60

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To optimize experimental parameters for the photosensitization of 5-aminolevulinic acid (ALA) in promyelocytic leukemia cell HL60 and compare them with normal human peripheral blood mononuclear cell (PBMC). Methods ALA incubation time, wavelength applied to irradiate, concentration of ALA incubated, irradiation fluence may modulate the effect of 5-aminolevulinic acid based Photodynamic Therapy (ALA-PDT).The high-pressure mercury lamps of 400W served as light source, the interference filter of 410nm, 432nm, 545nm, 577nm were used to select the specific wavelength. Fluorescence microscope was used to detect the fluorescence intensity and location of protoporphyrin IX (PpIX) endogenously produced by ALA. MTT assay was used to measure the survival of cell. Flow cytometry with ANNEXIN V FITC kit (contains annexin V FITC, binding buffer and PI) was used to detect the mode of cell death. Results ① 1mmol/L ALA incubated 1×105/mL HL60 cell line for 4 hours, the maximum fluorescence of ALA induced PpIX was detected in cytomembrane. ② Irradiated with 410nm for 14.4J/cm2 can result in the minimum survivability of HL60 cell. ③ The main mode of HL60 cell death caused by ALA-PDT is necrosis. Conclusion ALA for 1mmol/L, 4 hours for dark incubation time, 410nm for irradiation wavelength, 14.4J/cm2 for irradiation fluence were the optimal parameters to selectively eliminate promyelocytic leukemia cell HL60 by ALA based PDT. The photosensitization of ALA based PDT caused the necrosis of HL60 cell, so it could be used for inactivation of certain leukemia cells.

  3. Growth inhibition and differentiation of promyelocytic cells (HL-60) induced by BC-4, an active principle from Boswellia carterii Birdw.

    Science.gov (United States)

    Jing, Y; Xia, L; Han, R

    1992-03-01

    The induction of cell differentiation and growth inhibition of HL-60 cells by Bc-4, an active principle from Boswellia carterii, was studied in vitro and in vivo. The proliferation of HL-60 cells was found to be inhibited by Bc-4 at a concentration of 5-10 micrograms/ml. Under the action of Bc-4, the acid phosphatase and NBT reduction activities in HL-60 cells were increased, and phagocytosis of cells was also induced. All these activities were concentration dependent. The HL-60 cells were induced by Bc-4 to differentiate into more mature cells morphologically. The in vivo growth of HL-60 cells in mouse subrenal capsules (SRC) and in diffusion chambers inoculated into mice was inhibited by Bc-4 at a dose of 50 mg/kg. The morphology of HL-60 cells treated by Bc-4 in diffusion chambers exhibited characteristics of mature granulocytic cells.

  4. NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

    Science.gov (United States)

    Hu, Xiu-Xiu; Zhong, Liang; Zhang, Xi; Gao, Yuan-Mei; Liu, Bei-Zhong

    2014-01-01

    A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The

  5. Pinoresinol inhibits proliferation and induces differentiation on human HL60 leukemia cells.

    Science.gov (United States)

    Sepporta, Maria Vittoria; Mazza, Teresa; Morozzi, Guido; Fabiani, Roberto

    2013-01-01

    Pinoresinol (PIN), one of the simplest lignans, is the precursor of other dietary lignans that are present in whole-grain cereals, legumes, fruits, and other vegetables. Several experimental and epidemiological evidences suggest that lignans may prevent human cancer in different organs. In this study we investigated the chemopreventive properties of PIN on cell lines derived from different sites either expressing or not the functional tumor suppressor protein p53. It was found that PIN inhibited the proliferation of p53 wild type colon and prostate tumor cells (HCT116 and LNCaP) while in breast cells the inhibition of growth was observed only in p53 mutant cells (MDA-MB-231). A potent antiproliferative activity of PIN was also observed on p53 null cells HL60 (IC50% 8 μM), their multidrug resistant variant HL60R (IC50% 32 μM) and K562. On HL60 cells, PIN caused a block of cell cycle in the G0/G1 phase, induced a weak proapoptotic effect but it was a good trigger of differentiation (NBT reduction and CD11b expression). PIN caused an upregulation of the CDK inhibitor p21(WAF1/Cip1) both at mRNA and protein levels so suggesting that this could be a mechanism by which PIN reduced proliferation and induced differentiation on HL60 cells.

  6. Enhancement of cisplatin cytotoxicity by benzyl isothiocyanate in HL-60 cells.

    Science.gov (United States)

    Lee, Younghyun; Kim, Yang Jee; Choi, Young Joo; Lee, Joong Won; Lee, Sunyeong; Chung, Hai Won

    2012-07-01

    Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most widely used chemotherapeutic drugs, but its effectiveness is limited by tumor cell resistance and the severe side effects it causes. One strategy for overcoming this problem is the concomitant use of natural dietary compounds as therapeutic agents. Benzyl isothiocyanate (BITC) is a promising chemopreventive agent found in cruciferous vegetables and papaya fruits. The aim of this study was to investigate the effects of BITC on cisplatin-induced cytotoxicity in human promyelocytic leukemia cells and normal human lymphocytes. The combined treatment of HL-60 cells with BITC followed by cisplatin (BITC/cisplatin) caused a significant decrease in cell viability. BITC also increased apoptotic cell death compared to cisplatin treatment alone. In normal human lymphocytes, BITC did not enhance the cytotoxic effects of cisplatin. Cellular exposure to BITC/cisplatin increased reactive oxygen species (ROS) generation but decreased the total glutathione (GSH) level in HL-60 cells. Pretreatment of HL-60 cells with N-acetylcysteine or glutathione monoethyl ester effectively decreased BITC/cisplatin-induced cell death. The addition of the extracellular signal-regulated kinase (ERK) inhibitor PD98059 abolished BITC/cisplatin-induced apoptosis. Taken together, our results suggest that BITC enhances cisplatin-induced cytotoxicity through the generation of ROS, depletion of GSH, and ERK signaling in HL-60 cells.

  7. TIEG1对HL-60细胞凋亡及Bcl-2/Bax表达的影响%Influence of TIEG1 on Apoptosis of HL-60 Cells and Expression of Bcl-2/Bax

    Institute of Scientific and Technical Information of China (English)

    姚鲲; 杨莹; 胡荣; 苗苗; 廖爱军; 杨威; 刘卓刚

    2013-01-01

    This study was aimed to investigate the influence of TIEG1 on apoptosis of HL-60 cells and the expression of Bcl-2/Bax.Different concentration of TIEG1 were used to treat HL-60 cells,the cell growth inhibition rate was detected by MTT method.After treating HL-60 cells with 12.03 ng/ml TIEG1,cell apoptosis was detected with flow cytometry.Bcl-2 and Bax was detected with RT-PCR.The results showed that TIEG1 had inhibitory effect on HL-60 cell proliferation,and in time-and dose-dependent manners.The more obvious inhibitory effect was observed in HL-60 cells treated with TIEG1 of 12.03 ng/ml.During the course of cell apoptosis,Bax expression increasied,but Bcl-2 expression decreased(P < 0.05).It is concluded that TIEG1 inhibits HL-60 cell proliferation and induces apoptosis in time and dose-dependent manners.During the course of HL-60 cells apoptosis induced by TIEG1,Bcl-2/Bax are assosciated with HL-60 cell apoptosis induced by TIEG1.%本研究旨在观察TIEG1对HL-60细胞凋亡及Bcl-2/Bax表达的影响.用不同浓度的TIEG1处理HL-60细胞,采用MTT法检测细胞生长抑制率.用12.03 ng/ml TIEG1处理HL-60细胞,以流式细胞术检测细胞凋亡,以RT-PCR方法检测Bcl-2及Bax的表达变化.结果表明,TIEG1对HL-60细胞具有增殖抑制作用,其抑制效应呈时间及剂量依赖性.TIEG1 12.03 ng/ml增殖抑制作用较为明显.在细胞凋亡过程中,Bax表达呈升高趋势,而Bcl-2呈下降趋势(P<0.05).结论:TIEG1可以抑制HL-60细胞增殖,进而诱导其凋亡,且呈时间、剂量依赖性.在TIEG1诱导HL-60细胞凋亡的过程中,Bcl-2/Bax与TIEG1诱导HL-60细胞凋亡相关.

  8. Retinoic acid therapy resistance progresses from unilineage to bilineage in HL-60 leukemic blasts.

    Directory of Open Access Journals (Sweden)

    Holly A Jensen

    Full Text Available Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA is the standard therapeutic agent for acute promyelocytic leukemia, but 10-20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2 HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17-negative cells. Wild-type (WT HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3. Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or "precommitment" phase or subsequently (the late or "lineage-commitment" phase. HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness

  9. [Effects of baicalin on HL-60 cell xenografts in nude mice and its mechanism].

    Science.gov (United States)

    Zheng, Jing; Hu, Jian-Da; Huang, Yi; Chen, Ying-Yu; Li, Jing; Chen, Bu-Yuan

    2012-10-01

    This study was aimed to investigate the effects of baicalin on HL-60 cell xenografts in nude mice in vivo and explore its mechanism. Xenograft tumor model of HL-60 cells in nude mice was established, which was divided randomly into 6 groups: negative control group (injection of 5% NaHCO(3)), 25, 50 and 100 mg/kg baicalin groups, combination group (50 mg/kg baicalin + 2 mg/kg VP16) and positive control group (VP16 4 mg/kg). The nude mice with HL-60 cell xenografts were treated with drugs via intraperitoneal injection daily. After treatment for 14 days average weigh and inhibitory rate of transplanted tumor stripped from 5 nude mice in each group were calculated, and the ultrastructure change of xenografts cells were tested by transmission electron microscopy. Histopathologic examination was used to observed the change of main organs in nude mice. The expression of signaling molecular PI3K/Akt proteins extracted from xenografts was detected by Western blot. The effects of baicalin on overall survival time in nude mice with HL-60 cell xenografts were evaluated. The results showed that baicalin could inhibit the growth of transplanted tumors in dose-dependent manner. There were more necrotic and apoptotic cells in mice of baicalin-treated groups and combination group than that in mice of negative control group. Baicalin could inhibit the proliferation of HL-60 cells in vivo by down-regulating the PI3K/Akt/mTOR signal pathway, where the expressions of p-Akt, mTOR and p-mTOR proteins decreased compared with negative control group, and no significant difference of Akt expression was found between different groups. Compared with negative control group, the median survival time of mice in combination group was more prolongated (P HL-60 cell xenografts in nude mice, and prolong median survival time of nude mice. The possible mechanisms may be related to inhibition of Akt activity and down-regulation of the PI3K/Akt/mTOR signal pathway. The combination of baicalin and VP16

  10. Retinoic acid therapy resistance progresses from unilineage to bilineage in HL-60 leukemic blasts.

    Science.gov (United States)

    Jensen, Holly A; Bunaciu, Rodica P; Ibabao, Christopher N; Myers, Rebecca; Varner, Jeffrey D; Yen, Andrew

    2014-01-01

    Emergent resistance can be progressive and driven by global signaling aberrations. All-trans retinoic acid (RA) is the standard therapeutic agent for acute promyelocytic leukemia, but 10-20% of patients are not responsive, and initially responsive patients relapse and develop retinoic acid resistance. The patient-derived, lineage-bipotent acute myeloblastic leukemia (FAB M2) HL-60 cell line is a potent tool for characterizing differentiation-induction therapy responsiveness and resistance in t(15;17)-negative cells. Wild-type (WT) HL-60 cells undergo RA-induced granulocytic differentiation, or monocytic differentiation in response to 1,25-dihydroxyvitamin D3 (D3). Two sequentially emergent RA-resistant HL-60 cell lines, R38+ and R38-, distinguishable by RA-inducible CD38 expression, do not arrest in G1/G0 and fail to upregulate CD11b and the myeloid-associated signaling factors Vav1, c-Cbl, Lyn, Fgr, and c-Raf after RA treatment. Here, we show that the R38+ and R38- HL-60 cell lines display a progressive reduced response to D3-induced differentiation therapy. Exploiting the biphasic dynamic of induced HL-60 differentiation, we examined if resistance-related defects occurred during the first 24 h (the early or "precommitment" phase) or subsequently (the late or "lineage-commitment" phase). HL-60 were treated with RA or D3 for 24 h, washed and retreated with either the same, different, or no differentiation agent. Using flow cytometry, D3 was able to induce CD38, CD11b and CD14 expression, and G1/G0 arrest when present during the lineage-commitment stage in R38+ cells, and to a lesser degree in R38- cells. Clustering analysis of cytometry and quantified Western blot data indicated that WT, R38+ and R38- HL-60 cells exhibited decreasing correlation between phenotypic markers and signaling factor expression. Thus differentiation induction therapy resistance can develop in stages, with initial partial RA resistance and moderate vitamin D3 responsiveness (unilineage

  11. Pycnogenol induces differentiation and apoptosis in human promyeloid leukemia HL-60 cells.

    Science.gov (United States)

    Huang, W W; Yang, J S; Lin, C F; Ho, W J; Lee, M R

    2005-06-01

    Pycnogenol, rich of many phytochemicals of medical value, is a commercialized nutrient supplement extracted from the bark of European coastal pine. In this study, we investigated the anti-tumor effects of Pycnogenol on HL-60, U937 and K562 human leukemia cell lines. We found that Pycnogenol inhibited cell proliferation dose- and time-dependently, and the IC(50)s of Pycnogenol on HL-60, U937 and K562 cells were 150, 40 and 100 microg/ml, respectively. When HL-60 cells were incubated with low concentrations of Pycnogenol (50, 100 and 125 microg/ml) for 24 h, a prominent G0/G1 arrest was observed, followed by gradual accumulation of sub-G0/G1 nuclei. At 48 h of treatment, 50-70% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, induction of NSE activity, and increases of cell surface expression of CD11b. However, results from Annexin V/PI staining, DAPI staining and DNA fragmentation assay indicated that Pycnogenol induced HL-60, U937 and K562 cell apoptosis at their respective IC(50)s after 24 h of treatments. Pretreatment of z-DEVD-fmk, a caspase-3 specific inhibitor, not only decreased caspase-3 activity but also reduced the percentage of apoptotic cells induced by Pycnogenol. This indicated that caspase-3 activation was involved in Pycnogenol induced-apoptosis. In conclusion, Pycnogenol induced differentiation and apoptosis in leukemia cells. Our data suggest that Pycnogenol could serve as a potent cancer chemopreventive or chemotherapeutic agent for human leukemia.

  12. The role of AMP-activated protein kinase in quercetin-induced apoptosis of HL-60 cells.

    Science.gov (United States)

    Xiao, Jie; Niu, Guomin; Yin, Songmei; Xie, Shuangfeng; Li, Yiqing; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan

    2014-05-01

    Our previous studies have shown that quercetin inhibits Cox-2 and Bcl-2 expressions, and induces human leukemia HL-60 cell apoptosis. In order to investigate the role of AMP-activated protein kinase (AMPK) on quercetin-induced apoptosis of HL-60 cells, we used flow cytometry to detect cell apoptosis. The expressions of LKB1, phosphorylated AMPK (p-AMPK), and Cox-2 protein were detected in HL-60 cells and normal peripheral blood mononuclear cells (PBMCs) by western blot. The expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin. The expressions of p-AMPK were detected in HL-60 cells after culture with AMPK inhibitor Compound C. Then, the expressions of LKB1, p-AMPK, and Cox-2 were detected in HL-60 cells after culture with quercetin alone or quercetin + Compound C. It was found that there was no significant difference in LKB1 between PBMCs and HL-60. p-AMPK in PBMCs was higher than that in HL-60, while Cox-2 was lower. After culture of HL-60 with quercetin, p-AMPK was increased, Cox-2 was decreased, but LKB1 remained unchanged. After culture of HL-60 with Compound C, p-AMPK was decreased. There was no significant difference in LKB1 between the quercetin-alone and the quercetin + Compound C groups. p-AMPK decreased more significantly, while Cox-2 increased more significantly in the quercetin + Compound C groups than those in the quercetin-alone groups. Taken together, these findings suggested that quercetin activates AMPK expression in HL-60 cells independent of LKB1 activation, inhibits Cox-2 expression by activating AMPK, and further regulates the Bcl-2-dependent pathways of apoptosis to exert its anti-leukemia effect.

  13. Affection of Boswellia Carterii Birdw on Telomerase Activityexpression in HL60 Cells%乳香提取物对HL60细胞端粒酶活性影响的研究

    Institute of Scientific and Technical Information of China (English)

    齐振华; 谭柏林; 彭旭阳; 张国平

    2000-01-01

    为研究乳香提取物对HL60细胞端粒酶活性影响,应用端粒酶重复序列扩增技术及聚丙烯胺凝胶电泳对HL60细胞端粒酶活性的表达进行检测,并应用乳香提取物及全反式维甲酸下调HL60细胞端粒酶活性.结果乳香提取物能降低HL60细胞端粒酶活性.其降低HL60细胞端粒酶活性作用与全反式维甲酸相似.

  14. Effect of 8-Chloroadenosine on Undifferentiatied HL-60 Cell Line%8-氯腺苷对未分化HL-60细胞增殖的影响(图片更正)

    Institute of Scientific and Technical Information of China (English)

    崔景荣; 惠峪; 相幼青; 张礼和

    2004-01-01

    Aim To study the effect of 8-chloroadenosine (8-CA)on undifferentiatied HL-60 cell line.Methods The IC50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB).Morphology of HL-60 cells was observed under a scanning electron microscope and a transmission electron microscope.The differentiation of HL-60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay.The cycle of HL-60 cells was analyzed by flow cytometry.Results 8-CA inhibited proliferation of eight human cancer cell lines.The IC50 ranked in the following order: KB (0.05 μmol·L-1) < HL-60 (0.25 μmol·L-1) < Bel-7402 (0.56 μmol·L-1) < MCF-7 (0.65 μmol·L-1) < HCT (0.79 μmol·L-1) < HeLa (0.89 μmol·L-1) < BGC-823 (1.149 μmol·L-1) < PG (2.50 μmol·L-1).The scanning and transmission electron microscopy showed that the microvilli of HL-60 cell surface shortened, and the shape of HL-60 cells nuclei changed to kidney-shaped, horse shoe-shaped and bilob ated after treatment with 8-CA.Meanwhile, 8-CA promoted NBT reduction and increased activity of acid phosphatase in HL-60 cells in a time and concentration-dependent manner.Flow cytometry analysis indicated that 8-CA induced an appreciable increase of the cell population in G1 phase with a marked reduction in S phase.Conclusion 8-CA can induce differentiation of HL-60 cells and block the cells at G1 phase, thus inhibiting proliferation of HL-60 cells.

  15. Homogeneously staining region in anthracycline-resistant HL-60/AR cells not associated with MDR1 amplification.

    Science.gov (United States)

    Gervasoni, J E; Taub, R N; Yu, M T; Warburton, D; Sabbath, M; Gilleran, S; Coppock, D L; D'Alessandri, J; Krishna, S; Rosado, M

    1992-10-01

    Anthracycline-resistant HL-60/AR cells and their drug-sensitive HL-60/S counterparts were characterized by karyotypic analysis and examined for the overexpression of DNA and mRNA sequences coding for P-glycoprotein (Pgp). The HL-60/S cells were karyotypically stable over a 5-year period of study (1986-1991), except for an additional small Giemsa-positive band noted at 7q22 in cultures harvested in 1987, but not in 1986. This change did not affect drug sensitivity. The drug-resistant HL-60/AR cells examined in 1986, 1987, and 1991 demonstrated a very stable karyotype. The most striking feature was a large homogeneously staining region in the long arm of chromosome 7 (7q11.2), and translocation of the remainder of the long arm to another centromere. Other changes in the HL-60/AR cells included inversion in 9q, partial deletion of the short arm of chromosome 10p, addition of material to the p arm of der(16), loss of chromosome 22, and the appearance of a new marker chromosome. Both HL-60/S and the HL-60/AR cells were found not to amplify DNA or mRNA sequences coding for the Pgp. Thus, although the HL-60/AR cells possess the classical multidrug resistance phenotype and demonstrate a homogeneously staining region near the region of the MDR1 gene, their resistance is due to mechanisms other than those coded for by MDR1.

  16. [Effect of overexpression of CAV1 mediated by lentivirus on proliferation and apoptosis of HL-60 cells].

    Science.gov (United States)

    Ma, Wei; Wang, Di-Di; Wang, Zhao; Zhu, Gui-Ming; Zhang, Peng-Xia

    2013-08-01

    This study was purposed to explore the effect of lentivirus-mediated CAV1 overexpression on proliferation and apoptosis in HL-60 cells. Recombinant lentiviral expression vector pcDNA-EF1-CAV1 was constructed, and cotransfected the 293TN cells with a mixture of pPACK packaging plasmids. Then collecting virus suspension infects the HL-60 cells, which make CAV1 gene stable transfection and high expression in the cells. The CAV1 protein expression status in HL-60 cells transfected was evaluated through Western blot method. Proliferative activity and apoptosis of HL-60 cells before and after transfection were detected by CCK-8 method and flow cytometry, respectively. The results showed that the PCR-positive clone screening and results of nucleotide sequencing confirmed that the CAV1 gene inserted into the expression vector pcDNA-EF1-GFP correctly, recombinant lentiviral particles Lv-CAV1 transfected HL-60 cells successfully and with transfection rate up to 90%. The result of Western blot showed that CAV1 protein expression in HL-60 cells significantly increased at 48 hours after transfection. CCK-8 result indicated that cell proliferation activity increased at 48 h after transfection (P HL-60 cells obviously decreased after transfection (P HL-60 cells can inhibit cell proliferation activity and promote cell apoptosis.

  17. Caveolin-1 plays a key role in the oleanolic acid-induced apoptosis of HL-60 cells.

    Science.gov (United States)

    Ma, Wei; Wang, Di-Di; Li, Li; Feng, Yu-Kuan; Gu, Hong-Mei; Zhu, Gui-Ming; Piao, Jin-Hua; Yang, Yu; Gao, Xu; Zhang, Peng-Xia

    2014-07-01

    Our previous study found that caveolin-1 (CAV-1) protein expression is upregulated during oleanolic acid (OA)-induced inhibition of proliferation and promotion of apoptosis in HL-60 cells. CAV-1 is the main structural protein component of caveolae, playing important roles in tumorigenesis and tumor development. It has been shown that cav-1 expression is lower in leukemia cancer cell lines SUP-B15, HL-60, THP-1 and K562 and in chronic lymphocytic leukemia primary (CLP) cells when compared with normal white blood cells, with the lowest cav-1 expression level found in HL-60 cells. To study the effects of cav-1 in HL-60 cells and the effects of cav-1 overexpression on OA drug efficacy, cav-1 was overexpressed in HL-60 cells using lentiviral-mediated transfection combined with OA treatment. The results showed that cav-1 overexpression inhibited HL-60 cell proliferation, promoted apoptosis, arrested the cell cycle in the G1 phase and inhibited activation of the PI3K/AKT/mTOR signaling pathway. Overexpression of CAV-1 also increased HL-60 cell sensitivity to OA. To further verify whether OA affects HL-60 cells via the activation of downstream signaling pathways by CAV-1, cav-1 gene expression was silenced using RNAi, and the cells were treated with OA to examine its efficacy. The results showed that after cav-1 silencing, OA had little effect on cell activity, apoptosis, the cell cycle and phosphorylation of HL-60 cells. This study is the first to show that CAV-1 plays a crucial role in the effects of OA on HL-60 cells.

  18. Targeting thioredoxin reductase by plumbagin contributes to inducing apoptosis of HL-60 cells.

    Science.gov (United States)

    Zhang, Junmin; Peng, Shoujiao; Li, Xinming; Liu, Ruijuan; Han, Xiao; Fang, Jianguo

    2017-04-01

    Plumbagin (PLB), a natural naphthoquinone from the traditional folk medicines Plumbago zeylanica, Dionaea muscipula, or Nepenthes gracilis, has been documented possessing a wide variety of pharmacological activities. Although PLB demonstrates anticancer activity in multiple types of malignant cells, the cellular targets of PLB have not been well defined and remained only partially understood. We reported here that PLB selectively inhibits TrxR and elicits reactive oxygen species in human promyelocytic leukemia HL-60 cells, which leads to elevation of GSSG/GSH ratio and decrease of cellular thiol pool. As a consequence, PLB disturbs the cellular redox homeostasis, induces oxidative stress-mediated apoptosis and eventually selectively kills HL-60 cells. Inhibition of TrxR by PLB thus discloses an unprecedented mechanism underlying the anticancer efficacy of PLB, and sheds light in considering the usage of PLB as a promising cancer therapeutic agent. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. THE EFFECTS OF LOW CONCENTRATIONS OF ZINC ON ETOPOSIDE INDUCED HL-60 CELLS APOPTOSIS

    Institute of Scientific and Technical Information of China (English)

    盛晓阳; 沈立松; 徐翀; 吴湘如; 陈宏新; 洪照毅

    2001-01-01

    Objective Observation of the effects of lower concentrations of zinc (20~400μmol/L), which is nearby the physiological concentration, on etoposide induced HL-60 cells apoptosis. Methods Using the flow cytometry , DNA extraction, electrophoresis , and fiuoresence microscopic observation. Results We demonstrated that the low concentrations of zinc also affect cells apoptosis. If zinc was added at early time, even 200μmol/L could inhibit VP16 induced HL-60 cell apoptosis completely in 4h. But at this concentration, zinc also seems to have cell toxicity, not prolong the time of protection effect. Conclusion Zinc plays multiple roles in cellular functions and in protecting cells from exogenous deleterious factors. Zinc plays a complex, dose- and time-dependent role in apoptosis.

  20. Structure-activity relationship of lysophosphatidylcholines in HL-60 human leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Eun-heeLEE; Mi-ranYUN; Wei-hongWANG; JeeHJUNG; Dong-soonIM

    2004-01-01

    AIM: To explore the structure-activity relationship of lysophosphatidylcholine (LPC) and lysolipid molecules from a marine sponge and ladybirds. METHODS: We tested three synthetic LPCs and four natural lysolipids on Ca2+ mobilization in HL-60 human leukemia cells. RESULTS: We observed lysolipid-mediated Ca2+ mobilization. The activity was the same in both ester-and ether-linked lysolipids, and introduction of a double bond or methoxy group on the alkyl chain did not significantly modulate the activity. However, replacement of trimethylammonium moiety in the choline structure with ammonium moiety reduced the activity. Furthermore, change of the alkyl chain length influenced the Ca2+ response. CONCLUSION: LPC-induced Ca2+ mobilization might be dependent on the length of alkyl chain and the presence of choline moiety in HL-60 leukemia cells.

  1. Structure-activity relationship of lysophosphatidylcholines in HL-60 human leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Eun-hee LEE; Mi-ran YUN; Wei-hong WANG; Jee H JUNG; Dong-soon IM

    2004-01-01

    AIM: To explore the structure-activity relationship of lysophosphatidylcholine (LPC) and lysolipid molecules from a marine sponge and ladybirds. METHODS: We tested three synthetic LPCs and four natural lysolipids on Ca2+mobilization in HL-60 human leukemia cells. RESULTS: We observed lysolipid-mediated Ca2+ mobilization. The activity was the same in both ester- and ether-linked lysolipids, and introduction of a double bond or methoxy group on the alkyl chain did not significantly modulate the activity. However, replacement of trimethylammonium moiety in the choline structure with ammonium moiety reduced the activity. Furthermore, change of the alkyl chain length influenced the Ca2+ response. CONCLUSION: LPC-induced Ca2+ mobilization might be dependent on the length of alkyl chain and the presence of choline moiety in HL-60 leukemia cells.

  2. Apoptosis induction of Persicae Semen extract in human promyelocytic leukemia (HL-60) cells.

    Science.gov (United States)

    Kwon, Hee-Young; Hong, Seon-Pyo; Hahn, Dong-Hoon; Kim, Jeong Hee

    2003-02-01

    The major ingredient of Persicae Semen is a cynogenic compound, amygdalin (D-mandelonitrile-beta-gentiobioside). Controversial results on the anticancer activity of amygdalin were reported due to its conversion to its inactive isomer, neoamygdalin. In order to inhibit the epimerization of amygdalin, we used newly developed simple acid boiling method in preparation of Persicae Semen extract. HPLC analysis revealed most of amygdalin in Persicae Semen extract was active D-form. Persicae Semen extract was used to analyze its effect on cell proliferation and induction of apoptosis in human promyelocytic leukemia (HL-60) cells. Persicae Semen extract was cytotoxic to HL-60 cells with IC50 of 6.4 mg/mL in the presence of 250 nM of beta-glucosidase. The antiproliferative effects of Persicae Semen extract appear to be attributable to its induction of apoptotic cell death, as Persicae Semen extract induced nuclear morphology changes and internucleosomal DNA fragmentation.

  3. Induction of apoptosis by Citrus paradisi essential oil in human leukemic (HL-60) cells.

    Science.gov (United States)

    Hata, Tomona; Sakaguchi, Ikuyo; Mori, Masahiro; Ikeda, Norikazu; Kato, Yoshiko; Minamino, Miki; Watabe, Kazuhito

    2003-01-01

    Limonene is a primary component of citrus essential oils (EOs) and has been reported to induce apoptosis on tumor cells. Little is known about induction of apoptosis by citrus EOs. In this study, we examined induction of apoptosis by Citrus aurantium var. dulcis (sweet orange) EO, Citrus paradisi (grapefruit) EO and Citrus limon (lemon) EO. These EOs induced apoptosis in HL-60 cells and the apoptosis activities were related to the limonene content of the EOs. Moreover, sweet orange EO and grapefruit EO may contain components besides limonene that have apoptotic activity. To identify the components with apoptotic activity, grapefruit EO was fractionated using silica gel columns, and the components were analyzed by GC-MS. The n-hexane fraction contained limonene, and the dichloromethane fraction (DF) contained aldehyde compounds and nootkatone. Decanal, octanal and citral in the DF showed strong apoptotic activity, suggesting that the aldehyde compounds induced apoptosis strongly in HL-60 cells.

  4. Ethanolic rhizome extract from Kaempferia parviflora Wall. ex. Baker induces apoptosis in HL-60 cells.

    Science.gov (United States)

    Banjerdpongchai, Ratana; Suwannachot, Kittiphan; Rattanapanone, Viboon; Sripanidkulchai, Bungorn

    2008-01-01

    Kaempferia parviflora Wall. ex. Baker is a Thai herb containing many flavonoids that have anti-inflammatory, anti-allergic and antioxidant activities. The objective of this study was to demonstrate apoptotic effects of Kaempferia parviflora Wall. ex. Baker rhizome ethanolic extract on HL-60 cells in vitro. The extract suppressed HL-60 cell growth and decreased cell viability in a dose- and time-dependent manner. Apoptotic cell death was demonstrated by changes in cell morphology, externalization of phosphatidylserine on the cell surface, loss in mitochondrial transmembrane potential and activation of caspase 3. Apoptosis induced by K. parviflora Wall. ex. Baker rhizome ethanolic extract was enhanced by treatment with paclitaxel or doxorubicin, and inhibitors of Akt, PI3-K and MEK.

  5. Dose- and Time-Dependent Response of Human Leukemia (HL-60 Cells to Arsenic Trioxide Treatment

    Directory of Open Access Journals (Sweden)

    Paul B. Tchounwou

    2006-06-01

    Full Text Available The treatment of acute promyelocytic leukemia (APL has been based on the administration of all-trans retinoic acid plus anthracycline chemotherapy, which is very effective as first line therapy; however 25 to 30% of patients will relapse with their disease becoming refractory to conventional therapy. Recently, studies have shown arsenic trioxide to be effective in the treatment of acute promyelocytic leukemia. In this study, we used the human leukemia (HL-60 cell line as a model to evaluate the cytoxicity of arsenic trioxide based on the MTT assay. Data obtained from this assay indicated that arsenic trioxide significantly reduced the viability of HL-60 cells, showing LD50 values of 14.26 + 0.5μg/mL, 12.54 + 0.3μg/mL, and 6.4 + 0.6μg/mL upon 6, 12, and 24 hours of exposure, respectively; indicating a dose- and time-dependent response relationship. Findings from the present study indicate that arsenic trioxide is highly cytotoxic to human leukemia (HL-60 cells, supporting its use as an effective therapeutic agent in the management of acute promyelocytic leukemia.

  6. GENE EXPRESSION PROFILING OF HUMAN PROMYELOCYTIC LEUKEMIA HL-60 CELL TREATED BY AJOENE

    Institute of Scientific and Technical Information of China (English)

    方志俊; 黄文秀; 黄明辉; 梁润松; 崔景荣; 王夔; 杨梦苏

    2002-01-01

    Objective: Ajoene, a major compound extracted from crashed garlic, has been shown to have antitumor, antimycotic, antimicrobial, antimutagenic functions in vivo or in vitro and treated as a potential antitumor drug. However, the molecular mechanisms underlying the tumor cytotoxicity of ajoene and even garlic substances are poorly defined. In the present study, we aimed to generate gene expression profiles of HL-60 cell treated by ajoene. Methods: A cDNA microarray presenting 2400 of genes amplified from human leukocyte cDNA library was constructed and the gene expression profiles of HL-60 cell induced by ajoene were generated. Results: After data analysis, 28 differentially expressed genes were identified and sequenced. These genes include 21 known genes and 7 ESTs. Most of the known genes are related to cell apoptosis, such as secretory granule (PRG1), beta-2 microglobulin (B2M), 16S ribosomal RNA gene and ribosomal protein S12. Several genes are related to cell differentiation, including the genes similar to H3 histone and ribosomal protein L31. Northern blot analysis was used to verify and quantify the expression of selected genes. Conclusion: Ajoene can induce HL-60 cell apoptosis significantly and may play a role in differentiation. cDNA microarray technology can be a valuable tool to gain insight into molecular events of pharmacological mechanism of herbal medicine.

  7. Lipid Raft is required for PSGL-1 ligation induced HL-60 cell adhesion on ICAM-1.

    Directory of Open Access Journals (Sweden)

    Tingshuang Xu

    Full Text Available P-selectin glycoprotein ligand-1 (PSGL-1 and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD, we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk, a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.

  8. Endomorphins, endogenous opioid peptides, induce apoptosis in human leukemia HL-60 cells.

    Science.gov (United States)

    Lin, Xin; Chen, Qiang; Xue, Li-Ying; Ma, Xiao-Jun; Wang, Rui

    2004-11-01

    Opioids play a role in the apoptosis machinery. We studied the induction of apoptosis in endomorphin 1 (EM1) and endomorphin 2 (EM2), 2 newly isolated endogenous mu-opioid receptor agonists. These endomorphins were able to reduce the viability of cultured HL-60 cells. The antiproliferative properties of endomorphins appeared to be attributable to their induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 indicated down-regulation, but the Bax, Fas, and FasL expression showed up-regulation as compared with the untreated control cells. These data support the idea that endomorphins induce apoptosis in HL-60 cells through the activation of the Bcl-2-Bax and the Fas-FasL pathway. We suggest that endomorphins may play an important role in the regulation of tumor cell death.

  9. Effect of Bcl-2 and caspase-3 on calcium distribution in apoptosis of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there's a change of intracellular calcium distribution, moving from cytoplast especially Golgi's apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi's apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi's apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspas-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

  10. EGCG抑制HL-60细胞增殖作用的机制研究

    Institute of Scientific and Technical Information of China (English)

    欧阳琳; 姜浩

    2011-01-01

    目的探讨EGCG抑制白血病HL-60细胞增殖的作用机制。方法采用软琼脂集落形成实验和绘制细胞生长曲线,检测EGCG对HL-60细胞增殖的影响;FCM及DNA凝胶电泳法检测EGCG处理HL-60细胞后的凋亡情况;实时荧光定量RT-PCR和Wester blotting检测AKT1的表达。结果细胞生长曲线和软琼脂集落形成实验结果均显示EGCG呈浓度依赖性抑制HL-60细胞的增殖(P〈0.05);FCM及DNA凝胶电泳法结果显示EGCG呈浓度依赖性诱导HL-60细胞的凋亡(P〈0.05)。实时荧光定量RT-PCR和Wester blotting结果显示EGCG浓度升高,HL-60细胞中AKT1的表达降低。结论 EGCG可能通过下调AKT1的表达,促进细胞凋亡,发挥其对HL-60细胞增殖的抑制作用。

  11. Involvement of calreticulin in cell proliferation, invasion and differentiation in diallyl disulfide-treated HL-60 cells.

    Science.gov (United States)

    Yi, Lan; Shan, Jian; Chen, Xin; Li, Guoqing; Li, Linwei; Tan, Hui; Su, Qi

    2016-09-01

    Diallyl disulfide (DADS) has shown potential as a therapeutic agent in various cancers. Previously, calreticulin (CRT) was found to be downregulated in differentiated HL-60 cells treated with DADS. The present study investigated the role of CRT proteins in DADS-induced proliferation, invasion and differentiation in HL-60 cells. The present study demonstrated that DADS treatment significantly changed the morphology of HL-60 cells and caused the significant time-dependent downregulation of CRT. Small interfering RNA (siRNA)-mediated knockdown of CRT expression significantly inhibited proliferation, decreased invasion ability, increased the expression of cluster of differentiation (CD)11b and reduced the expression of CD33 in DADS-treated HL-60 cells. DADS also significantly affected cell proliferation, invasion and differentiation in CRT-overexpressed HL-60 cells. Nitroblue tetrazolium (NBT) reduction assays showed decreased NBT reduction activity in the CRT overexpression group and increased NBT reduction in the CRT siRNA group. Following treatment with DADS, the NBT reduction abilities in all groups were increased. In conclusion, the present study clearly demonstrates the downregulation of CRT during DADS-induced differentiation in HL-60 cells and indicates that CRT is involved in cell proliferation, invasion and differentiation in DADS-treated HL-60 cells.

  12. [Effect of mPGES-1 inhibitor MK886 on apoptosis and drug resistance of HL-60/A cells].

    Science.gov (United States)

    Li, Yi-Qing; Yin, Song-Mei; Nie, Da-Nian; Xie, Shuang-Feng; Ma, Li-Ping; Wang, Xiu-Ju; Wu, Yu-Dan

    2012-08-01

    This study was aimed to investigate the effect of MK886, a mPGES-1 inhibitor, on apoptosis and drug resistance of leukemia HL-60/A cell line. Expression of mPGES-1 was assayed by QT-PCR and Western blot. The effect of MK886 on HL-60/A cell proliferation was assayed by CCK-8 method, and flow cytometry was used to detect cell apoptosis. The expression of Akt and P-Akt was detected by Western blot. PGE2 was measured by ELISA. Effect of MK886 (10 µmol/L) on the chemotherapeutic sensitivity of HL-60/A cells and expression of mdr-1 mRNA and P170 protein were investigated too. The results indicated the expression of mPGES-1 was higher in HL-60/A cells. MK886 inhibited HL-60/A cell proliferation and induced apoptosis in a time- and concentration-dependent manner. Expression of mPGES-1 and P-Akt and synthesis of PGE2 decreased significantly. MK886 reduced expression of mdr-1 and P170 protein and enhanced the sensitivity of HL-60/A cells to chemotherapeutic drugs. It is concluded that MK886 can inhibit HL-60/A cell proliferation, induce apoptosis and enhance sensitivity to chemotherapeutic drugs, the mechanism of which possibly associates to down-regulation of mPGES-1/PGE2 synthesis, reduction P-Akt expression and decreasing mdr-1 and P170 protein expression.

  13. Effect of Eucheuma gelatinae polysaccharide on apoptosis of human leukemia HL-60 cells%琼枝麒麟菜多糖对白血病细胞株HL-60凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    吴显劲; 欧超伟; 孟庆勇

    2007-01-01

    目的 观察琼枝麒麟菜多糖(EGP)对白血病细胞株HL-60凋亡的影响.方法 分别采用100、200、400 mg/L的EGP处理HL-60细胞.采用荧光显微镜观察HL-60细胞形态、MTT法测定细胞株的增殖抑制情况,流式细胞仪测定细胞凋亡率,逆转录-聚合酶链反应(RT-PCR)检测凋亡相关基因Bcl-2、Fas表达情况.结果 200、400 mg/kg的EGP作用后HL-60细胞出现典型的凋亡形态学变化,HL-60细胞抑制率增加;凋亡抑制基因Bcl-2表达下调,促凋亡基因Fas表达上调.结论 琼枝麒麟菜多糖能诱导HL-60细胞发生典型凋亡.

  14. Utilization of the human cell line HL-60 for chemiluminescence based detection of microorganisms and related substances

    DEFF Research Database (Denmark)

    Timm, Michael; Hansen, Erik W; Moesby, Lise;

    2006-01-01

    species (ROS) when challenged with pyrogenic substances. In a luminol enhanced chemilumimetric assay the responsiveness of differentiated HL-60 cells is tested towards Salmonella typhimurium, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, lipopolysaccharide (LPS) and lipoteichoic acid (LTA...

  15. Antiproliferative activity of various Uncaria tomentosa preparations on HL-60 promyelocytic leukemia cells.

    Science.gov (United States)

    Pilarski, Radosław; Poczekaj-Kostrzewska, Magdalena; Ciesiołka, Danuta; Szyfter, Krzysztof; Gulewicz, Krzysztof

    2007-01-01

    The woody Amazonian vine Uncaria tomentosa (cat's claw) has been recently more and more popular all over the world as an immunomodulatory, antiinflammatory and anti-cancer remedy. This study investigates anti-proliferative potency of several cat's claw preparations with different quantitative and qualitative alkaloid contents on HL-60 acute promyelocytic human cells by applying trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay (MTT). By standardization and statistical comparison of the obtained results pteropodine and isomitraphylline are indicated to be most suitable for standardization of medical cat's claw preparations.

  16. Effect of DNA methylation on protein-DNA interaction of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    何忠效; 白坚石; 张昱

    1999-01-01

    HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.

  17. EFFECT OF ARSENIC TRIOXIDE OR ATRA ON PRIMARY APL CELL OR HL-60 CELLS AND THEIR VALUE ANALYSIS IN HYPERLEUKOCYTOSIS

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To detect the effect of arsenic trioxide or ATRA on APL cells or HL-60 cells and to investigate the mechanism of the hyperleukocytosis and detect the cross resistance between ATRA and arsenic trioxide. Methods: The number of promyelocytes or more matured granulocytes were counted by regular method, MTT test was used to measure the proliferation of HL-60 cells or APL cells, flow cytometry analysis to measure the apoptosis, NBT method to detect the differentiation of HL-60 cells or APL cells. Results: The proliferation of primary APL cells or HL-60 cells could be inhibited in vitro by either arsenic trioxide or ATRA, which could induce obvious apoptosis or obvious differentiation of primary APL cells or HL-60 cells. Inhibition of proliferation or apoptosis of ATRA resistant HL-60 cells were achieved by exposure toarsenic trioxide in vitro. On the other hand, the results of in vivo treatment showed that arsenic trioxide also induce of hyperleukocytosis. Conclusion: The results indicated that the hyperleukocytosis induced by ATRA is not contributed to the mechanism of more differentiation than apoptosis, there was not cross resistance between ATRA and arsenic trioxide.

  18. Multidrug resistance protein 1 (ABCC1) confers resistance to arsenic compounds in human myeloid leukemic HL-60 cells.

    Science.gov (United States)

    Xu, Shi; Zhang, Yan Fang; Carew, Micheal W; Hao, Wen Hui; Loo, Jacky Fong Chuen; Naranmandura, Hua; Le, X Chris

    2013-06-01

    Arsenic trioxide (As(2)O(3)) is established as one of the most effective drugs for treatment of patients with acute promyelocytic leukemia, as well as other types of malignant tumors. However, HL-60 cells are resistant to As(2)O(3), and little is known about the underlying resistance mechanism for As(2)O(3) and its biomethylation products, namely, monomethylarsonous acid (MMA(III)) on the treatment of tumors. In the present study, we investigated the molecular mechanisms underlying iAs(III) and its intermediate metabolite MMA(III)-induced anticancer effects in the HL-60 cells. Here, we show that the HL-60 cells exhibit resistance to inorganic iAs(III) (IC(50) = 10 μM), but are relatively sensitive to its intermediate MMA(III) (IC(50) = 3.5 μM). Moreover, we found that the multidrug resistance protein 1 (MRP1), but not MRP2, is expressed in HL-60 cells, which reduced the intracellular arsenic accumulation, and conferred resistance to inorganic iAs(III) and MMA(III). Pretreatment of HL-60 with MK571, an inhibitor of MRP1, significantly increased iAs(III) and MMA(III)-induced cytotoxicity and arsenic accumulations, suggesting that the expression of MRP1/4 may lead to HL-60 cells resistance to trivalent arsenic compounds.

  19. NPM1基因沉默的HL-60及其耐药细胞株的建立%Establishment of Nucleophosmin Gene Silenced HL-60 and Its Resistant Cell Line

    Institute of Scientific and Technical Information of China (English)

    林敏辉; 胡建达

    2011-01-01

    本研究旨在构建针对HL-60细胞及其耐药细胞株(HL-60/ADR)的NPM1-RNAi的细胞模型,为研究NPM1基因在白血病耐药过程中的作用奠定实验基础.通过针对NPM1基因的shRNA与线性化的pGCSIL-GFP 载体进行连接转化,对获得的重组子(pGCSIL-GFP-NPM1-shRNA)进行PCR和测序鉴定.采用慢病毒载体系统将pHelper 1.0载体、pHelper 2.0载体与pGCSIL-GFP-NPM1-shRNA共转染293T细胞,包装成NPM1 -RNAi-LV,将慢病毒载体感染HL-60HL-60/ADR细胞.采用实时定量RT-PCR和Western blot法分别从mRNA和蛋白水平验证建立的细胞株的转染效率.结果表明,成功构建了重组真核表达载体pGCSIL-GFP-NPM1-shRNA,并通过慢病毒载体系统包装成NPM-1-RNAi-LV;将NPM1-RNAi-LV转染至HL-60HL-60/ADR细胞后,从mRNA水平上看,对细胞的NPM1 mRNA表达有显著抑制,达到90%以上(p<0.05);从蛋白水平上看,对细胞的NPM蛋白表达具有显著的抑制效果,表明转染后的HL-60HL-60/ ADR细胞的NPM1基因特异性沉默.NPM1基因沉默后,HL-60/ADR对阿霉素的耐药性有一定程度的下降.结论:成功构建了NPM1-RNAi的HL-60HL-60/ADR细胞模型,为进一步研究NPM1基因在白血病耐药过程中的作用建立了良好的实验基础.%This study was aimed to construct model cell line of NPMl-RNAi in HL-60 cells and its resistant line (HL-60/ADR) so as to provide a experimental basis for investigating the potential role of NPM1 gene in leukemia drug resistance. The shRNA targeting to NPM1 was ligated into linear pGCSIL-GFP vector, and transformed into E. Coli DH5a. Positive clone was identified by PCR and DNA sequencing. pHelper 1.0, pHelper 2. 0 and pGCSIL-GFP-NPMl-shRNA were cotransformed into 293T cells by lentivius vector system. NPMl-RNAi-LV was transfected into HL-60 and HL-60/ADR cell lines. The efficiency of NPM-RNAi-LV was detected by using real-time quantitative RT-PCR and Western blot. The results showed that the recombinant eukaryotic

  20. Scavenging of superoxide anions by lecithinized superoxide dismutase in HL-60 cells.

    Science.gov (United States)

    Ishihara, Tsutomu; Shibui, Misaki; Hoshi, Takaya; Mizushima, Tohru

    2016-01-01

    Superoxide dismutase covalently bound to four lecithin molecules (PC-SOD) has been found to have beneficial therapeutic effects in animal models of various diseases. However, the mechanism underlying these improved therapeutic effects has not yet been elucidated. It has previously been shown that PC-SOD localizes on the plasma membrane and in the lysosomes of cells. In this study, we evaluated the superoxide anion-scavenging activity of PC-SOD in HL-60 human promyelocytic leukemia cells. Compared to SOD, PC-SOD had only 17% scavenging activity in cell-free systems. Nevertheless, by analyzing enzyme activities in cell suspensions containing PC-SOD or SOD, PC-SOD and SOD showed almost equal activity for scavenging extracellular superoxide anions produced by HL-60 cells. Furthermore, the activity for scavenging extracellular superoxide anions increased with increased amount of PC-SOD on the plasma membrane. Moreover, PC-SOD exhibited no obvious inhibitory effect on the scavenging of intracellular superoxide anions. These results suggested that the association of PC-SOD with the plasma membrane plays a key role in its beneficial therapeutic effects. Thus, this finding may provide a rationale for selecting target diseases for PC-SOD treatment.

  1. Molecular events induced in the HL 60 cell line following treatment with gamma-interferon.

    Science.gov (United States)

    Dubreuil, P; Courcoul, M; Mannoni, P

    1988-01-01

    Gamma-interferon (gamma IFN), like the phorbol ester TPA, is able to induce the differentiation of the HL 60 human promyelocytic cell line in the monocytic pathway. Morphological and serological data show a differential expression of cell surface markers upon treatment with these two inducers. It was reported that TPA treatment alters the expression of some protooncogenes, like c-myc and c-fos, involved in the induction of cell proliferation, and c-fos and c-fms (CSF.1 receptor), linked to monocytic differentiation. We have analyzed the variations in the levels of expression of these oncogenes upon gamma IFN treatment of HL 60 cells. c-myc expression was unchanged during the whole induction period. The early increase reported in c-fos expression was not observed; however, c-fos levels increased upon 24 hr of gamma IFN treatment. These results suggest either that TPA and gamma IFN act at different steps, or, alternatively, that different pathways exist in the monocytic differentiation.

  2. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells.

    Science.gov (United States)

    Smith, James; Bunaciu, Rodica P; Reiterer, Gudrun; Coder, David; George, Thaddeus; Asaly, Michael; Yen, Andrew

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  3. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Smith, James; Bunaciu, Rodica P.; Reiterer, Gudrun [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Coder, David; George, Thaddeus [Amnis Corporation, Seattle, Washington (United States); Asaly, Michael [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Yen, Andrew, E-mail: ay13@cornell.edu [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States)

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  4. Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The events of cell death and the expression of nuclear matrix protein(NMP)have been investigated in a promyelocytic leukemic cell line HL-60 induced with etoposide.By means of TUNEL assay,the nuclei displayed a characteristic morphology change,and the amount of apoptotic cells increased early and reached maximun about 39% after treatment with etoposide for 2 h.Nucleosomal DNA fragmentation was observed after treatment for 4 h.The morphological change of HL-60 cells,thus,occurred earlier than the appearance of DNA ladder.Total nuclear matrix proteins were analyzed by 2-dimensional gel electrophoresis.Differential expression of 59 nuclear matrix proteins was found in 4 h etoposide treated cells.Western blotting was then performed on three nuclear matrix acssociated proteins,PML,HSC70 and NuMA.The expression of the suppressor PML protein and heat shock protein HSC70 were significantly upregulated after etoposide treatment,while NuMA,a nuclear mitotic apparatus protein,was down regulated.These results demonstrate that significant biochemical alterations in nuclear matrix proteins take place during the apoptotic process.

  5. Antileukemic Potential of Momordica charantia Seed Extracts on Human Myeloid Leukemic HL60 Cells

    Directory of Open Access Journals (Sweden)

    Ramani Soundararajan

    2012-01-01

    Full Text Available Momordica charantia (bitter gourd has been used in the traditional system of medicine for the treatment of various diseases. Anticancer activity of M. charantia extracts has been demonstrated by numerous in vitro and in vivo studies. In the present study, we investigated the differentiation inducing potential of fractionated M. charantia seed extracts in human myeloid HL60 cells. We found that the HL60 cells treated with the fractionated seed extracts differentiated into granulocytic lineage as characterized by NBT staining, CD11b expression, and specific esterase activity. The differentiation inducing principle was found to be heat-stable, and organic in nature. The differentiation was accompanied by a downregulation of c-myc transcript, indicating the involvement of c-myc pathway, at least in part, in differentiation. Taken together these results indicate that fractionated extracts of M. charantia seeds possess differentiation inducing activity and therefore can be evaluated for their potential use in differentiation therapy for leukemia in combination with other inducers of differentiation.

  6. Extracellular stress stimuli alter galectin expression profiles and adhesion characteristics of HL-60 cells.

    Science.gov (United States)

    Timoshenko, A V; Lanteigne, J; Kozak, K

    2016-02-01

    Galectins, a family of soluble β-galactoside-binding proteins, are involved in the regulation of various cellular functions, which are essential for adaptive cellular stress responses (CSRs). Although expression patterns of galectins and galectin-binding glycans change during tissue development and cancer, the requirement and role of galectin networks in the CSRs are not completely understood. In this study, we report that the treatment of human promyelocytic HL-60 cells with stimuli mimicking hypoxia (CoCl2), inducing the endoplasmic reticulum stress (tunicamycin), and stimulating cell differentiation, result in stress-specific differential expression of galectin transcripts. In addition, we show that CoCl2 increases the expression of cell surface glycans recognized by both β-galactoside- and GlcNAc-binding lectins. Thus, microenvironmental stress changes the glycobiological status of cells representing expression profiles of endogenous lectins and corresponding glycans. These findings introduce a novel classification of galectins in HL-60 cells, which suggests diverse functions of galectin members in CSRs.

  7. Targeting miR-155 suppresses proliferation and induces apoptosis of HL-60 cells by targeting Slug/PUMA signal.

    Science.gov (United States)

    Liang, Hui; Dong, Ziyan; Liu, Jiang-Feng; Chuang, Wei; Gao, Li-Zhen; Ren, Yu-Guo

    2016-10-27

    Recent studies have shown that high miR-155 expression was associated with poor prognosis in patients with acute myelogeneous leukemia (AML). Furthermore, targeting miR-155 results in monocytic differentiation and apoptosis. However, the exact role and mechanisms of miR-155 in human AML remains speculative. HL-60 cells were treated with anti-miR-155 for 72 h. Cell growth and apoptosis in vitro were detected by MTT, BrdU proliferation, colony formation and flow cytometry assay. The effect of anti-miR-155 on growth of HL-60 cells was also evaluated in a leukemia mouse model. Slug cDNA and PUMA siRNA trannsfection was used to assess the signal pathway. Different protein expression was detected by western blot assay and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay. The results shown that targeting miR-155 resulted in a 24-fold decrease of miR-155 expression compared to negative control in the HL-60 cells. Targeting miR-155 significantly downregulated Slug and upregulated PUMA expression, and decreased HL-60 cell growth by 70% , impaired colony formation by approximately 60%, and increased HL-60 cell apoptosis by 45%. Targeting PUMA reversed miR-155 sliencing-induced proliferation and apoptosis of HL-60 cells. Restoration of Slug decreased PUMA expression. In murine engraftment models of HL-60 cells, we showed that targeting miR-155 was able to reduce tumor growth. This was accompanied with decreased Slug expression and increased PUMA expression in these tumors. Collectively, our findings strongly suggest targeting miR-155 exhibited in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of Slug expression and increase of PUMA expression.

  8. Isoliquiritigenin-induced effects on Nrf2 mediated antioxidant defence in the HL-60 cell monocytic differentiation.

    Science.gov (United States)

    Chen, Hongmei; Zhang, Bo; Yuan, Xuan; Yao, Ying; Zhao, Hong; Sun, Xiling; Zheng, Quisheng

    2013-11-01

    To evaluate the role of redox homeostasis in differentiation in human promyelocytic leukemia cells (HL-60) induced by isoliquiritigenin (ISL) through modulation of the nuclear erythroid-related factor 2/antioxidant responsive element (Nrf2/ARE) pathway. Morphological changes, cell surface markers CD11b/CD14, and nitroblue tetrazolium (NBT)-reducing ability were used to determine the differentiation of HL-60, and 2,7-dichlorofluorescein was used to detect the level of intracellular reactive oxygen species (ROS). Thiobarbituric acid test was utilised to determine the levels of malondialdehyde production in ISL-treated HL-60. The study determines and presents the redox state of the ratio of reduced/oxidised glutathione as a consequence of progression from differentiation in HL-60. Expression levels of the Nrf2/ARE downstream target genes were determined by quantitative polymerase chain reaction. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) inhibitors, apocynin (APO), and diphenyleneiodonium (DPI) were used for the preliminary study to determine the potential downstream targets regulated by NADPH oxidase in ISL-induced HL-60 differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of HL-60 differentiation, namely, morphology changes, NBT reductive activities, and expression levels of surface antigens CD11b/CD14. Intercellular redox homeostasis changes toward oxidation during drug exposure are necessary to support ISL-induced differentiation. The unique expression levels of the Nrf2/ARE downstream target genes in the differentiation of HL-60 recorded a statistically significant and dose-dependent increase (P < 0.05), which were suppressed by NADPH oxidase inhibitor, APO, and DPI. ISL as a differentiation-inducing agent with mechanisms involved in the Nrf2/ARE pathway to modulate intercellular redox homeostasis, and thus, facilitate differentiation.

  9. 银杏外种皮多糖对HL-60细胞的体外实验研究%Experimental Study of Ginkgo biloba Exocarp Polysaccharides on HL-60 Cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    许爱华; 陈华圣; 孙步蟾

    2004-01-01

    目的:研究银杏外种皮多糖对体外HL-60细胞的作用.方法:运用MTT方法和流式细胞技术,检测HL-60细胞体外增殖、凋亡以及相关基因的表达.结果:银杏外种皮多糖(40~160 m/L,48h)可抑制HL-60细胞增殖及增殖促进基因c-myc的表达,可诱导HL-60细胞凋亡并下调凋亡抑制基因bcl-2的表达.结论:银杏外种皮多糖对HL-60细胞增殖及凋亡的影响涉及增殖促进基因c-myc和凋亡抑制基因bcl-2.

  10. Reversal of chemoresistance with small interference RNA (siRNA) in etoposide resistant acute myeloid leukemia cells (HL-60).

    Science.gov (United States)

    Kachalaki, Saeed; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi; Shanehbandi, Dariush; Mohammadinejad, Sina; Mansoori, Behzad

    2015-10-01

    Overexpression of ATP-binding cassette (ABC) drug transporters is a major barrier in the success of cancer chemotherapy. One way to overcome overexpression of ABC drug transporter-mediated chemoresistance in acute myeloid leukemia is to suppress ABC drug transporter genes expression by small interference RNA (siRNA). In this study was assessed the involvement of ABCB1 gene in the mechanisms of resistance to etoposide in AML cells. The etoposide-resistant HL-60 cells were generated by stepwise exposure increasing concentrations of etoposide. The etoposide-resistant HL-60 cells were transfected with siRNAs using Transfection Reagent. The ABCB1 mRNA expression were assessed by real-time quantitative PCR. The MDR1/P-gp levels were measured by Western blotting. The sensitivity of resistant HL-60 cells to etoposide after transfection was determined using MTT assay. Apoptosis of resistant HL-60 cells after transfection was detected by flow cytometer. It was found that siRNA effectively inhibited ABCB1 expression at both mRNA and protein levels. Knockdown of the ABCB1 gene correlated with increased sensitivity of the resistant HL-60 cells to etoposide and was observed to lower the cytotoxic index (IC50 etoposide value) after transfection. Our results indicate that product of the ABCB1 gene have effective role in resistance to etoposide in acute myeloid leukemia cells. Copyright © 2015. Published by Elsevier Masson SAS.

  11. NADPH oxidase-dependent production of reactive oxygen species induces endoplasmatic reticulum stress in neutrophil-like HL60 cells.

    Directory of Open Access Journals (Sweden)

    Wilson Mitsuo Tatagiba Kuwabara

    Full Text Available Reactive oxygen species (ROS primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60 differentiated into neutrophil-like cells (dHL60 induces ER stress and activates the unfolded protein response (UPR. To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.

  12. NADPH oxidase-dependent production of reactive oxygen species induces endoplasmatic reticulum stress in neutrophil-like HL60 cells.

    Science.gov (United States)

    Kuwabara, Wilson Mitsuo Tatagiba; Zhang, Liling; Schuiki, Irmgard; Curi, Rui; Volchuk, Allen; Alba-Loureiro, Tatiana Carolina

    2015-01-01

    Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.

  13. Proapoptotic activity of heterocyclic compounds containing succinimide moiety in the promyelocytic leukemia cell line HL-60.

    Science.gov (United States)

    Kuran, Bozena; Krawiecka, Mariola; Kossakowski, Jerzy; Koronkiewicz, Mirosława; Chilmonczyk, Zdzisław

    2013-01-01

    In the present paper, we describe proapoptotic activity of several heterocyclic compounds 9, 12, 18, 19 and 20 possessing succinimide (as well as succinimide related) moieties. The compounds properties were examined with the aid of flow cytometry on the promyelocytic leukemia cell line HL-60. The highest proapoptotic activity exhibited compound 12 (4-{4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl}-1,7-diethyl-8,9-diphenyl-4-azatricyklo[5.2.1.0(2,6)]-dec-8-ene-3,5,10-trione). The synthesis of compounds 1-17 is also described. The structures of obtained compounds were characterized by 1H NMR, 13C NMR, ESI MS and/or elemental analyses.

  14. Isolation and reconstitution of the N-formylpeptide receptor from HL-60 derived neutrophils.

    Science.gov (United States)

    Hoyle, P C; Freer, R J

    1984-02-27

    A multifunctional receptor for N-formylpeptides exists on the membranes of neutrophils. This receptor has now been isolated from neutrophils derived from HL-60 promyelocytic leukemia cells. After solubilization by Nonidet-P40 and purification by affinity chromatography and HPLC the isolated receptor was reconstituted into egg phosphatidylcholine vesicles by SM-2 Bio-Bead removal of the Nonidet-P40. Analysis of the affinity and selectivity of the receptor was done by direct binding of two high-affinity ligands, formyl-Met-Leu-[3H]Phe-OH and formyl-Nle-Leu-Phe-[3H]Tyr-OH. The data suggest that the receptor can be isolated and reconstituted without apparent alteration of its binding affinity and selectivity, and that there appear to be no co-factors or subunits upon which these binding characteristics are dependent.

  15. Synthesis of minoxidil conjugates and their evaluation as HL-60 differentiation agents.

    Science.gov (United States)

    Stoica, Sonia; Magoulas, George E; Antoniou, Antonia I; Suleiman, Sherif; Cassar, Analisse; Gatt, Lucienne; Papaioannou, Dionissios; Athanassopoulos, Constantinos M; Schembri-Wismayer, Pierre

    2016-02-15

    Activation of minoxidil (MNX) with N,N'-carbonyldiimidazole and coupling with natural polyamines (PAs) and commercially available aliphatic or aromatic amines provided a series of new conjugates which were evaluated for their ability to induce differentiation to HL-60 acute myeloid leukemia cancer cells, using a modified NBTZ reduction test. Although neither MNX nor 4,4'-methylenedianiline (MDA) or 2,7-diaminofluorene (DAF), alone or in combination, had any effect, the MNX-spermine (SPM) conjugate (11) and the conjugates 7 and 8 of MNX with MDA and DAF exhibited a differentiation-inducing effect at a concentration of 10 μM without being toxic on proliferating human peripheral blood mononuclear cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821: Phosphoproteomic Analysis

    Directory of Open Access Journals (Sweden)

    Barbora Šalovská

    2014-07-01

    Full Text Available DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs: Ataxia teleangiectasia mutated (ATM, DNA-dependent protein kinase (DNA-PK and ATM and Rad3-related kinase (ATR. Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonylphenyl-N-phenylpyrazine-2-carboxamide, has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative. Hydrophilic interaction liquid chromatography (HILIC-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.

  17. Ganoderma lucidum polysaccharide exerts anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells.

    Science.gov (United States)

    Yang, Guohua; Yang, Lei; Zhuang, Yun; Qian, Xifeng; Shen, Yunfeng

    2016-01-01

    In this study, we investigated the anti-tumor activity both in vitro and in vivo of a polysaccharide obtained from Ganoderma lucidum on HL-60 acute myeloid leukemia cells, and focused on its targeting effect on mitogen-activated protein kinase (MAPK) pathways. It was found by the methods such as western blot and flow cytometry (FCM), that G. lucidum polysaccharide (GLP) blocked the extracellular signal-regulated kinase/MAPK signaling pathway, simultaneously activated p38 and JNK MAPK pathways, and therefore regulated their downstream genes and proteins, including p53, c-myc, c-fos, c-jun, Bcl-2, Bax, cleaved caspase-3 and cyclin D1. As a result, cycle arrest and apoptosis of HL-60 cells were induced. Therefore, GLP exerted anti-tumor activity via MAPK pathways in HL-60 acute leukemia cells.

  18. [Combination induction of cell differentiation of HL-60 cells by daidzein (S86019) and BC-4 or Ara-C].

    Science.gov (United States)

    Jing, Y K; Han, R

    1993-01-01

    Experiments demonstrated that the cell differentiation of HL-60 cells induced by low concentrations of daidzein (S86019), BC-4 (active principle of Boswellia carterii Birdw) and Ara-C was not impressive. However, when they were used in combination 80% of HL-60 cells exhibited NBT reduction and 82% of the cells showed phagocytosis after four days exposure to daidzein and BC-4. When HL-60 cells were exposed to daidzein and Ara-c, 70% of the cells exhibited NBT reduction and phagocytosis. Flow cytometry indicated that the majority of the cells were blocked at G1 phase under the induction of combination of daidzein with BC-4 or Ara-C.

  19. Stimulation by leukotriene D4 of increases in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells.

    Science.gov (United States)

    Baud, L; Goetzl, E J; Koo, C H

    1987-10-01

    The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) evoked increases in the cytosolic concentration of intracellular calcium ([Ca+2]i) in dimethylsulfoxide-differentiated HL-60 cells, as assessed by the fluorescence of quin-2. The increases in [Ca+2]i reached a peak within 15-90 s, attained 50% of the maximum level at 1.2 nM LTD4 and 60 nM LTC4, were greater in maximal magnitude for LTD4 than LTC4, and subsided in 5-7 min. Flow cytometric evaluation of the LTD4-induced increases in [Ca+2]i, reflected in increases in the fluorescence of intracellular indo-1, revealed that a mean of 77% of differentiated HL-60 cells responded, as contrasted with lesser increases in only 50% of undifferentiated HL-60 cells. The capacity of pretreatment of HL-60 cells with LTD4 to prevent subsequent responses of [Ca+2]i to LTC4 and LTD4, and the finding that the serine-borate inhibitor of conversion of LTC4 to LTD4 suppressed concurrently both LTC4-induced rises in [Ca+2]i and increases in adherence to Sephadex G-25 indicated that the responses of HL-60 cells to LTC4 required conversion to LTD4. That pertussis toxin and a chemical antagonist of LTD4 reduced the [Ca+2]i response suggested a dependence on LTD4 receptors. The LTD4-induced increases in [Ca+2]i were dependent on extracellular calcium and diminished by lanthanum, but not affected by nifedipine nor associated with changes in membrane potential, as measured with the fluorescent probe 3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2]i in HL-60 cells, which is coupled to an increase in adherence, appears to involve LTD4 receptor-specific and voltage-independent calcium channels in the plasma membrane.

  20. Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells

    DEFF Research Database (Denmark)

    Stervbo, Ulrik; Vang, Ole; Bonnesen, Christine

    2006-01-01

    , an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration......- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity....

  1. 肉苁蓉总苷在HL-60细胞中的抗氧化活性研究%Antioxidant property of general cistanosides in human HL-60 cell line

    Institute of Scientific and Technical Information of China (English)

    木合布力·阿布力孜; 毛新民; 热娜·卡斯木; 马淑燕; Leininger-Muller B; Gérard SIEST

    2008-01-01

    目的 观察肉苁蓉总苷(general cistanosides,GCs)在HL-60细胞中的抗氧化生物活性.方法 GCs从新疆盐生肉苁蓉中提取得到;利用HL-60细胞中的氧化系统,对GCs的抗氧化活性进行测定研究.HL-60细胞中自由基诱发程度由探针物DCFH-DA的氧化产物DCF的浓度来检测.银杏叶提取物EGB 761用作阳性对照物.结果 GCs以0.25、0.50 g·L-1和0.75 g·L-1浓度,对自由基的抑制率分别为56%、62%和67%.其抗氧化活性与EGB 761类似.结论 GCs在HL-60细胞氧化系统中具有很强的抗自由基氧化活性.

  2. 肉桂醛对HL-60细胞的生长抑制及诱导凋亡作用研究%Proliferation Inhibitory and Apoptosis-inducing Effects of Cinnamaldehyde on HL-60 Cells

    Institute of Scientific and Technical Information of China (English)

    魏练平; 罗美; 蒋瑾; 蒋立科

    2011-01-01

    Objective: To determine the proliferation inhibitory and apoptosis-inducing effects of cinnamaldehyde on HL-60 cells.Methods: The proliferation inhibitory effect was evaluated using trypan-blue staining and MTT assay,and the apoptosis-inducing effect using flow cytometry,scanning electron microscope(SEM) and transmission electron microscope(TEM).Results: Cinnamaldehyde had an oblivious inhibitory effect on the proliferation of HL-60 cells with dependence upon its concentration and treatment time and the LC50,median lethal concentration was 34.87 μg/mL.The rate of apoptosis in HL-60 cells was 32.6 % after being treated with cinnamaldehyde at the dose of 20 μg/mL.for 16 h.Typical apoptotic morphological changes in the cells was observed under fluorescence microscopy,SEM and TEM.Conclusion: cinnamaldehyde cna notably inhibit the proliferation and induce the apoptosis of HL-60 cells.%目的:探讨肉桂醛对HL-60细胞的增殖及凋亡的影响。方法:采用台盼蓝细胞计数和四甲基偶氮唑蓝(MTT)法研究肉桂醛对HL-60细胞增殖的抑制作用;采用流失细胞仪(FCM)、荧光显微镜(FM)、扫描电镜(SEM)以及透射电镜(TEM)探讨肉桂醛对HL-60细胞的诱导凋亡作用。结果:台盼蓝细胞计数和MTT法结果表明:肉桂醛对HL-60细胞的增殖有明显抑制作用,抑制作用与肉桂醛质量浓度及作用时间呈依赖关系,其半数致死质量浓度为34.87μg/mL;流式细胞仪检测结果表明:肉桂醛(终质量浓度20μg/mL)作用16h后,HL-60细胞早期凋亡比例为32.6%;该组细胞在荧光显微镜、扫描电镜以及透射电镜观察下均呈现早期凋亡特征。结论:肉桂醛能抑制HL-60细胞增殖并促进其凋亡。

  3. Examining the lateral displacement of HL60 cells rolling on asymmetric P-selectin patterns.

    Science.gov (United States)

    Lee, Chia-Hua; Bose, Suman; Van Vliet, Krystyn J; Karp, Jeffrey M; Karnik, Rohit

    2011-01-01

    The lateral displacement of cells orthogonal to a flow stream by rolling on asymmetrical receptor patterns presents a new opportunity for the label-free separation and analysis of cells. Understanding the nature of cell rolling trajectories on such substrates is necessary to the engineering of substrates and the design of devices for cell separation and analysis. Here, we investigate the statistical nature of cell rolling and the effect of pattern geometry and flow shear stress on cell rolling trajectories using micrometer-scale patterns of biomolecular receptors with well-defined edges. Leukemic myeloid HL60 cells expressing the PSGL-1 ligand were allowed to flow across a field of patterned lines fabricated using microcontact printing and functionalized with the P-selectin receptor, leveraging both the specific adhesion of this ligand-receptor pair and the asymmetry of the receptor pattern inclination angle with respect to the fluid shear flow direction (α = 5, 10, 15, and 20°). The effects of the fluid shear stress magnitude (τ = 0.5, 1, 1.5, and 2.0 dyn/cm(2)), α, and P-selectin incubation concentration were quantified in terms of the rolling velocity and edge tracking length. Rolling cells tracked along the inclined edges of the patterned lines before detaching and reattaching on another line. The detachment of rolling cells after tracking along the edge was consistent with a Poisson process of history-independent interactions. Increasing the edge inclination angle decreased the edge tracking length in an exponential manner, contrary to the shear stress magnitude and P-selectin incubation concentration, which did not have a significant effect. On the basis of these experimental data, we constructed an empirical model that predicted the occurrence of the maximum lateral displacement at an edge angle of 7.5°. We also used these findings to construct a Monte Carlo simulation for the prediction of rolling trajectories of HL60 cells on P

  4. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  5. Identification and cloning of the SNARE proteins VAMP-2 and syntaxin-4 from HL-60 cells and human neutrophils.

    Science.gov (United States)

    Smolen, J E; Hessler, R J; Nauseef, W M; Goedken, M; Joe, Y

    2001-08-01

    Degranulation and membrane fusion by neutrophils are essential to host defense. We sought homologues of neuron-specific fusion proteins in human neutrophils and in their precursors, the promyelocytic cell line HL-60. We screened a differentiated HL-60 library and obtained an 848 bp sequence with a 351 bp open reading frame, identical to that published for human VAMP-2 and including 5' and 3' untranslated regions. RNA from HL-60 cells during differentiation into the neutrophil lineage was subjected to Northern blot analysis. which revealed a transcript of approximately 1050 bp at all stages of differentiation. The amount of these transcripts increased approximately threefold during differentiation, a finding confirmed by quantitative RT-PCR. We also detected mRNA for VAMP-2 in human neutrophils and monocytes using RT-PCR. In like fashion, transcripts of syntaxin-4, another fusion protein, were recovered from a neutrophil cDNA library. As with VAMP-2, expression of syntaxin-4 (determined by Northern blots) also increased, but by only 50%, during differentiation of HL-60 cells. These studies demonstrate that neutrophils and their progenitors possess mRNA for the fusion proteins VAMP-2 and syntaxin-4, and that their transcription increases during differentiation, concurrent with the functional maturation of myeloid cells.

  6. Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells.

    Science.gov (United States)

    Parker, Amber; Cuddihy, Sarah L; Son, Tae G; Vissers, Margreet C M; Winterbourn, Christine C

    2011-10-01

    Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H(2)O(2) to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H(2)O(2) was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.

  7. Effect of bixin on DNA damage and cell death induced by doxorubicin in HL60 cell line.

    Science.gov (United States)

    Santos, G C; Almeida, M R; Antunes, Lmg; Bianchi, Mlp

    2016-12-01

    Bixin is a natural red pigment extracted from annatto. Although it is widely used as a coloring agent in food, there are few studies about the effect of this carotenoid on DNA. This study aimed to investigate the effects of bixin on cytotoxicity and genotoxicity induced by doxorubicin in HL60 cells. At concentrations above 0.3 μg/mL, bixin demonstrated cytotoxic effects in HL60 cells. Furthermore, this carotenoid was neither mutagenic nor genotoxic to HL60 cells and reduced the DNA damage induced by doxorubicin. Bixin and doxorubicin showed no apoptotic effect in HL60 cells, but the simultaneous combined treatments showed an increase in the percentage of apoptotic cells. In conclusion, our results showed that bixin modulates the cytotoxicity of doxorubicin via induction of apoptosis. The results of this study provide more knowledge about the toxic effects of anticancer treatments and how the natural compounds can be useful on these therapeutic approaches. © The Author(s) 2016.

  8. Role of Extracelluar Regulated Protein Kinases in FTY720-induced Apoptosis of Leukemia Cell Lines HL-60 and U937

    Institute of Scientific and Technical Information of China (English)

    李登举; 张瑶珍; 胡向荣; 曹文静; 黄伟

    2004-01-01

    Summary: The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL-60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL-60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragment analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL-60 and U937cells effectively in a dose-dependent manner. After incubation with FTY720 for 24 h, apoptosis was observed in HL-60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down-regulated and the distribution of ERK1/2 protein in cell' nuclear was reduced during FTY720-induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720-induced apoptosis and proliferation inhibition of leukemia cells.

  9. The preparation of HL-60 cells vaccine expressing BCG heat shock protein 70 and detection of its immunogenicity in vitro.

    Science.gov (United States)

    Li, Xiao-Ling; Zhao, Yan-Xia; Sun, Li-Rong; Yang, Jing; Xu, Hui-Juan

    2012-10-01

    Gene-modified cell vaccines are the best way to achieve the immunotherapy for all types of acute leukemia. In this study, the recombinant eukaryotic expression vector (pDisplay-HSP70) of heat shock protein 70 (HSP70) of Bacille Calmette-Guérin (BCG) was constructed by amplifying the whole BCG HSP70 gene using polymerase chain reaction (PCR) and sub-cloning into the polyclone endonuclease sites in pDisplay. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection and its anti-tumor effect and mechanism were further studied. Results showed that the fragment of BCG HSP70 was consistent with Mycobacterium tuberculosis HSP70 gene published in GeneBank. DNA sequencing showed that the recombinant vector was correctly constructed and named pDisplay-HSP70. After BCG HSP70 gene transfection, the yellow-green fluorescence on the HL-60 cells surface was observed under a fluorescence microscope. The immunogenicity of HSP70-transfected HL-60 cells exhibited upregulated proliferation of lymphocytes, increased cytokine secretion (IFN-γ) and enhanced killing activity. These results suggested that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells. It may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.

  10. Dynamic effects of autophagy on arsenic trioxide-induced death of human leukemia cell line HL60 cells

    Institute of Scientific and Technical Information of China (English)

    Ya-ping YANG; Zhong-qin LIANG; Bo GAO; Yan-li JIA; Zheng-hong QIN

    2008-01-01

    Aim: To evaluate the contribution of an autophagic mechanism to the As2O3-induced death of human acute myeloid leukaemia cell line HL60 cells. Methods: The growth inhibition of HL60 cells induced by As2O3 was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazohum bromide colorimetric assay. The ac-tivation of autophagy was determined with monodansylcadaverine labeling and transmission electron microscope. The role of autophagy in the As2O3-induced death of HL60 cells was assessed using autophagic and lysosomal inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Results: After treatment with As2O3, the proliferation of HL60 cells was significantly inhibited and the formation of autophagosomes increased. The blockade of autophagy maturation with the autophagy-specific inhibitor 3-methyladenine (3-MA) or the lysosome-neutraliz-ing agent NH4C11 h before As2O3 potentiated the As2O3-induced death of HL60 cells. In contrast, 3-MA attenuated As2O3-induced death when administered 30 min after As2O3. 3-MA and NH4Cl also inhibited As2O3-induced upregulation of microtubule-associated protein 1 light chain 3, the protein required for autophagy in mammalian cells. Following As2O3, lysosomes were activated as indicated by increased levels of cathepsins B and L. The apoptotic response of HL60 cells to As2O3 was suggested by the collapse of mitochondrial membrane potential, re-lease of cytochrome c from mitochondria, and the activation of caspase-3. Pre-treatment with 3-MA prior to As2O3 amplified these apoptotic signals, while post-treatment with 3-MA 30 min after As2O3 attenuated the apoptotic pathways. Conclusion: Autophagy plays complex roles in the As2O3-induced death of HL60 cells; it inhibits As2O3-induced apoptosis in the initiation stage, but amplifies the AS2O3-mediated apoptotic program if it is persistently activated.

  11. SENSITIVITY OF LEUKEMIC CELL LINE HL-60 TO COMBINATION OF NEFERINE AND ARSENIC

    Institute of Scientific and Technical Information of China (English)

    LIU Ge-xiu; ZHANG Yuan; HE Dong-mei

    2006-01-01

    Objective: To determine whether neferine (Nef) enhances the sensitivity of human myeloid leukemia HL-60 cells to arsenic trioxide (ATO). Methods: Apoptosis was detected by DNA electrophoresis. Giemsa staining was used to observe the apoptotic cells under microscope. The apoptotic rates of cells were analyzed using flow cytometry. The inhibitory rates of cell growth were assayed by MTT, and the expression of P-gp was determined by flow cytometry. Results: Low doses of ATO (1.0 (mol/L) only partially inhibitory percentage of cell growth at 72 h was (9.92(3.03) % (P>0.05, vs control). The combination of 1.0 (mol/L ATO with 2.0 (mol/L Nef inhibited (45.27(4.93) % of cell growth, and induced apoptosis in leukemic cells more significantly than wither ATO or Nef on their own (P<0.01). Moreover, ATO-induced expression of P-gp in leukemic cells was inhibited significantly by Nef. Conclusion: These results indicate that Nef significantly increases sensitivity of leukemic cells to ATO, which might be associated with inhibitory expression of P-gp gene. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.

  12. Effect of fractionation and rate of radiation dose on human leukemic cells, HL-60

    Energy Technology Data Exchange (ETDEWEB)

    Rhee, J.G.; Song, C.W.; Kim, T.H.; Levitt, S.H.

    1985-03-01

    The capacity of HL-60 cells, human acute promyelocytic leukemic cells established in culture, to repair sublethal radiation damage was estimated from the response of the cells to fractionated irradiation or to a single irradiation at difference dose rates. After exposure of cells to a single dose of X rays at a dose rate of 78 rad/min, the survival curve was characterized by n = 2.5, D/sub q/ = 80 rad, and D/sub 0/ = 83.2 rad. Split-dose studies demonstrated that the cells were able to repair a substantial portion of sublethal radiation damage in 2 hr. The response of the cells to irradiation at different dose rates decreased with a decrease in the dose rates, which could be attributed to repair of sublethal radiation damage. The possibility that some of the malignant hemopoietic cells, if not all, may possess a substantial capacity to repair sublethal radiation damage should not be underestimated in planning total-body irradiation followed by bone marrow transplantation.

  13. Flow-Through Electroporation of HL-60 White Blood Cell Suspensions using Nanoporous Membrane Electrodes.

    Science.gov (United States)

    Chen, Zhiqiang; Akenhead, Michael A; Sun, Xinghua; Sapper, Harrison; Shin, Hainsworth Y; Hinds, Bruce J

    2016-08-01

    A flow-through electroporation system, based on a novel nanoporous membrane/electrode design, for the delivery of cell wall-impermeant molecules into model leukocytes, HL-60 promyelocytes, was demonstrated. The ability to apply low voltages to cell populations, with nm-scale concentrated electric field in a periodic array, contributes to high cell viability. With applied biases of 1-4V, delivery of target molecules was achieved with 90% viability and up to 65% transfection efficiency. More importantly, the system allowed electrophoretic pumping of molecules from a microscale reservoir across the membrane/electrode system into a microfluidic flow channel for transfection of cells, a design that can reduce reagent amount by eightfold compared to current strategies. The flow-through system, which forces intimate membrane/electrode contact by using a 10μm channel height, can be easily scaled-up by adjusting the microfluidic channel geometry and/or the applied voltage pulse frequency to control cell residence times at the cell membrane/electrode interface. The demonstrated system shows promise in clinical applications where low-cost, high cell viability and high volume transfection methods are needed without the risk of viral vectors. In particular genetic modification of freely mobile white blood cells to either target disease cells or to express desired protein/enzyme biomolecules is an important target platform enabled by this device system. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Loss of TRF2 by radiation-induced apoptosis in HL60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Nakagami, Yoshihiro; Ito, Megumi; Hara, Takamitsu; Inoue, Tomio [Yokohama City Univ. (Japan). School of Medicine

    2002-06-01

    A telomere consists of the short tandem DNA repeats of (T2AG3)n localized to the distal ends of chromosomes. Telomere repeat binding factor 2 (TRF2) has been implicated in the protection of chromosome ends. Recently, it has been reported that the loss of TRF2 induces apoptosis by various stimuli or genetic technique, however, the effects of radiation are not known. Therefore, this study investigated the interaction between TRF2 and radiation. Western blot analysis, immunohistochemistry, and a DNA fragmentation assay for the detection of apoptosis were performed. The interaction between elastase and TRF2 was also investigated in vitro. Western blot analyses and immunohistochemistry showed that {gamma}-rays induce the temporary accumulation and subsequent loss of TRF2 protein in the nuclei of irradiated HL60 cells. Following DNA fragmentation, the loss of TRF2 could be detected. TRF2 was broken down by elastase, which was translocated into the nucleus before the loss of TRF2. The results of the study showed that irradiation first induces activation of TRF2, consequently protecting the end of the chromosome. Subsequently, translocation of elastase into the nucleus results in the breakdown of TRF2 after DNA fragmentation has occurred. (author)

  15. Regulation of shear stress on rolling behaviors of HL-60 cells on P-selectin

    Science.gov (United States)

    Ling, YingChen; Fang, Ying; Yang, XiaoFang; Li, QuHuan; Lin, QinYong; Wu, JianHua

    2014-10-01

    Circulating leukocytes in trafficking to the inflammatory sites, will be first tether to, and then roll on the vascular surface. This event is mediated through specific interaction of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1), and regulated by hemodynamics. Poor data were reported in understanding P-selectin-mediated rolling. With the flow chamber technique, we herein observed HL-60 cell rolling on P-selectin with or without 3% Ficoll at various wall shear stresses from 0.05 to 0.4 dyn/cm2. The results demonstrated that force rather than transport regulated the rolling, similar to rolling on L- and E-selectin. The rolling was accelerated quickly by an increasing force below the optimal shear threshold of 0.15 dyn/cm2 first and then followed by a slowly decelerating phase starting at the optimum, showing a catch-slip transition and serving as a mechanism for the rolling. The catch-slip transition was completely reflected to the tether lifetime and other rolling parameters, such as the mean and fractional stop time. The narrow catch bond regime stabilized the rolling quickly, through steeply increasing fractional stop time to a plateau of about 0.85. Data presented here suggest that the low shear stress threshold serves as a mechanism for most cell rolling events through P-selectin.

  16. Bioreductive activation of mitoxantrone by NADPH cytochrome P450 reductase does not change its apoptotic stimuli properties in regard to sensitive and multidrug resistant leukaemia HL60 cells.

    Science.gov (United States)

    Kostrzewa-Nowak, Dorota; Tarasiuk, Jolanta

    2013-12-05

    The objective of this study was to examine the effect of bioreductive activation of antitumour drug, mitoxantrone (MX), by liver NADPH cytochrome P450 reductase (CPR) on inducing apoptosis of human promyelocytic sensitive leukaemia HL60 cell line and its multidrug resistance (MDR) sublines exhibiting two different phenotypes of MDR related to the overexpression of P-glycoprotein (HL60/VINC) or MRP1 (HL60/DOX). It was found that non-activated as well as CPR-activated form of MX used at IC90 were able to influence cell cycle of sensitive HL60 as well as resistant cells and induce apoptosis. Interestingly, it was evidenced that HL60/VINC cells were more susceptible to undergo caspase-3/caspase-8-dependent apoptosis induced by both studied forms of MX compared to HL60 and HL60/DOX cells. However, the examined agent did not change the expression of Fas receptors on the surface of HL60 sensitive as well as resistant cells regardless of its form used in the study. Obtained results suggest that CPR-dependent reductive activation of MX does not change its apoptotic stimuli properties in regard to sensitive HL60 and multidrug resistant (HL60/VINC and HL60/DOX) leukaemia cells. Nevertheless, taking into account that side toxic effects observed in course of patient treatment with antitumour drugs are dose-dependent, it seems that the reported increase in antiproliferative activity and ability to induce apoptosis of MX after its reductive activation by exogenous CPR against the MDR cells overexpressing both P-glycoprotein and MRP1 at much more lower concentrations of this drug could be of clinical importance for the treatment of tumours resistant to classical chemotherapy. © 2013 Elsevier B.V. All rights reserved.

  17. Mitochondrial DNA damage and oxidative damage in HL-60 cells exposed to 900MHz radiofrequency fields.

    Science.gov (United States)

    Sun, Yulong; Zong, Lin; Gao, Zhen; Zhu, Shunxing; Tong, Jian; Cao, Yi

    2017-03-01

    HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900MHz radiofrequency fields (RF) at 120μW/cm(2) power intensity for 4h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2'-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Force-dependent bond dissociation govern rolling of HL-60 cells through E-selectin.

    Science.gov (United States)

    Li, Quhuan; Fang, Ying; Ding, Xiaoru; Wu, Jianhua

    2012-08-15

    E-selectin-mediated rolling on vascular surface of circulating leukocyte on vascular surface is a key initial event during inflammatory response and lymphocyte homing. This event depends not only on the specific interactions of adhesive molecules but also on the hemodynamics of blood flow. Little is still understood about whether wall shear stress or shear rate regulates the rolling. With flow chamber techniques, we here measured the effects of transport, shear stress and cell deformation on rolling of both unfixed and fixed HL-60 cells on E-selectin either in the absence or in the presence of 3% Ficoll in medium at various wall shear stresses from 0.05 to 0.7 dyn/cm(2). The results demonstrated a triphasic force-dependent rolling, that is, as increasing of force, the rolling would be accelerated firstly, then followed a decelerating phase occurred at the initial shear threshold of about 0.1 dyn/cm(2), and lastly returned to an accelerating process starting at the optimal shear threshold of 0.35 dyn/cm(2) approximately. The catch bond regime was completely reflected to rolling behaviors, such as tether lifetime, cell stop time and rolling velocity, meaning that the dominant factor to govern rolling is force. The initial shear threshold might be the minimum level of wall shear stress to sustain a stationary rolling, and the optimal shear threshold would make rolling to the most stable and regular. These findings strongly elucidate the catch bond mechanism for flow-enhanced rolling through E-selectin since longer bond lifetimes led to slower and stabler rolling.

  19. Induction of apoptosis by furanodiene in HL60 leukemia cells through activation of TNFR1 signaling pathway

    Institute of Scientific and Technical Information of China (English)

    MA En-long; WANG Xiao-long; LI Yan-chun; TAI Wen-jiao; LI Te; GUO Tao

    2008-01-01

    Objective Curcuma wenyujin, named Ezhu in Chinese, a traditional Chinese medicine, which has been shown to possess antiearcinogenie activity and used for the treatment of tumor in China,for example, hepatic cancer and leukemia. β-elemene, a component of Curcuma wenyujin, was largely reported as a main active component for anti-tumor effect of Curcuma wenyujin. However, furanodiene, another main component of Curcuma wenyujin, was seldom investigated for its anti-tumor activities. In the present study, we aim to investigate the effect of furanodiene on human leukemia HL60 cells and to study the accurate molecular mechanisms of its action. Methods Trypan blue exclusion experiment was used for cell growth inhibition assay. The apoptotic characterizations were assessed by flow cytometry analysis, AO/EB staining assay and agarose gel eleetrophoresis assay. The expression of apoptosis-related proteins and the release of cytochrome c from mitochondrial were detected by western blotting, and the mRNA levels of TNF-α and TNFR1 were probed by RT-PCR. Formation of TNFR1 complex was analyzed by using immunoprecipitation of TNFR1 with TRRF1/2 and RIP. Results Furanodiene (10-100μM) treatment inhibited the growth of HL60 cells in a concentration-dependent manner. The effect of furanodiene on HL60 cells was associated with the induction of apoptosis, which was characterized by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-8 and caspase-9. In the Bcl-2 family proteins, Bid protein (a substrate of caspase-8) was activated by furanodiene, but Bcl-2, Bax and Bcl-xL proteins were not inuenced by furanodiene stimulation. Furanodiene elicited cytochrome c release from mitochondria into the cytosol. Moreover, furanodiene treatment caused the upregulation of TNFR1, the formation of TNFR1 complex and an obvious production of TNF-a in HL60 cells. The soluble TNFR1 receptor effectively inhibited furanodiene-induced apoptosis. Conclusions Furanodiene

  20. Characterization of a receptor for interleukin-5 on human eosinophils and the myeloid leukemia line HL-60

    Energy Technology Data Exchange (ETDEWEB)

    Ingley, E.; Young, I.G. (Medical Molecular Biology Group, John Curtin School of Medical Research, Australian National University, Canberra (Australia))

    1991-07-15

    Interleukin-5 (IL-5) promotes the growth and differentiation of human eosinophils and may regulate the selective eosinophilia and eosinophil activation seen in certain diseases. Radiolabeled recombinant human IL-5 (hIL-5) was used to characterize the IL-5 receptor present on normal human eosinophils and on the myeloid leukemia line HL-60, which can be induced to differentiate into eosinophilic cells. Binding studies with eosinophils and HL-60 cells grown under alkaline conditions demonstrated similar high-affinity binding sites for hIL-5 on both cell types with kd values of approximately 400 pmol/L. The binding observed was specific in that it was not inhibited by hIL-3, human granulocyte-macrophage colony-stimulating factor, or hIL-2. Binding studies with a number of other human cell lines, including a B-lymphoma line, and with lymphocyte and neutrophil preparations were also performed, but IL-5 receptors were not detectable on these cells. The number of hIL-5 receptors on HL-60 cells could be correlated with its propensity to differentiate towards an eosinophilic cell type. Expression of hIL-5 receptors on HL-60 cells was upregulated by butyric acid under alkaline conditions, downregulated by hIL-3, virtually eliminated by dimethyl sulfoxide and hIL-5, while hIL-2 had no detectable effect. One major 125I-hIL-5-crosslinked complex of 75 to 85 Kd in Mr was detected on HL-60 cells using crosslinking agents giving a molecular mass of 55 to 60 Kd for the hIL-5 receptor itself. Studies using cellular autoradiography showed that IL-5 receptors were evenly distributed on eosinophils but that receptor distribution on HL-60 cells was noticeably heterogeneous. Eosinophils were the only cells in slides prepared from peripheral blood that had detectable levels of IL-5 receptors in agreement with the specific action of IL-5 on the human eosinophil lineage.

  1. Apoptosis-inducing potential of Myrothamnus flabellifolius, an edible medicinal plant, on human myeloid leukemia HL-60 cells

    Directory of Open Access Journals (Sweden)

    J. Dhillon

    2014-02-01

    Full Text Available Summary. Conventional therapies for treating acute myeloid leukemia involve chemotherapy and radiation. This approach causes damage to both normal and cancerous cells resulting in several side effects. There is a dire need to discover novel drugs that selectively targets only the cancer cells with minimal effects on normal cells. Our research is an effort to identify a novel plant based drug which is edible and selectively targets only the leukemic cells with negligible effects on the normal cells. In this study, extracts from Myrothamnus flabellifolius, a South African resurrection plant was used against human leukemic cells (HL-60. M. flabellifolius is known for its anti-viral, anti-microbial and anti-inflammatory properties. Extracts from this plant also contain derivatives of galloyl and quinic acid. In literature, galloyl and quinic acid have been demonstrated to show anti-cancerous effects. Here, we investigated the anti-cancerous effects of the methanolic and petroleum ether extract of this plant on human leukemic cells (HL-60 compared to non-leukemic lymphocytes (TK6. The methanolic extract depicted reduced HL-60 cell viability while the petroleum ether extract did not. The loss in HL-60 viability in response to the methanolic extract was accompanied by the induction of caspase-dependent apoptosis by way of caspase-7 and Poly (ADP-ribose polymerase cleavage. This study establishes an IC50 of 62.5 µg/ml of dry Myrothamnus extract on HL-60 leukemic cells.Industrial Relevance. The outcome of our study depicts the potential of M. flabellifolius as a cancer drug due to its selective biological activity against cancer cells. The anti-cancer effects of this plant extract did not manifest toxic side effects as it did not harm the normal lymphocytic cells. The edible nature of M. flabellifolius marks it as having a potential role in cancer treatment as a complementary medicine to the existing treatment options.Keywords. Myrothamnus

  2. TRIM32 promotes retinoic acid receptor {alpha}-mediated differentiation in human promyelogenous leukemic cell line HL60

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Tomonobu [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan); Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 (Japan); Okumura, Fumihiko [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan); Iguchi, Akihiro; Ariga, Tadashi [Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 (Japan); Hatakeyama, Shigetsugu, E-mail: hatas@med.hokudai.ac.jp [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer TRIM32 enhanced RAR{alpha}-mediated transcriptional activity even in the absence of RA. Black-Right-Pointing-Pointer TRIM32 stabilized RAR{alpha} in the human promyelogenous leukemic cell line HL60. Black-Right-Pointing-Pointer Overexpression of TRIM32 in HL60 cells induced granulocytic differentiation. Black-Right-Pointing-Pointer TRIM32 may function as a coactivator for RAR{alpha}-mediated transcription in APL cells. -- Abstract: Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor {alpha} (RAR{alpha}). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RAR{alpha} and enhances transcriptional activity of RAR{alpha} in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RAR{alpha}, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RAR{alpha}-mediated transcriptional activity even in the absence of RA and stabilizes RAR{alpha} in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RAR{alpha}-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL.

  3. Ultraviolet light-emitting diode irradiation-induced cell death in HL-60 human leukemia cells in vitro

    Science.gov (United States)

    XIE, DONG; SUN, YAN; WANG, LINGZHEN; LI, XIAOLING; ZANG, CHUANNONG; ZHI, YUNLAI; SUN, LIRONG

    2016-01-01

    Ultraviolet (UV) radiation is considered to be a potent cell-damaging agent in various cell lineages; however, the effect of UV light-emitting diode (LED) irradiation on human cells remains unclear. The aim of the present study was to examine the effect of UV LED irradiation emitting at 280 nm on cultured HL-60 human leukemia cells, and to explore the underlying mechanisms. HL-60 cells were irradiated with UV LED (8, 15, 30 and 60 J/m2) and incubated for 2 h after irradiation. The rates of cell proliferation and apoptosis, the cell cycle profiles and the mRNA expression of B-cell lymphoma 2 (Bcl-2) were detected using cell counting kit-8, multicaspase assays, propidium iodide staining and reverse transcription-quantitative polymerase chain reaction, respectively. The results showed that UV LED irradiation (8–60 J/m2) inhibited the proliferation of HL-60 cells in a dose-dependent manner. UV LED at 8–30 J/m2 induced dose-dependent apoptosis and G0/G1 cell cycle arrest, and inhibited the expression of Bcl-2 mRNA, while UV LED at 60 J/m2 induced necrosis. In conclusion, 280 nm UV LED irradiation inhibits proliferation and induces apoptosis and necrosis in cultured HL-60 cells. In addition, the cell cycle arrest at the G0/G1 phase and the downregulation of Bcl-2 mRNA expression were shown to be involved in UV LED-induced apoptosis. PMID:26820261

  4. Differentiation of HL-60 promyelocytes to granulocytes induced via the activation of protein kinase-C by benzene

    Energy Technology Data Exchange (ETDEWEB)

    Carlson, C.; O' Connor, A.; Kalf, G. (Rutgers-the State Univ., Piscataway, NJ (United States) Thomas Jefferson Univ., Philadelphia, PA (United States))

    1991-03-15

    Benzene is a hematotoxin which affects the development of bone marrow progenitor cells and a leukemogen which causes acute myelogenous leukemia. The authors studied the effect of benzene on the differentiation of progenitors of the myeloid lineage, using HL-60 promyelocytic leukemia cells which can be induced to differentiate to granulocytes via the activation of protein kinase-C (PKC) by DMSO and retinoic acid. Exposure of HL-60 cells to 5 mM benzene for 5 min. results in the activation of PKC as measured by an increases in the phosphorylation of cellular proteins in a whole cell assay including proteins pp17 and pp27 reported by Feuerstein and Cooper to be involved in HL-60 cell differentiation. The increase in protein phosphorylation observed with benzene was equally as great as that observed with 100 ng/mL PMA, used as a control. Under the same conditions, benzene induces differentiation of the promyelocytes into granulocytes as measured by the acquisition of superoxide production and granulocyte morphology. Preincubation with 40 {mu}M sphinganine, a PKC inhibitor, prevents the benzene-induced increase in cellular protein phosphorylation and the differentiation to granulocytes. These results indicate that benzene, by activation of PKC, can affect myeloid differentiation which may play a role in the ability of benzene to cause acute myelogenous leukemia.

  5. Ascorbic acid inhibits TPA-induced HL-60 cell differentiation by decreasing cellular H₂O₂ and ERK phosphorylation.

    Science.gov (United States)

    Yiang, Giou-Teng; Chen, Jen-Ni; Wu, Tsai-Kun; Wang, Hsueh-Fang; Hung, Yu-Ting; Chang, Wei-Jung; Chen, Chinshuh; Wei, Chyou-Wei; Yu, Yung-Luen

    2015-10-01

    Retinoic acid (RA), vitamin D and 12-O‑tetradecanoyl phorbol-13-acetate (TPA) can induce HL-60 cells to differentiate into granulocytes, monocytes and macrophages, respectively. Similar to RA and vitamin D, ascorbic acid also belongs to the vitamin family. High‑dose ascorbic acid (>100 µM) induces HL‑60 cell apoptosis and induces a small fraction of HL‑60 cells to express the granulocyte marker, CD66b. In addition, ascorbic acid exerts an anti‑oxidative stress function. Oxidative stress is required for HL‑60 cell differentiation following treatment with TPA, however, the effect of ascorbic acid on HL‑60 cell differentiation in combination with TPA treatment remains to be fully elucidated. The aim of the present study was to investigate the cellular effects of ascorbic acid treatment on TPA-differentiated HL-60 cells. TPA-differentiated HL-60 cells were used for this investigation, this study and the levels of cellular hydrogen peroxide (H2O2), caspase activity and ERK phosphorylation were determined following combined treatment with TPA and ascorbic acid. The results demonstrated that low‑dose ascorbic acid (5 µM) reduced the cellular levels of H2O2 and inhibited the differentiation of HL‑60 cells into macrophages following treatment with TPA. In addition, the results of the present study further demonstrated that low‑dose ascorbic acid inactivates the ERK phosphorylation pathway, which inhibited HL‑60 cell differentiation following treatment with TPA.

  6. The Comparative PDT Experiment of the Inactivation of HL60 on Modified TiO2 Nanoparticles

    Directory of Open Access Journals (Sweden)

    Kaiqi Lu

    2015-01-01

    Full Text Available Four samples of modified titanium dioxide (TiO2, Fe/TiO2 (2 wt%, Fe/TiO2 (5 wt%, and 5-ALA/TiO2, were experimented in photodynamic therapy (PDT on leukemia cells HL60, performing promising photocatalytic inactivation effect. Fe/TiO2 and 5-ALA/TiO2 were synthesized in methods of precipitation and ultrasonic methods, respectively. X-ray diffraction spectra and UV-Vis spectra were studied for the samples’ crystalline phase and redshift of absorption peak. Further, FTIR spectra and Raman spectra were obtained to examine the combination of 5-aminolevulinic (5-ALA and TiO2 nanoparticles. The toxicity of these four kinds of nanoparticles was studied through darkroom experiments. And based on the concentration which caused the same toxic effect (90% on HL60, PDT experiments of TiO2, Fe/TiO2 (2%, Fe/TiO2 (5%, and ALA/TiO2 were done, resulting in the fact that the photokilling efficiency was 69.7%, 71.6%, 72%, and 80.6%, respectively. Scanning electron microscope (SEM images of the samples were also taken to study the morphology of HL60 cells before and after PDT, resulting in the fact the activation of the modified TiO2 from PDT was the main cause of cell apoptosis.

  7. Chemical modulation of the ultra-weak photon emission from Saccharomyces cerevisiae and differentiated HL-60 cells

    Science.gov (United States)

    Červinková, Kateřina; Nerudová, Michaela; Hašek, Jiří; Cifra, Michal

    2015-01-01

    The ultra-weak photon emission (UPE) is a universal phenomenon common to all cells with active oxidative metabolism. Generally accepted mechanism of the origin of the ultra-weak photon emission considers reactions of radical or nonradical reactive oxygen species (ROS) with biomolecules such as lipids and proteins which lead to the formation of electron excited species. During the transition to the ground state the excess energy is released as a photon with a wavelength in the visible range of the electromagnetic spectrum. Since the intensity of the light is very low it is possible to be measured only by highly sensitive devices. We used Hamamatsu Photonics PMT module H7360-01 mounted into a light-tight chamber for the purposes of this work. The goal of our research is to delineate an origin of UPE from two model organisms; differentiated HL-60 cells (human promyelocytic leukemia) and yeast cells Saccharomyces cerevisiae. While the UPE from the yeast cells arises spontaneously during the growth without any external stimuli, UPE from HL-60 is induced by phorbol 12-myristate, 13-acetate (PMA). It is possible to modulate the UPE production by certain antioxidants which scavenge ROS formed during the metabolism (yeast cells) or respiratory burst (HL-60 cells). The experiments are focused on the description of effects caused by antioxidants. Several kinds of antioxidants (ascorbic acid, mannitol, glutathione) with different concentration were used and we studied the changes in the UPE intensities of and the temporal developments of the optical signal.

  8. Regulated expression of the MRP8 and MRP14 genes during terminal differentiation of human promyelocytic leukemic HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Warner-Bartnicki, A.L.; Murao, S.; Collart, F.R.; Huberman, E.

    1992-02-14

    The calcium-binding proteins MRP8 and MRP14 are induced during monomyelocytic cell maturation and may mediate the growth arrest in differentiating HL-60 cells. We determined the levels of a protein complex (PC) containing MRP8 and MRP14 and investigated the mechanism by which the genes encoding these proteins are regulated in HL-60 cells treated with the differentiation-inducing agent mycophenolic acid. Elevated levels of the PC were found to directly parallel gains in the steady-state levels of MRP8 and MRP14 mRNA. Transcription studies with the use of nuclear run-on experiments revealed increased transcription initiation at the MRP8 and MRP14 promoters after MPA treatment. 1{alpha},25-Dihydroxyvitamin D{sub 3}, which induces HL-60 cell differentiation by another mechanism, was also found to increase transcription initiation at the MRP8 and MRP14 promoters, suggesting that this initiation is the major control of MRP8 and MRP14 gene expression during terminal differentiation of human promyelocytic cells.

  9. The effect of cytoplasmic regions of LIF receptor on activating MAPK p42/44 in HL-60 cell

    Institute of Scientific and Technical Information of China (English)

    LIU Hou-qi; SHAN Ji-kuan; TANG Shu-ping; LIU Shan-rong; JI Kai-hong

    2001-01-01

    Objective: To study the relation between the cytoplasmic region of each subunit of leukemia inhibitory factor (LIF) receptor and mitogen-activated protein kinase (MAPK) in human leukemia line HL-60 for studying the mechanism of leukemia cell proliferation disorder. Methods: We prepared the chimerical receptor with exchanged cytoplasmic domain (190/130,130/190), expressed it on the membrane of HL-60, and then bound LIF with the wild type receptor competitively. The cytoplasmic region homodimerization (190cyt- 190cyt, 130cyt- 130cyt) initiated intracellular signal transduction, cell proliferation and differentiation. MAPK phosphorylation level was observed by means of immunoblotting and immunobiochemistry. Results: Higher level ofMAPK p42/p44 phosphorylation (Thr202Tyr204) and faster cell proliferation were determined in group 190/130 compared with the wild type receptor. Those in group 130/190 were lower than the others. Conclusion: The cytoplasmic domain ofgpl30 involves in the induction of MAPK activation and the cell proliferation of HL-60.

  10. Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals.

    Science.gov (United States)

    Khan, Saifur R; Baghdasarian, Argishti; Nagar, Prarthna H; Fahlman, Richard; Jurasz, Paul; Michail, Karim; Aljuhani, Naif; Siraki, Arno G

    2015-09-01

    In this study, the cellular effects resulting from the metabolism of aminoglutethimide by myeloperoxidase were investigated. Human promyelocytic leukemia (HL-60) cells were treated with aminoglutethimide (AG), an arylamine drug that has a risk of adverse drug reactions, including drug-induced agranulocytosis. HL-60 cells contain abundant amounts of myeloperoxidase (MPO), a hemoprotein, which catalyzes one-electron oxidation of arylamines using H2O2 as a cofactor. Previous studies have shown that arylamine metabolism by MPO results in protein radical formation. The purpose of this study was to determine if pathways associated with a toxic response could be determined from conditions that produced protein radicals. Conditions for AG-induced protein radical formation (with minimal cytotoxicity) were optimized, and these conditions were used to carry out proteomic studies. We identified 43 proteins that were changed significantly upon AG treatment among which 18 were up-regulated and 25 were down-regulated. The quantitative proteomic data showed that AG peroxidative metabolism led to the down-regulation of critical anti-apoptotic proteins responsible for inhibiting the release of pro-apoptotic factors from the mitochondria as well as cytoskeletal proteins such as nuclear lamina. This overall pro-apoptotic response was confirmed with flow cytometry which demonstrated apoptosis to be the main mode of cell death, and this was attenuated by MPO inhibition. This response correlated with the intensity of AG-induced protein radical formation in HL-60 cells, which may play a role in cell death signaling mechanisms.

  11. Proliferation inhibition, cell cycle arrest and apoptosis induced in HL-60 cells by a natural diterpene ester from Daphne mucronata

    Science.gov (United States)

    Nouri, K.; Yazdanparast, R.

    2011-01-01

    Background and the purpose of the study Gnidilatimonoein (Gn), a new diterpene ester from Daphne mucronata, possesses strong anti-metastasis and anti-tumor activities. In this study, its apoptosis and differentiation capabilities were evaluated by using the leukemia HL-60 cell line. Material and methods Cell prolifaration inhibition was estimated by MTT assay. The occurrence of apoptosis was evaluated by EtBr/AO double staining technique, cell cycle analyses and detection of apoptotic cells by Annexin V-FITC and propodium iodide (PI). Differentiation of the cells was determined by NBT reduction assay and the expression of specific cell surface markers such as CD14 and CD11b, were analyzed by flow cytometry. Results The drug decreased the growth of the cells dose- and time-dependently and the IC50 was found to be 1.3 µM. Our data suggested that Gn induced both monocytic differentiation and apoptosis among HL-60 cells. In addition, cell cycle analyses showed an increase in G1 phase population by 24 hrs, which was gradually replaced by Sub-G1 cell population (apoptotic cells) by 72 hrs. Conclusion Based on these data, the Gn-treated HL-60 cells displayed differentiation-dependent apoptosis. Thus, Gn might be a good candidate for differentiation therapy of leukemia, pending full biological evaluation of the compound among the wide array of leukemia cells. PMID:22615651

  12. Cytotoxic and apoptotic effects of different extracts of Artemisia biennis Willd. on K562 and HL-60 cell lines

    Directory of Open Access Journals (Sweden)

    Zahra Tayarani-Najaran

    2017-02-01

    Full Text Available Objective(s: Artemisia is a genus of herbs and small shrubs forms an important part of natural vegetation in Iran. It has been reported that several Artemisia species possess anti-proliferative effects. Considering the value of this genus in anti-cancer researches we have chosen Artemisia biennis for cytotoxic and mechanistic studies. Materials and Methods:In this study we have investigated the cytotoxic and apoptotic effects of petroleum ether, dichloromethane, ethyl acetate, ethanol, and ethanol:water (1:1 v/v extracts of A. biennis Willd. on two cancer human cell lines (K562 and HL-60 and J774 as normal cells. Results: CH2Cl2 extract was found to have the highest anti-proliferative effect on cancer cells. IC50 values obtained in AlamarBlue® assay for CH2Cl2 extract were 64.86 and 54.31 µg/ml on K562 and HL-60 cells respectively. In flow cytometry histogram of the cells treated with CH2Cl2 extract, sub-G1 peak was induced. DNA fragmentation, increased in the level of Bax and cleavage of PARP protein all showed the induction of apoptosis with CH2Cl2 extract after 48 hr contact with cells. Conclusion: The results can corroborate the cytotoxic and apoptotic effects of the CH2Cl2 extract of A. biennis on the K562 and HL-60 cancer cell lines.

  13. Using cell fate attractors to uncover transcriptional regulation of HL60 neutrophil differentiation

    Directory of Open Access Journals (Sweden)

    Kauffman Stuart A

    2009-02-01

    Full Text Available Abstract Background The process of cellular differentiation is governed by complex dynamical biomolecular networks consisting of a multitude of genes and their products acting in concert to determine a particular cell fate. Thus, a systems level view is necessary for understanding how a cell coordinates this process and for developing effective therapeutic strategies to treat diseases, such as cancer, in which differentiation plays a significant role. Theoretical considerations and recent experimental evidence support the view that cell fates are high dimensional attractor states of the underlying molecular networks. The temporal behavior of the network states progressing toward different cell fate attractors has the potential to elucidate the underlying molecular mechanisms governing differentiation. Results Using the HL60 multipotent promyelocytic leukemia cell line, we performed experiments that ultimately led to two different cell fate attractors by two treatments of varying dosage and duration of the differentiation agent all-trans-retinoic acid (ATRA. The dosage and duration combinations of the two treatments were chosen by means of flow cytometric measurements of CD11b, a well-known early differentiation marker, such that they generated two intermediate populations that were poised at the apparently same stage of differentiation. However, the population of one treatment proceeded toward the terminally differentiated neutrophil attractor while that of the other treatment reverted back toward the undifferentiated promyelocytic attractor. We monitored the gene expression changes in the two populations after their respective treatments over a period of five days and identified a set of genes that diverged in their expression, a subset of which promotes neutrophil differentiation while the other represses cell cycle progression. By employing promoter based transcription factor binding site analysis, we found enrichment in the set of divergent

  14. Expression of costimulatory molecule CD86 in HL-60 cells induced by MG132 and its effect on allogeneic mixed lymphocyte reaction.

    Science.gov (United States)

    Yu, Mei-Xia; Liu, Xun; Zhou, Yong-Ming; Cheng, Yan-Xiang; Cheng, Jing; Qiu, Yu-Zhen; Xing, Xiao-Lei; Yao, Chun-Hong; Bai, Ru-Jun

    2014-10-01

    This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P HL-60 treated with MG132 was up-regulated time-dependently (P HL-60 cells treated with MG132 and reached its peak when the concentration

  15. EGCG抑制HL-60细胞增殖作用的机制研究%Study of EGCG Inhibited Proliferation of HL-150 Cells

    Institute of Scientific and Technical Information of China (English)

    欧阳琳; 姜浩

    2011-01-01

    目的 探讨EGCG抑制白血病HL-60细胞增殖的作用机制.方法 采用软琼脂集落形成实验和绘制细胞生长曲线,检测EGCG对HL-60细胞增殖的影响;FCM及DNA凝胶电泳法检测ECCG处理HL-60细胞后的凋亡情况;实时荧光定量RT-PCR和Wester blotting检测AKT1的表达.结果 细胞生长曲线和软琼脂集落形成实验结果均显示EGCG呈浓度依赖性抑制HL-60细胞的增殖(P<0.05);FCM及DNA凝胶电泳法结果显示EGCG呈浓度依赖性诱导HL-60细胞的凋亡(P<0.05).实时荧光定量RT-PCR和Wester blotting结果显示EGCG浓度升高,HL-60细胞中AKT1的表达降低.结论 EGCG可能通过下调AKT1的表达,促进细胞凋亡,发挥其对HL-60细胞增殖的抑制作用.

  16. Diallyl disulfide suppresses growth of HL-60 cell through increasing his-tone acetylation and p21WAF1 expression in vivo and in vitro

    Institute of Scientific and Technical Information of China (English)

    Jie ZHAO; Wei-guo HUANG; Jie HE; Hui TAN; Qian-jin LIAO; Qi SU

    2006-01-01

    Aim: To examine the differentiation induction and growth inhibition of HL-60 cells by diallyl disulfide (DADS), and its relationship with the alterations of histone acetylation and p21WAF1 expression in vitro and in vivo. Methods: Differentiation was studied by nitroblue tetrazolium (NET) reduction of HL-60 cell in vitro. HL-60 cells 5xl06 were injected into the right side of the peritoneal cavity of severe combined immunodeficiency (SCID) mice. When the peritoneal neoplasms were detected, the SCID mice were randomly divided into 3 groups and received an ip injection of vehicle alone (NS), DADS or sodium butyrate (SB). The growth inhibition of peritoneal neoplasms induced by DADS was observed by a growth curve. The cycle distribution of HL-60 cells in SCID mice was monitored by flow cytometry. The expression of acetylated histone H3, H4 and p21WAF1 were measured by Western blot. Results: After treatment with DADS for 0-72 h, the NET reduction ability of HL-60 cells increased in a time-dependent manner, compared with no treatment of HL-60 cells. In the HL-60 cells treated with DADS for 24 h, the expression of acetylated histone H3, H4, and p21WAF1 increased obviously. After treatment with DADS, tumor growth was markedly suppressed. HL-60 cells from mice treated with DADS were blocked in the G1 phase, from 25.4% to 63.4%. The tumors from the mice treated with DADS showed an increase of acetylated histone H3, H4, and p21WAF1. Conclusion: DADS could induce differentiation and inhibit the growth of HL-60 cells through increasing the expression of acetylated histone H3, H4, and p21WAF1 in vitro and in vivo.

  17. The Effect of Telomerase Antisense RNA on Telomerase Activity and Cell Proliferation of HL-60%端粒酶反义RNA 对HL-60细胞端粒酶活性及细胞增殖的影响

    Institute of Scientific and Technical Information of China (English)

    刘利; 孙秉中; 梁英民; 张传山; 张惠中; 阎严; 张方信

    2001-01-01

    Objective To study the effect of telomerase antisense RNA ontelomerase activity and cell proliferation of HL-60 cells.Methods By using lipofectin mediated DNA transfection,telomerase antisense RNA was successfully introduced into HL-60 cells.By means of TRAP、dynamics of cell growth、flucytometry the changes of HL-60 cells in aspect of telomerase activity,proliferation were detected.Results Telomerase antisense RNA can significantly inhibit the telomerase activity and the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells.Conclusion It seems to be a new methods to treat leukemia by using telomerase antisense to inhibit telomerase activity and the proliferation of HL-60 cells.telomerase is a new latent target to treat leukemia.%目的 探讨端粒酶反义RNA对HL-60细胞端粒酶活性及细胞增殖的影响。方法 用脂质体介导法将pBBS212-hTR(反义端粒酶RNA真核表达载体)导入人髓性白血病细胞系HL-60中,采用TRAP法测定端粒酶活性,细胞生长动力学检测,FCM分析等方法观察其对HL-60细胞的端粒酶活性及细胞增殖影响。结果 端粒酶反义RNA能显著抑制HL-60细胞的端粒酶活性,抑制HL-60细胞的增殖并诱导其凋亡。结论 以端粒酶反义RNA抑制端粒酶活性是治疗白血病的潜在途径。

  18. 24- and 26-homo-1,25-dihydroxyvitamin D/sub 3/: preferential activity in inducing differentiation of human leukemia cells HL-60 in vitro inducing differentiation of human leukemia cells HL-60 in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Ostrem, V.K.; Tanaka, Y.; Prahl, J.; DeLuca, H.F.; Ikekawa, N.

    1987-05-01

    1,25-Dihydroxyvitamin D/sub 3/, the hormonal form of vitamin D/sub 3/, promotes the differentiation of HL-60 human promyelocytic leukemia cells into monocytes. Differentiation changes include the induction of phagocytosis, the initiation of nitroblue tetrazolium-reducing activity, and the appearance of nonspecific acid esterase. The authors have found that the 24-homo- and 26-homo-1,25-dihydroxyvitamin D/sub 3/ and their ..delta../sup 22/ analogues are 10-fold more potent than 1,25-dihydroxyvitamin D/sub 3/ in inducing differentiation of HL-60 cells in vitro. In vivo, these analogues show activity similar to 1,25-dihydroxy-vitamin D/sub 3/ in stimulating intestinal calcium transport in vitamin D-deficient rats. The 24-homoanalogues are significantly less active, whereas the 26-homo derivatives are more active than the natural hormone in mobilizing calcium from bone. This unusual activity pattern cannot be explained on the basis of the affinity of these analogues for the 1,25-dihydroxy-vitamin D/sub 3/ intracellular receptor: both 24-homo- and 26-homo-1,25-dihydroxyvitamin D/sub 3/ have the same effectiveness as 1,25-dihydroxyvitamin D/sub 3/ in displacing the tritiated hormone from its receptor in rat intestine of HL-60 cells. These analogues of 1,25-dihydroxyvitamin D/sub 3/ may be of some interest as possible therapeutic substances, or as tools in understanding the action of 1,25-dihydroxyvitamin D/sub 3/ in inducing differentiation.

  19. Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells

    National Research Council Canada - National Science Library

    Lee, Wei-Jiunn; Chen, Yun-Ru; Tseng, Tsui-Hwa

    2011-01-01

    .... In the present study, by evaluation of fragmentation of DNA, poly (ADP-ribose) polymerase (PARP) and procaspases, we found that quercetin was able to induce apoptosis of human leukemia HL-60 cells in a dose-dependent manner...

  20. EFFECTS OF QUERCETIN ON CELL MORPHOLOGY AND EXPRESSION OF PML mRNA AND PROTEIN OF NB4 AND HL-60 CELLS

    Institute of Scientific and Technical Information of China (English)

    钟璐; 陈芳源; 韩洁英; 邵念贤; 欧阳仁荣

    2001-01-01

    Objective To investigate the effects of quercetin on cell morphology, expression of promyelocytic leukemia ( PML ) mRNA and PML protein localization of NB4, HL-60 cells. Methods Cells morphology assayed by Wright's stain, fluorescence stain, and PML mRNA expression by RT-PCR , PML protein localization by immuno-fluorescence. Results The typical apoptosis was found in NB4 and HL-60 cells after treatment with quercetin . Immuno-fluorescence analysis showed after treatment with quercetin , the fusion protein disappeared in NB4 cells, PML protein relocated, then degraded, and that also seen in HL-60 cells. The expression of PML mRNA is not changed in quercetin-treated cells. Conclusion PML play the role of apoptosis inducer in leukemia cells at the translational level, quercetin can inhibit the proliferation of leukemia cells, and induce NB4, HL-60

  1. The induction of monocytopoiesis in HL-60 promyelocytic leukemia cells is inhibited by hydroquinone, a toxic metabolite of benzene

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, N.L.

    1992-01-01

    Chronic exposure of humans to benzene has been shown to have a cytotoxic effect on hematopoietic progenitor cells in intermediate stages of differentiation which can lead to aplastic anemia and acute myelogenous leukemia. This thesis examined the effect of hydroquinone, a toxic metabolite of benzene found in the bone marrow, on the human promyelocytic leukemia cell line (HL-60) which can be induced to differentiate to both monocyte and myeloid cells, and thus has been used as a surrogate for a granulocyte/macrophage progenitor cell. Exposure of HL-60 cells to noncytotoxic concentrations of hydroquinone for three hours prior to induction with 12-O-tetradecanoyl phorbol-13-acetate caused a dose-dependent inhibition of the acquisition of characteristics of monocytic differentiation. These included adherence, nonspecific esterase activity and phagocytosis. Hydroquinone had no effect on cell proliferation. Hydroquinone appeared to be affecting maturation beyond the monoblast/promonocyte stages. Hydroquinone also prevented differentiation induced by 1, 25-dihydroxy vitamin D[sub 3], however, the block occurred after the acquisition of adherence. Hydroquinone at concentrations that inhibited monocytic differentiation had no effect on differentiation to granulocytes, suggesting that the block in the differentiation of these bipotential cells is at a step unique to the monocytic pathway. Hydroquinone was unable to prevent differentiation induced by the macrophage-derived cytokine interleukin-1, a differentiation factor for cells of the monocytic lineage. These data demonstrate that treatment of Hl-60 cells with hydroquinone prior to induction of differentiation prevents the acquisition of the monocytic phenotype induced by TPA or 1, 25(OH)[sub 2]D[sub 3] by a mechanism which at present is unknown, but which appears to be specific for the monocytic pathway. These results are of considerable significance for benzene hematotoxicity.

  2. Mangiferin increases Nrf2 protein stability by inhibiting its ubiquitination and degradation in human HL60 myeloid leukemia cells.

    Science.gov (United States)

    Zhao, Jie; Zhang, Benping; Li, Shanshan; Zeng, Linglan; Chen, Yan; Fang, Jun

    2014-05-01

    The nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signaling pathway is a key target for cancer chemoprevention. Recent studies have that Nrf2 activation may be the result of an increase in Nrf2 protein stability. Mangiferin (MA), a compound monomer extracted from the mango plant, has antioxidant and cytoprotective activities. Our previous study demonstrated that MA increased Nrf2 expression and activated Nrf2 signaling in hematopoietic cells. Thus, in the present study, we aimed to investigate the mechanisms by which MA increases Nrf2 expression in human HL60 myeloid leukemia cells in vitro. Our western blot analysis results revealed that MA markedly increased Nrf2 expression in dose- and time-dependent manner. However treatment with MA did not affect the Nrf2 mRNA level. The results of cycloheximide (CHX)-chase analysis demonstrated that the Nrf2 protein half-life was prolonged to 58 min when the HL60 cells were pre-incubated with 50 µM MA for 4 h, whereas its half-life was only 20 min in the non-MA treated control cells. Further experiments revealed that MA mainly enhanced non-ubiquitinated Nrf2 protein levels when increasing Nrf2 protein stability; these effects differed from those induced by the proteasome inhibitor, MG132. Subsequent immunoprecipitation experiments confirmed that MA inhibited Nrf2 ubiquitination in HL60 cells. These results provide evidence that MA increases Nrf2 protein stability by inhibiting its ubiquitination and degradation in hematopoietic cells. This may be one of the mechanisms through which MA activates the Nrf2-mediated antioxidant response and exerts cytoprotective effects.

  3. BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

    Science.gov (United States)

    Balcer-Kubiczek, E. K.; Zhang, X. F.; Han, L. H.; Harrison, G. H.; Davis, C. C.; Zhou, X. J.; Ioffe, V.; McCready, W. A.; Abraham, J. M.; Meltzer, S. J.

    1998-01-01

    We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

  4. Cytotoxic effects of tetracycline analogues (doxycycline, minocycline and COL-3 in acute myeloid leukemia HL-60 cells.

    Directory of Open Access Journals (Sweden)

    Hairong Song

    Full Text Available Tetracycline analogues (TCNAs have been shown to inhibit matrix metalloproteinases and to induce apoptosis in several cancer cell types. In the present study, the cytotoxic effects of TCNAs doxycycline (DOXY, minocycline (MINO and chemically modified tetracycline-3 (COL-3 were investigated in the human acute myeloid leukemia HL-60 cell line. Cells were incubated with TCNAs in final concentrations of 0.5-100 µg/ml for 24 h. Viability of the leukemic cells was inhibited in a concentration-dependent manner using resazurin assay. The estimated IC50s were 9.2 µg/ml for DOXY, 9.9 µg/ml for MINO and 1.3 µg/ml for COL-3. All three TCNAs induced potent cytotoxic effects and cell death. Apoptosis, which was assessed by morphological changes and annexin V positivity, was concentration- and time-dependent following incubation with any one of the drugs. TCNAs induced DNA double strand breaks soon after treatment commenced as detected by γH2AX and western blot. The loss of mitochondrial membrane potential (Δψm, caspase activation and cleavage of PARP and Bcl-2 were observed; however, the sequence of events differed among the drugs. Pancaspase inhibitor Z-VAD-FMK improved survival of TCNAs-treated cells and decreased TCNAs-induced apoptosis. In summary, we demonstrated that TCNAs had a cytotoxic effect on the HL-60 leukemic cell line. Apoptosis was induced via mitochondria-mediated and caspase-dependent pathways in HL-60 cells by all three TCNAs. COL-3 exerted the strongest anti-proliferative and pro-apoptotic effects in concentrations that have been achieved in human plasma in reported clinical trials. These results indicate that there is a therapeutic potential of TCNAs in leukemia.

  5. Cytotoxic effects of tetracycline analogues (doxycycline, minocycline and COL-3) in acute myeloid leukemia HL-60 cells.

    Science.gov (United States)

    Song, Hairong; Fares, Mona; Maguire, Kim R; Sidén, Ake; Potácová, Zuzana

    2014-01-01

    Tetracycline analogues (TCNAs) have been shown to inhibit matrix metalloproteinases and to induce apoptosis in several cancer cell types. In the present study, the cytotoxic effects of TCNAs doxycycline (DOXY), minocycline (MINO) and chemically modified tetracycline-3 (COL-3) were investigated in the human acute myeloid leukemia HL-60 cell line. Cells were incubated with TCNAs in final concentrations of 0.5-100 µg/ml for 24 h. Viability of the leukemic cells was inhibited in a concentration-dependent manner using resazurin assay. The estimated IC50s were 9.2 µg/ml for DOXY, 9.9 µg/ml for MINO and 1.3 µg/ml for COL-3. All three TCNAs induced potent cytotoxic effects and cell death. Apoptosis, which was assessed by morphological changes and annexin V positivity, was concentration- and time-dependent following incubation with any one of the drugs. TCNAs induced DNA double strand breaks soon after treatment commenced as detected by γH2AX and western blot. The loss of mitochondrial membrane potential (Δψm), caspase activation and cleavage of PARP and Bcl-2 were observed; however, the sequence of events differed among the drugs. Pancaspase inhibitor Z-VAD-FMK improved survival of TCNAs-treated cells and decreased TCNAs-induced apoptosis. In summary, we demonstrated that TCNAs had a cytotoxic effect on the HL-60 leukemic cell line. Apoptosis was induced via mitochondria-mediated and caspase-dependent pathways in HL-60 cells by all three TCNAs. COL-3 exerted the strongest anti-proliferative and pro-apoptotic effects in concentrations that have been achieved in human plasma in reported clinical trials. These results indicate that there is a therapeutic potential of TCNAs in leukemia.

  6. Effects of PCB126 and PCB153 on telomerase activity and telomere length in undifferentiated and differentiated HL-60 cells.

    Science.gov (United States)

    Xin, Xing; Senthilkumar, P K; Schnoor, Jerald L; Ludewig, Gabriele

    2016-02-01

    PCBs are persistent organic pollutants that are carcinogenic and immunotoxic and have developmental toxicity. This suggests that they may interfere with normal cell maturation. Cancer and stem/progenitor cells have telomerase activity to maintain and protect the chromosome ends, but lose this activity during differentiation. We hypothesized that PCBs interfere with telomerase activity and the telomere complex, thereby disturbing cell differentiation and stem/progenitor cell function. HL-60 cells are cancer cells that can differentiated into granulocytes and monocytes. We exposed HL-60 cells to PCB126 (dioxin-like) and PCB153 (nondioxin-like) 6 days before and during 3 days of differentiation. The differentiated cells showed G0/G1 phase arrest and very low telomerase activity. hTERT and hTR, two telomerase-related genes, were downregulated. The telomere shelterins TRF1, TRF2, and POT1 were upregulated in granulocytes, and TRF2 was upregulated and POT1 downregulated in monocytes. Both PCBs further reduced telomerase activity in differentiated cells, but had only small effects on the differentiation and telomere-related genes. Treatment of undifferentiated HL-60 cells for 30 days with PCB126 produced a downregulation of telomerase activity and a decrease of hTERT, hTR, TRF1, and POT1 gene expression. With PCB153, the effects were less pronounced and some shelterin genes were increased after 30 days of exposure. With each PCB, no differentiation of cells was observed and cells continued to proliferate despite reduced telomerase activity, resulting in shortened telomeres after 30 days of exposure. These results indicate cell-type and PCB congener-specific effects on telomere/telomerase-related genes. Although PCBs do not seem to strongly affect differentiation, they may influence stem or progenitor cells through telomere attrition with potential long-term consequences for health.

  7. Arachidonic acid enhances TPA-induced differentiation in human leukemia HL-60 cells via reactive oxygen species-dependent ERK activation.

    Science.gov (United States)

    Chien, Chih-Chiang; Wu, Ming-Shun; Shen, Shing-Chuan; Yang, Liang-Yo; Wu, Wen-Shin; Chen, Yen-Chou

    2013-04-01

    The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent stimulator of differentiation in human leukemia cells; however, the effects of arachidonic acid (AA) on TPA-induced differentiation are still unclear. In the present study, we investigated the contribution of AA to TPA-induced differentiation of human leukemia HL-60 cells. We found that treatment of HL-60 cells with TPA resulted in increases in cell attachment and nitroblue tetrazolium (NBT)-positive cells, which were significantly enhanced by the addition of AA. Stimulation of TPA-induced intracellular reactive oxygen species (ROS) production by AA was detected in HL-60 cells via a DCHF-DA analysis, and the addition of the antioxidant, N-acetyl-cysteine (NAC), was able to reduce TPA+AA-induced differentiation in accordance with suppression of intracellular peroxide elevation by TPA+AA. Furthermore, activation of extracellular-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) by TPA+AA was identified in HL-60 cells, and the ERK inhibitor, PD98059, but not the JNK inhibitor, SP600125, inhibited TPA+AA-induced NBT-positive cells. Suppression of TPA+AA-induced ERK protein phosphorylation by PD98059 and NAC was detected, and AA enhanced ERK protein phosphorylation by TPA was in HL-60 cells. AA clearly increased TPA-induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression, which was inhibited by NAC and PD98059 addition. Eicosapentaenoic acid (EPA) as well as AA showed increased intracellular peroxide production and differentiation of HL-60 cells elicited by TPA. Evidence of AA potentiation of differentiation by TPA in human leukemia cells HL-60 via activation of ROS-dependent ERK protein phosphorylation was first demonstrated herein.

  8. The Comparative PDT Experiment of the Inactivation of HL60 on Modified TiO2 Nanoparticles

    OpenAIRE

    Kaiqi Lu; Qiyun He; Li Chen; Baoquan Ai; Jianwen Xiong

    2015-01-01

    Four samples of modified titanium dioxide (TiO2), Fe/TiO2 (2 wt%), Fe/TiO2 (5 wt%), and 5-ALA/TiO2, were experimented in photodynamic therapy (PDT) on leukemia cells HL60, performing promising photocatalytic inactivation effect. Fe/TiO2 and 5-ALA/TiO2 were synthesized in methods of precipitation and ultrasonic methods, respectively. X-ray diffraction spectra and UV-Vis spectra were studied for the samples’ crystalline phase and redshift of absorption peak. Further, FTIR spectra and Raman spec...

  9. Immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated HL-60 cells, granulocytes, and monocytes

    DEFF Research Database (Denmark)

    Boje, Alex; Moesby, Lise; Timm, Michael;

    2012-01-01

    that testosterone at pharmacological doses reduced the production of interleukin-8 and reactive oxygen species from differentiated HL-60 cells in a concentration dependent manner without affecting phagocytosis. The cells were stimulated with zymosan, lipopolysaccharide, or Bacillus subtilis. At the highest...... concentration of testosterone (120 µM), interleukin-8 secretion was reduced 42-80%, and production of reactive oxygen species was reduced 32-46%. Flutamide, an antagonist of the classical intracellular androgen receptor, was unable to antagonize the immunosuppressive effect of testosterone. We further...

  10. Bryostatin, an activator of the calcium phospholipid-dependent protein kinase, blocks phorbol ester-induced differentiation of human promyelocytic leukemia cells HL-60

    Energy Technology Data Exchange (ETDEWEB)

    Kraft, A.S.; Smith, J.B.; Berkow, R.L.

    1986-03-01

    Phorbol esters bind to and activate a calcium phospholipid-dependent protein kinase (C kinase). Some researchers believe that activation of C kinase is necessary for the induction of phorbol ester biologic effects. The authors' research indicates that bryostatin, a macrocyclic lactone that binds to the phorbol ester receptor in human polymorphonuclear leukocytes, also binds to this receptor in the human promyelocytic leukemia cell line, HL-60. Bryostatin activates partially purified C kinase from HL-60 cells in vitro, and when applied to HL-60 cells in vivo, it decreases measurable cytoplasmic C kinase activity. Unlike the phorbol esters, bryostatin is unable to induce a macrophage-like differentiation of HL-60 cells; however, bryostatin, in a dose-dependent fashion, blocks phorbol ester-induced differentiation of HL-60 cells and, if applied within 48 hr of phorbol esters, halts further differentiation. These results suggest that activation of the C kinase by some agents is not sufficient for induction of HL-60 cell differentiation and imply that some of the biologic effects of phorbol esters may occur through a more complex mechanism than previously thought.

  11. CD14 mediated endogenous TNF-alpha release in HL60 AML cells: a potential model for CD14 mediated endogenous cytokine release in the treatment of AML.

    Science.gov (United States)

    Treon, S P; Anand, B; Ulevitch, R; Broitman, S A

    1994-01-01

    In previous studies, HL60 AML cells treated with tumor necrosis factor-alpha (TNF), interferon-gamma (IFN), and lipopolysaccharides (LPS) displayed decreased growth and viability, enhanced monocytic pathway differentiation and endogenous TNF release. Endogenous TNF release by LPS/TNF/IFN treated HL60 cells was postulated to play a role with the above findings. In these studies, HL60 cells expressed CD14 when treated with TNF, IFN, and LPS. CD14 mediates TNF release in monocytes/macrophages in response to binding of LPS with LPS binding protein (LBP). CD14 was not expressed in either untreated or LPS only treated HL60 cells. CD14 expression was present and greater with HL60 cells cultured with LPS/TNF/IFN vs TNF/IFN (47.47% vs 9.07% positive, respectively) suggesting synergism for LPS in CD14 induction. CD14 expression was associated with endogenous TNF release, and with significantly higher levels by HL60 cells treated with LPS/TNF/IFN vs TNF/IFN (p < 0.001). Addition of anti-CD14 antibody significantly reduced release of TNF in TNF/IFN (p < 0.001) and LPS/TNF/IFN (p = 0.0013) treated cells. KG1 and U937 AML cells treated with LPS, TNF, and IFN did not express CD14, nor release TNF. A model for inducing release of endogenous growth inhibitory cytokines by CD14 bearing AML cells is proposed as an approach to AML therapy.

  12. Propofol Treatment Inhibits Constitutive Apoptosis in Human Primary Neutrophils and Granulocyte-Differentiated Human HL60 Cells

    Science.gov (United States)

    Hsing, Chung-Hsi; Chen, Chia-Ling; Lin, Wei-Chieh; Lin, Chiou-Feng

    2015-01-01

    Apoptosis regulation is essential for neutrophil homeostasis. We previously demonstrated that a process involving glycogen synthase kinase (GSK)-3β determines neutrophil apoptosis. As for this apoptotic process, an overdose of propofol (2,6-Diisopropylphenol; 25 μg/ml or 140 μM) also causes GSK-3β-mediated macrophage apoptosis; however, the early deactivation of GSK-3β with low-dose propofol has been shown. Therefore, we hypothesize that low-dose propofol may induce neutrophil survival via GSK-3β inactivation. Following in vitro culture, the therapeutic concentration of propofol (10 μg/ml or 56 μM) treatment decreased constitutive apoptosis in isolated human primary neutrophils and in granulocyte-differentiated HL60 cells after all-trans retinoic acid (1 μM) treatment. The inactivation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT and the activation of GSK-3β results in myeloid cell leukemia 1 (Mcl-1) down-regulation, the loss of the mitochondrial transmembrane potential, and caspase-3 activation in these cells, which is accompanied by apoptosis. Notably, propofol treatment attenuates these effects in a PI3-kinase-regulated manner. We found that propofol initiates PI3-kinase/AKT-mediated GSK-3β inactivation and Mcl-1 stabilization, rescuing the constitutive apoptosis in primary neutrophils and granulocyte-differentiated acute promyelocytic leukemia HL60 cells. PMID:26061531

  13. Effect of myeloperoxidase inhibition on gene expression profiles in HL-60 cells exposed to 1,2,4,-benzenetriol.

    Science.gov (United States)

    Miyahara, Emiko; Nishikawa, Takuro; Takeuchi, Toru; Yasuda, Kaori; Okamoto, Yasuhiro; Kawano, Yoshifumi; Horiuchi, Masahisa

    2014-03-20

    While it is known that benzene induces myeloid leukemia in humans, the mechanism has yet to be clarified. Previously, we suggested that myeloperoxidase (MPO) was the key enzyme because it promotes generation of powerful oxidant hypochlorous acid (HOCl) which, reacting with DNA, causes leukemogenesis. In this study, using a whole-human-genome oligonucleotide microarray to clarify the relationships between myelotoxicity of benzene and MPO, we analyzed the genome-wide expression profiles of HL-60 human promyelocytic cell lines exposed to 1,2,4-benzenetriol (BT) with or without MPO inhibition. The microarray analysis revealed that short (1 h) and longer (4 h) exposure to BT changed the expression in HL-60 cells of 1,213 or 1,214 genes associated with transcription, RNA metabolic processes, immune response, apoptosis, cell death, and biosynthetic processes (|Z-score|> 2.0), and that these changes were dramatically lessened by MPO-specific inhibition. The presence of functionally important genes and, specifically, genes related to apoptosis, carcinogenesis, regulation of transcription, immune responses, oxidative stress, and cell-cycle regulation were further validated by real-time RT-PCR. Gene expression profiles along with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis suggest that BT-induced DNA halogenation by MPO is a primary reaction in the leukemogenesis associated with benzene.

  14. A novel triazole derivative of betulinic acid induces extrinsic and intrinsic apoptosis in human leukemia HL-60 cells.

    Science.gov (United States)

    Khan, Imran; Guru, Santosh K; Rath, Santosh K; Chinthakindi, Praveen K; Singh, Buddh; Koul, Surrinder; Bhushan, Shashi; Sangwan, Payare L

    2016-01-27

    In an attempt to arrive at more potent cytotoxic agent than the bioactive natural product betulinic acid, influence of small structural modifications of its 1, 2, 3 triazole derivatives tethered at C-28 and both C3, C-28 using click chemistry approach has been studied. The chemically characterized triazoles have been screened for in vitro cytotoxicity against four human cancer cell lines HL-60, MiaPaCa-2, PC-3 and A549 which has allowed to identify triazole derivative 28{1N (4-fluoro phenyl)-1H-1, 2, 3-triazol-4-yl} methyloxy betulinic ester having better potency profile than the parent compound with IC50 values in the range of 5-7 μM. It caused disruption of mitochondrial membrane potential, rendered Bcl-2 cleavage, Bax translocation and decrease Bcl-2/Bax ratio. These events are accompanied by activation of caspases -9, -3, which cleave the PARP-1. It also induces caspase-8, which is involved in extrinsic apoptotic pathway. Therefore, it induces apoptosis through both intrinsic and extrinsic pathways in human leukemia HL-60 cells.

  15. Effects of Quercetin and Resveratrol combined Reversing Drug Resistance of Leukemia Cell Line HL-60-ADM%槲皮素联合白藜芦醇逆转白血病细胞株HL-60-ADM耐药性的影响

    Institute of Scientific and Technical Information of China (English)

    张复华; 尹松梅; 牛国敏; 杨国雷; 凌奕文; 李蕊; 徐嘉愉

    2014-01-01

    Objective The recent study showed that querctin and resveratrol can reverse multidug resistance in leukemia. The aim of this study was to investigate the reversal multidug resistance effect by querctin and resveratrol. Methods CCK-8 assay was used to compare the toxic effect of querstin or resveratrol alone with that in combination with adriamycin on HL-60-A cells. Results Compared to HL-60 cell line, the IC50 value of adriamycin for HL-60-A increased signiifcantly, it showed that HL-60-A was multidug resistance cell line;querctin or resveratrol could inhibite cell proliferation of HL-60-A in a dose-dependent pattern, while the toxic effect of querctin combined with resveratrol was greater than that of the cells treated with querstin or resveratrol alone;the low-dose of querctin and resveratrol could enhance the sensitivity of adriamycin on HL-60-A, and querstin combined with resveratrol could further enhance the toxic effect of adriamycin. Conclusion Querctin and resveratrol both illustrated signiifcant reversal effect on the leukemia cell line, while the reversal effect of querctin was greater than resveratrol, the synergistic reversal effect of querstin combined with resveratrol was determined.%目的:研究槲皮素(Quercetin,Que)及白藜芦醇(Resveratrol,Res)单用及联用对HL-60-A细胞株耐药性的影响。方法不同浓度阿霉素(adriamycin,ADM)孵育HL-60HL-60-A细胞48 h后CCK-8法测定细胞存活率;Que、Res单用或联用,及加用ADM 48 h测定HL-60-A细胞存活率。结果 ADM作用48 h后,HL-60-A较HL-60细胞IC50明显增高增加提示为耐药细胞株;Que及Res在12.5~150μmol/L时呈剂量依赖性杀伤HL-60-A细胞,Que毒性作用更强,二者可以进一步增强杀伤效应;Que及Res低剂量时可增强ADM对HL-60-A敏感性,二者联用进一步增强ADM的杀伤效应。结论 Que及Res均可逆转白血病细胞耐药性,Que作用较强,二者联合可更有效逆转肿瘤耐药。

  16. MNP-端粒酶反义寡核苷酸复合物诱导HL-60细胞凋亡%The apoptosis of HL - 60 cells induced by magnetic nano - particulate with telomerase antisense oligonucleotides complex

    Institute of Scientific and Technical Information of China (English)

    程呈; 忻茜; 陈祥峰; 李兴玉

    2008-01-01

    反义寡核苷酸具有抑制细胞基因表达作用,对于病毒感染及癌症的治疗具有潜在疗效,然而由于其在生物介质中的不稳定性和低穿透率,常需要修饰于一定的载体上,纳米粒子便是其良好的载体,修饰后可提高反义寡核苷酸的稳定性.研究应用FACS,MTT比色法,电子显微镜,原子力显微镜,细胞集落法研究MNP-端粒酶反义寡核苷酸复合物体外诱导HL-60细胞(人急性早幼粒细胞白血病细胞系)细胞凋亡情况.研究结果表明:通过MTT比色法检测可知不同的药物浓度对细胞的生长有明显的抑制影响;实验组细胞在诱导后于流式细胞术检测中产生了亚二倍体峰(凋亡峰);通过集落形成及电镜观察都有明显的凋亡现象产生,因此认为MNP-端粒酶反义寡核苷酸复合物具有诱导HL-60细胞凋亡的作用.

  17. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells

    Directory of Open Access Journals (Sweden)

    Choi Jung-Hye

    2009-12-01

    Full Text Available Abstract Background Compound K [20-O-β-(D-glucopyranosyl-20(S-protopanaxadiol], a metabolite of the protopanaxadiol-type saponins of Panax ginseng C.A. Meyer, has been reported to possess anti-tumor properties to inhibit angiogenesis and to induce tumor apoptosis. In the present study, we investigated the effect of Compound K on apoptosis and explored the underlying mechanisms involved in HL-60 human leukemia cells. Methods We examined the effect of Compound K on the viabilities of various cancer cell lines using MTT assays. DAPI assay, Annexin V and PI double staining, Western blot assay and immunoprecipitation were used to determine the effect of Compound K on the induction of apoptosis. Results Compound K was found to inhibit the viability of HL-60 cells in a dose- and time-dependent manner with an IC50 of 14 μM. Moreover, this cell death had typical features of apoptosis, that is, DNA fragmentation, DNA ladder formation, and the externalization of Annexin V targeted phosphatidylserine residues in HL-60 cells. In addition, compound-K induced a series of intracellular events associated with both the mitochondrial- and death receptor-dependent apoptotic pathways, namely, (1 the activation of caspases-3, -8, and -9; (2 the loss of mitochondrial membrane potential; (3 the release of cytochrome c and Smac/DIABLO to the cytosol; (4 the translocation of Bid and Bax to mitochondria; and (5 the downregulations of Bcl-2 and Bcl-xL. Furthermore, a caspase-8 inhibitor completely abolished caspase-3 activation, Bid cleavage, and subsequent DNA fragmentation by Compound K. Interestingly, the activation of caspase-3 and -8 and DNA fragmentation were significantly prevented in the presence of cycloheximide, suggesting that Compound K-induced apoptosis is dependent on de novo protein synthesis. Conclusions The results indicate that caspase-8 plays a key role in Compound K-stimulated apoptosis via the activation of caspase-3 directly or indirectly through

  18. Monocytoid differentiation of freshly isolated human myeloid leukemia cells and HL-60 cells induced by the glutamine antagonist acivicin.

    Science.gov (United States)

    Nichols, K E; Chitneni, S R; Moore, J O; Weinberg, J B

    1989-10-01

    Previously we showed that starvation of HL-60 promyelocytic leukemia cells for a single essential amino acid induced irreversible differentiation into more mature monocyte-like cells. Although not an essential amino acid, glutamine is important in the growth of normal and neoplastic cells. The glutamine analogue, alpha S,5S-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) inhibits several glutamine-utilizing enzymes and therefore depletes cells of certain metabolic end products. The current study was designed to examine in vitro the effects of acivicin on growth and differentiation of several established human myeloid leukemia cell lines, including the HL-60 cell line, and of freshly isolated cells from patients with acute nonlymphocytic leukemia (ANLL). Four-day culture of HL-60 cells with acivicin at concentrations of 0.1 to 10.0 micrograms/mL (0.56 to 56 nmol/L) decreased cell growth by 33% to 88% as compared with untreated control cells. Viability of cells was greater than 92% for untreated cells and 93% to 41% for acivicin-treated cells. Cells treated with acivicin differentiated along a monocytic pathway as shown by increased H2O2 production and alpha-naphthyl butyrate esterase (NSE) content. Differentiation was time and dose dependent, and was irreversible. Changes in H2O2 production and NSE content were partially abrogated by co-culture with 10 mmol/L exogenous cytidine and guanosine but not by co-culture with other nucleosides or glutamine. At these concentrations of acivicin, differentiation was associated with expression of the N-formyl-methyl-leucyl-phenylalanine-receptor (FMLP-R) on 8% to 29% of cells as compared with 8% for control cells. Acivicin potentiated the differentiating effects of interferon-gamma, tumor necrosis factor, dihydroxyvitamin D3, dimethylsulfoxide, and retinoic acid. Culture of cells from the U937 (monoblastic), K562 (erythroleukemia), and KG-1 (myeloblastic) cell lines resulted in decreased growth and viability

  19. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  20. Diesel exhaust particle-induced cell death of human leukemic promyelocytic cells HL-60 and their variant cells HL-NR6.

    Science.gov (United States)

    Matsuo, M; Uenishi, R; Shimada, T; Yamanaka, S; Yabuki, M; Utsumi, K; Sagai, M

    2001-04-01

    The cytotoxicity of diesel exhaust particles (DEPs) toward human leukemic promyelocytic cells HL-60 was examined. DEPs were toxic and cytotoxicity increased in a dose-dependent manner. All cells died with 750 microg/ml DEPs in culture media. Apoptosis occurred in HL-60 cells exposed to DEPs. The cytotoxicity of DEP extracts with organic solvents was much lower than those of DEPs and organic solvent-washed residual DEPs. HL-NR6 cells, an HL-60 variant cell line, having higher superoxide dismutase and catalase activities than HL-60 cells, were more resistant to DEP cytotoxicity. When preincubated with the fluorescent probe diacetoxymethyl 6-carboxy-2',7'-dichlorodihydrofluorescinate diacetate and then exposed to DEPs, HL-60 cells emitted green fluorescence under blue illumination, indicating that reactive oxygen species were generated within the cells. The DEP cytotoxicity correlated inversely with the cellular concentration of reduced glutathione (GSH), which had been attenuated with L-buthionine-(R,S)-sulfoximine, a gamma-glutamylcysteine synthetase inhibitor, and was lowered with ethyl reduced glutathionate, a GSH carrier across biomembranes. Further, DEPs themselves decreased the cellular concentration of GSH in a dose-dependent manner. The alpha-tocopherol model compound 2,2,5,7,8-pentamethylchroman-6-ol decreased DEP cytotoxicity, while alpha-tocopherol had no effect. In addition, quinacrine, an endocytosis inhibitor, decreased DEP cytotoxicity. These results show that DEPs are cytotoxic and suggest that the cytotoxicity results from generation of reactive oxygen species by DEPs which have been incorporated into cells.

  1. A polysaccharide from the fruiting bodies of Agaricus blazei Murill induces caspase-dependent apoptosis in human leukemia HL-60 cells.

    Science.gov (United States)

    Li, Xiaohui; Zhao, Xin; Wang, Hongmin; Han, Junqing; Liu, Li

    2014-09-01

    Polysaccharides are the major active ingredients of fungus Agaricus blazei for treating and preventing cancer. However, there are no reports showing anti-tumor activity of A. blazei polysaccharides (ABP) on human leukemia (HL)-60 cells in vitro and in vivo. In this study, we demonstrated that ABP efficiently inhibited proliferation of cultured HL-60 cells, which was associated with the induction of apoptosis. The increase in ABP-induced apoptosis was accompanied by loss of mitochondria membrane potential (∆Ψm), cytochrome c release from the mitochondria, activation of caspase-3, degradation of poly(ADP-ribose) polymerase (PARP), and the elevated ratio of Bcl-2-associated X (Bax)/B-cell lymphoma 2 (Bcl-2). Moreover, z-DEVD-fmk, a caspase-3 inhibitor, reversed the cytotoxic effects and apoptotic characteristics induced by ABP in HL-60 cells. Furthermore, we confirmed that ABP could obviously inhibit the solid cancer growth of leukemia HL-60 in tumor xenograft model. These data demonstrated that ABP effectively induced the apoptosis of HL-60 cells via a signaling cascade of mitochondrial caspase-3-dependent pathway.

  2. Antrodia camphorata induces G(1) cell-cycle arrest in human premyelocytic leukemia (HL-60) cells and suppresses tumor growth in athymic nude mice.

    Science.gov (United States)

    Yang, Hsin-Ling; Kumar, K J Senthil; Kuo, Ya-Ting; Chang, Hebron C; Liao, Jiunn-Wang; Hsu, Li-Sung; Hseu, You-Cheng

    2014-09-01

    Antrodia camphorata is a well-known medicinal mushroom in Taiwan. The broth from a fermented culture of Antrodia camphorata (AC) has been shown to induce apoptosis in cultured human premyelocytic leukemia (HL-60) cells. In the present study, we examined the effects of AC on cell cycle arrest in vitro in HL-60 cells and on tumor regression in vivo using an athymic nude mouse model. We found that AC (20-80 μg mL(-1)) treatment significantly induced G1 cell-cycle arrest in HL-60 cells by reducing the levels of cyclin D1, CDK4, cyclin E, CDK2, cyclin A, and phosphorylation of retinoblastoma protein (p-Rb). Moreover, AC treatment led to significantly increased protein expression levels of CDK inhibitors, including p21(WAF1) and p15(NIK4B). Additionally, AC treatment markedly induced intracellular ROS generation and mitochondrial dysfunction in HL-60 cells. Furthermore, the in vivo study results revealed that AC treatment was effective in terms of delaying the tumor incidence in nude mice that had been inoculated with HL-60 cells as well as in reducing the tumor burden. Histological analysis confirmed that AC treatment significantly modulated the xenografted tumor progression as demonstrated by a reduction in mitotic cells. Our data strongly suggest that Antrodia camphorata could be an anti-cancer agent for human leukemia.

  3. Monitoring magnesium efflux cyclic AMP-induced in HL60 cells by using a new hydroxyquinoline fluorescent chemosensor

    Directory of Open Access Journals (Sweden)

    Chiara Marraccini

    2014-01-01

    Full Text Available Cellular homeostasis of magnesium is still unclear. Several studies documented the occurrence of fluxes of magnesium across the plasmamembrane within minutes from the application of metabolic or hormonal stimuli. These fluxes, however, result in limited variation of free Mg2+ intracellular concentration and large changes in total Mg content. It has been reported that a stimulation with cyclic AMP caused a movement of total magnesium within 10 min after treatment in cardiomyocytes. In this study we tested this hypothesis in HL60 leukemic cells, not excitable but highly proliferating cell model. We evaluated Mg flux by DCHQ5, the phenyl-derivative of hydroxyquinoline fluorescent probe family. We observed a drastic decrease of intracellular total magnesium in the first 3 min. We also verified that at least 10% of the total intracellular amount of magnesium moved in the supernatant of stimulated cells.

  4. A quantitative method for measurement of HL-60 cell apoptosis based on diffraction imaging flow cytometry technique.

    Science.gov (United States)

    Yang, Xu; Feng, Yuanming; Liu, Yahui; Zhang, Ning; Lin, Wang; Sa, Yu; Hu, Xin-Hua

    2014-07-01

    A quantitative method for measurement of apoptosis in HL-60 cells based on polarization diffraction imaging flow cytometry technique is presented in this paper. Through comparative study with existing methods and the analysis of diffraction images by a gray level co-occurrence matrix algorithm (GLCM), we found 4 GLCM parameters of contrast (CON), cluster shade (CLS), correlation (COR) and dissimilarity (DIS) exhibit high sensitivities as the apoptotic rates. It was further demonstrated that the CLS parameter correlates significantly (R(2) = 0.899) with the degree of nuclear fragmentation and other three parameters showed a very good correlations (R(2) ranges from 0.69 to 0.90). These results demonstrated that the new method has the capability for rapid and accurate extraction of morphological features to quantify cellular apoptosis without the need for cell staining.

  5. Genotoxicity of alkene epoxides in human peripheral blood mononuclear cells and HL60 leukaemia cells evaluated with the comet assay.

    Science.gov (United States)

    Fabiani, Roberto; Rosignoli, Patrizia; De Bartolomeo, Angelo; Fuccelli, Raffaela; Morozzi, Guido

    2012-08-30

    Volatile organic compounds (VOCs) exert their carcinogenic activity through the production of epoxide metabolites. Because of their high reactivity some epoxides are also produced in the chemical industry for the synthesis of other compounds. Therefore, human exposure to VOCs epoxides does occur and may be an important human health concern. In this study, the in vitro genotoxic potential of epoxides originating from 1,3-butadiene (3,4-epoxy-1-butene: EB; 1,2:3,4-diepoxybutane: DEB), isoprene (3,4-epoxy-2-methyl-1-butene: IO), styrene (styrene-7,8-oxide: SO), propylene (propylene oxide: PO) and 1-butene (1,2-epoxy-butane: BO) in human peripheral blood mononuclear cells (PBMCs) and promyelocytic leukaemia cells (HL60) was measured with the comet assay (single-cell gel electrophoresis, SCGE). The effect of inclusion of foetal calf serum (FCS, 5%) in the cell-culture medium and different durations of exposure (2h, 24h) were also investigated. All epoxides tested produced DNA damage in a concentration range that did not reduce cell viability. HL60 cells were more resistant than PBMCs to the DNA damage induced by the different epoxides. With the exception of IO, the treatment for 24h resulted in an increase of DNA damage. FCS slightly protected PBMCs from the genotoxic effects induced by IO and BO, whilst no such effect was noted for the other compounds. Overall, the dose-dependent effects that were seen allowed us to define a genotoxicity scale for the different epoxides as follows: SO>EB>DEB>IO>PO>BO, which is in partial agreement with the International Agency for Research on Cancer (IARC) classification of the carcinogenic hazards.

  6. Effects of red orpiment on cell morphology and expression of PML mRNA and protein in NB4 and HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    钟璐; 陈芳源; 韩洁英; 邵念贤; 欧阳仁荣

    2003-01-01

    Objective To investigate the effects of red orpiment on cell morphology, expression of promyelocytic leukemia (PML) mRNA and its protein localization in NB4 and HL-60 cell lines.Methods Cell morphology was assayed by Wright's staining and fluorescence staining, while PML mRNA expression was determined by RT-PCR. PML protein localization by evaluated by immunofluorescence staining. Results The typical apoptosis was found in NB4 and HL-60 cells after treatment with red orpiment. The fusion protein was no longer observed in NB4 cells, PML protein was relocated, and then degraded. In HL-60 cells, PML protein underwent a similar progress. The expression of promyelocytic leukemia (PML) mRNA was not changed in the treated cells.Conclusion Red orpiment inhibits the proliferation of leukemia cells by inducing them to undergo apoptosis.

  7. Effect of frankincense extract on VEGF and its receptor-1 expression in acute promyelocytic leukemia cell lines HL-60 cells%乳香提取物对HL-60细胞株VEGF分泌及其Flt-1受体表达的影响

    Institute of Scientific and Technical Information of China (English)

    张勇; 齐振华; 李乐赛; 符晓华; 彭小宁; 张娜; 于才红

    2011-01-01

    目的:探索不同浓度乳香提取物作用于HL-60细胞后,白血病细胞株HL-60细胞VEGF mRNA表达及VEGF蛋白分泌及受体Flt-1的变化.方法:MTT法检测乳香提取物对细胞增殖的抑制作用,选择适当的药物浓度及作用时间:RT-PCR法检测乳香提取物处理前后HL-60细胞中VEGF mRNA表达水平;Western Blot法检测乳香提取物处理前后HL-60细胞VEGF分泌和受体Flt-1蛋白表达水平.结果:乳香提取物在15.0 mg/L时在体外能下调HL-60细胞VEGF mRNA的表达和VEGF蛋白的分泌并呈时间与剂量依赖性(P<0.05).结论:乳香提取物在体外可以抑制HL-60细胞分泌VEGF,同时抑制HL-60细胞内VEGF及其受体Flt-1 mRNA及蛋白表达下降,具有潜在抑制急性早幼粒细胞白血病血管新生的作用,是一种可供选择的抗白血病药物.%Objective To assess the effects of Frankincense extract on VEGF and Fit-1 expression in acute promyelocytic leukemia Cell Lines HL-60 cells. Methods HL-60 cells were treated with Frankincense extract, then cell proliferation were examined by the method of MTT. The level of VEGF-mRNA expression was distinguished by semi -quantitative RT -PCR technique. Western Blot was applied to examine the protein expression of VEGF and Fit-1. Results Within the concentration of (10.0~15.0) mg/L, Frankincense extract strongly inhibited proliferation of HL-60 cells. When the drug is 15.0 mg/L, α-boswellic acid can inhibit the mRNA expression of VEGF and the protein expression of VEGF and Fit-1. Conclusion Within a certain drug concentration, Frankincense extract can inhibit HL-60 cells proliferation. Its role of inhibiting angiogenesis maybe relate with down regulating the expression of VEGF and Fit-1.

  8. Inhibition of NF-κb by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-XL expression

    Institute of Scientific and Technical Information of China (English)

    曹文静; 张瑶珍; 张东华; 李登举; 唐锦治

    2004-01-01

    Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB) activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism.Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. The cytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner.Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the anti-leukemia effects of TNF-α or even of other cytotoxic agents.

  9. c-myc but not Hif-1α-dependent downregulation of VEGF influences the proliferation and differentiation of HL-60 cells induced by ATRA.

    Science.gov (United States)

    Song, Guanhua; Li, Yanmei; Zhang, Zhiyong; Ren, Xia; Li, Hongjiang; Zhang, Wen; Wei, Ruoying; Pan, Sufei; Shi, Lulu; Bi, Kehong; Jiang, Guosheng

    2013-06-01

    Vascular endothelial growth factor (VEGF) plays an important role in solid tumor growth, progression and metastasis as well as in the proliferation and differentiation of hematological malignancies. However, the molecular mechanism that modulates VEGF expression and secretion in leukemia cells has not yet to be elucidated. The purpose of the present study was to investigate the role of the signal pathway in modulating the expression of VEGF in HL-60 cells. Specific siRNAs targeting VEGF were transfected into HL-60 cells and the VEGF expression was measured with reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay. The cell proliferation of HL-60 cells was detected by the cell counting kit-8 (CCK-8) assay and the differentiation of HL-60 cells induced by all-trans-retinoic acid (ATRA) was detected by the RT-PCR assay and flow cytometry assay for CD11b. The upstream transcription factors that were related to VEGF expression such as P53, SP-1, c-jun, VHL, cox-2, c-myc and stat3 were detected by RT-PCR assay. In addition, the chromatin immunoprecipitation (ChIP) assay was used to reveal the role of c-myc by binding the target gene VEGF. The results demonstrated the hypoxia-inducible factor 1α-related signaling pathway, not the same as in solid tumors, might not play a key role in modulating VEGF expression. c-myc contributes to the modulation of VEGF expression by targeting the promoter of VEGF, which was indicated by the ChIP assay. In conclusion, our data demonstrate that VEGF plays an important role in the differentiation and proliferation of HL-60 cells; c-myc-dependent downregulation of VEGF induced by ATRA contributes to the differentiation of HL-60 cells.

  10. Involvement of the histamine H4 receptor in clozapine-induced hematopoietic toxicity: Vulnerability under granulocytic differentiation of HL-60 cells.

    Science.gov (United States)

    Goto, Aya; Mouri, Akihiro; Nagai, Tomoko; Yoshimi, Akira; Ukigai, Mako; Tsubai, Tomomi; Hida, Hirotake; Ozaki, Norio; Noda, Yukihiro

    2016-09-01

    Clozapine is an effective antipsychotic for treatment-resistant schizophrenia, but can cause fatal hematopoietic toxicity as agranulocytosis. To elucidate the mechanism of hematopoietic toxicity induced by clozapine, we developed an in vitro assay system using HL-60 cells, and investigated the effect on hematopoiesis. HL-60 cells were differentiated by all-trans retinoic acid (ATRA) into three states according to the following hematopoietic process: undifferentiated HL-60 cells, those undergoing granulocytic ATRA-differentiation, and ATRA-differentiated granulocytic cells. Hematopoietic toxicity was evaluated by analyzing cell survival, cell proliferation, granulocytic differentiation, apoptosis, and necrosis. In undifferentiated HL-60 cells and ATRA-differentiated granulocytic cells, both clozapine (50 and 100μM) and doxorubicin (0.2µM) decreased the cell survival rate, but olanzapine (1-100µM) did not. Under granulocytic differentiation for 5days, clozapine, even at a concentration of 25μM, decreased survival without affecting granulocytic differentiation, increased caspase activity, and caused apoptosis rather than necrosis. Histamine H4 receptor mRNA was expressed in HL-60 cells, whereas the expression decreased under granulocytic ATRA-differentiation little by little. Both thioperamide, a histamine H4 receptor antagonist, and DEVD-FMK, a caspase-3 inhibitor, exerted protection against clozapine-induced survival rate reduction, but not of live cell counts. 4-Methylhistamine, a histamine H4 receptor agonist, decreased the survival rate and live cell counts, as did clozapine. HL-60 cells under granulocytic differentiation are vulnerable under in vitro assay conditions to hematopoietic toxicity induced by clozapine. Histamine H4 receptor is involved in the development of clozapine-induced hematopoietic toxicity through apoptosis, and may be a potential target for preventing its occurrence through granulocytic differentiation.

  11. 6-C-methyl flavonoids isolated from Pinus densata inhibit the proliferation and promote the apoptosis of the HL-60 human promyelocytic leukaemia cell line.

    Science.gov (United States)

    Yue, Rongcai; Li, Bo; Shen, Yunheng; Zeng, Huawu; Li, Bo; Yuan, Hu; He, Yiren; Shan, Lei; Zhang, Weidong

    2013-08-01

    Three structurally related 6-C-methyl flavonoids isolated from Pinus densata, including 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone (PD1), 5,7,4'-trihydroxy-3,8-dimethoxy-6-C-methylflavone (PD2), and 5,7,4'-trihydroxy-3-methoxy-6-C-methylflavone (PD3), were tested for their ability to inhibit the proliferation and promote the apoptosis of the HL-60 human leukaemia cell line. Cytotoxicity assays in the HL-60 human cancer cell line demonstrated that 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone exhibited the most potent cytotoxicity of the three structurally related 6-C-methyl flavonoids. 5,4'-Dihydroxy-3,7,8-trimethoxy-6-C-methylflavone inhibited the proliferation of HL-60 cells in a dose-dependent manner with an IC₅₀ of 7.91 µM (48 h treatment). Furthermore, 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone-induced apoptosis was associated with mitochondrial membrane disruption and cytochome c release. Flow cytometry analyses revealed an increase in the hypodiploid population in 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone-treated HL-60 cells. Treatment with a concentration of 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone that induced apoptosis activated caspase-3 but did not activate caspase-1. A caspase-3 inhibitor (Ac-DEVD-CHO), but not a caspase-1 inhibitor (Ac-YVAD-CHO), reversed the cytotoxic effects of 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone in HL-60 cells. These data demonstrated that 5,4'-dihydroxy-3,7,8-trimethoxy-6-C-methylflavone effectively induced the apoptosis of HL-60 cells and exhibited significant anticancer activity via the mitochondrial caspase-3-dependent apoptosis pathway.

  12. 4H-Chromene-based anticancer agents towards multi-drug resistant HL60/MX2 human leukemia: SAR at the 4th and 6th positions.

    Science.gov (United States)

    Puppala, Manohar; Zhao, Xinghua; Casemore, Denise; Zhou, Bo; Aridoss, Gopalakrishnan; Narayanapillai, Sreekanth; Xing, Chengguo

    2016-03-15

    4H-Chromene-based compounds, for example, CXL017, CXL035, and CXL055, have a unique anticancer potential that they selectively kill multi-drug resistant cancer cells. Reported herein is the extended structure-activity relationship (SAR) study, focusing on the ester functional group at the 4th position and the conformation at the 6th position. Sharp SARs were observed at both positions with respect to cellular cytotoxic potency and selectivity between the parental HL60 and the multi-drug resistant HL60/MX2 cells. These results provide critical guidance for future medicinal optimization. Copyright © 2016. Published by Elsevier Ltd.

  13. Simultaneous multi-parameter observation of Harring-tonine-treating HL-60 cells with both two-photon and confo-cal laser scanning microscopy

    Institute of Scientific and Technical Information of China (English)

    张春阳; 李艳平; 马辉; 李素文; 薛绍白; 陈瓞延

    2001-01-01

    Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluores-cent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simulta-neously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell.

  14. The enhancement of sensitivity of HL-60 cells to arsenic trioxide by Bcl-2 siRNA%以Bcl-2为靶标siRNA提高HL-60细胞对三氧化二砷敏感性的探讨

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To study whether the small-interference RNA (siRNA) targeting against Bcl-2 gene can enhance sensitivity of HL-60 cell to arsenic trioxide. Methods: SiRNA was transferred into the HL-60 cells. At 6 h after transfection, the cells were cultured with arsenic trioxide. The cell growth of the HL-60 cells was detected using MTT at 24, 48 and 72 h, respectively.The levels of the Bcl-2 protein and reactive oxygen species (ROS), as well as of the membrane potential of the mitochondrion were determined by flow cytometry. Results: The Bcl-2 siRNA significantly increased the inhibitory action of arsenic trioxide on growth of HL-60 cells. The combination of siRNA with arsenic trioxide resulted in decrease of the Bcl-2 protein level and increase of the ROS level, as well as significant descending of the membrane potential of mitochondrion of HL-60 (P < 0.05).Conclusion: The siRNAtargeting Bcl-2 can increase the sensitivity of the HL-60 leukemia cells to arsenic trioxide by inhibiting the expression of Bcl-2 protein.

  15. Upregulation of CD54 and downregulation of HLA‑ABC contribute to the novel enhancement of the susceptibility of HL-60 cells to NK cell-mediated cytolysis induced by ATRA plus VPA.

    Science.gov (United States)

    Zou, Huijuan; Li, Lianlian; Han, Yang; Ma, Ruiping; Liao, Qiong; Tian, Jing; Zhang, Xiaoyu; Ren, Xia; Song, Guanhua; Guo, Qiang; Li, Xia; Ding, Huifang; Jiang, Guosheng

    2017-01-01

    Enhancement of the susceptibility of HL-60 cells to NK cell-mediated cytolysis induced by all-trans-retinoic acid (ATRA) plus valproate (VPA) was evaluated. In addition to the synergistic effect of ATRA plus VPA on HL-60 cells, the optimal concentration of 1 mM VPA plus 0.5 µM ATRA increased the cytotoxic sensitivity of HL-60 cells to NK cells. The expression of the activated receptors NKp30 and NKG2D on NK-92 cells was higher compared with the levels noted for the other receptors, and the expression of NKG2D ligands MICA/B on HL-60 cells was not significantly upregulated in the ATRA plus VPA goup compared with the control. Moreover, it was observed that the ligands of NKp30 on HL-60 cells presented the same variation trend. As to the co-stimulatory and adhesion molecules on NK-92 and their ligands on HL-60 cells post exposure to ATRA and VPA alone or their combination, there was no obvious change in the expression of CD112, CD48 and CD70 on the HL-60 cells. However, the expression of CD54 on HL-60 cells was significantly upregulated. In contrast, the expression of NKG2A ligands HLA-ABC on HL-60 cells was obviously downregulated. In addition, the expression of HLA-E on the HL-60 cells in the group treated with ATRA plus VPA was not significantly increased. In conclusion, the combination of VPA and ATRA not only induced the differentiation of HL-60 cells, but also induced enhancement of the sensitivity of HL-60 cells to NK cells by downregulating the expression of HLA-ABC and upregulating the expression of CD54, but not MICA/MICB. The results provide experimental and theoretical basis for the clinical combination of a low-dose of ATRA plus VPA for the treatment of leukemia.

  16. BCL-x{sub L}/MCL-1 inhibition and RARγ antagonism work cooperatively in human HL60 leukemia cells

    Energy Technology Data Exchange (ETDEWEB)

    Perri, Mariarita; Yap, Jeremy L.; Yu, Jianshi [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine Street, Baltimore, MD 21201 (United States); Cione, Erika [Department of Pharmacy, Health and Nutritional Sciences, Ed. Polifunzionale, University of Calabria, 87036 Rende, CS (Italy); Fletcher, Steven [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine Street, Baltimore, MD 21201 (United States); Kane, Maureen A., E-mail: mkane@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine Street, Baltimore, MD 21201 (United States)

    2014-10-01

    The acute promyelocytic leukemia (APL) subtype of acute myeloid leukemia (AML) is characterized by chromosomal translocations that result in fusion proteins, including the promyelocytic leukemia–retinoic acid receptor, alpha fusion protein (PML–RARα). All-trans retinoic acid (atRA) treatment is the standard drug treatment for APL yielding cure rates >80% by activating transcription and proteasomal degradation of retinoic acid receptor, alpha (RARα). Whereas combination therapy with As{sub 2}O{sub 3} has increased survival further, patients that experience relapse and are refractory to atRA and/or As{sub 2}O{sub 3} is a clinically significant problem. BCL-2 family proteins regulate apoptosis and over-expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL-2) family proteins has been associated with chemotherapeutic resistance in APL including impairment of the ability of atRA to induce growth arrest and differentiation. Here we investigated the novel BH3 domain mimetic, JY-1-106, which antagonizes the anti-apoptotic BCL-2 family members B-cell lymphoma-extra large (BCL-x{sub L}) and myeloid cell leukemia-1 (MCL-1) alone and in combination with retinoids including atRA, AM580 (RARα agonist), and SR11253 (RARγ antagonist). JY-1-106 reduced cell viability in HL-60 cells alone and in combination with retinoids. The combination of JY-1-106 and SR11253 had the greatest impact on cell viability by stimulating apoptosis. These studies indicate that dual BCL-x{sub L}/MCL-1 inhibitors and retinoids could work cooperatively in leukemia treatment. - Highlights: • Novel Bcl-x{sub L}/Mcl-1 inhibitor JY-1-106 reduces HL60 cell viability. • JY-1-106 is investigated in combination with retinoic acid, AM580, and SR11253. • AM580 is an RARα agonist; SR11253 is an RARγ antagonist. • Combined use of JY-1-106/SR11253 exhibited the greatest cell viability reduction. • JY-1-106 alone or in combination with retinoids induces apoptosis.

  17. The Critical Role of Redox Homeostasis in Shikonin-Induced HL-60 Cell Differentiation via Unique Modulation of the Nrf2/ARE Pathway

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2012-01-01

    Full Text Available Among various cancer cell lines, the leukemia cell line HL-60 was most sensitive to Shikonin, with evidence showing both the prooxidative activities and proapoptotic effects of micromolar concentrations of Shikonin. However, the mechanism involved in the cytotoxicity of Shikonin in the submicromolar range has not been fully characterized. Using biochemical and free radical biological experiments in vitro, we identified the prodifferentiated profiles of Shikonin and evaluated the redox homeostasis during HL-60 differentiation. The data showed a strong dose-response relationship between Shikonin exposure and the characteristics of HL-60 differentiation in terms of morphology changes, nitroblue tetrazolium (NBT reductive activity, and the expression level of surface antigens CD11b/CD14. During drug exposure, intercellular redox homeostasis changes towards oxidation are necessary to support Shikonin-induced differentiation, which was proven by additional enzymatic and non-enzymatic redox modulators. A statistically significant and dose-dependent increase (P<0.05 was recorded with regard to the unique expression levels of the Nrf2/ARE downstream target genes in HL-60 cells undergoing late differentiation, which were restored with further antioxidants employed with the Shikonin treatment. Our research demonstrated that Shikonin is a differentiation-inducing agent, and its mechanisms involve the Nrf2/ARE pathway to modulate the intercellular redox homeostasis, thus facilitating differentiation.

  18. The Photocatalytic Inactivation Effect of Fe-Doped TiO2 Nanocomposites on Leukemic HL60 Cells-Based Photodynamic Therapy

    Directory of Open Access Journals (Sweden)

    Kangqiang Huang

    2012-01-01

    Full Text Available The Fe-doped TiO2 nanocomposites synthesized by a deposition-precipitation method were characterized by X-ray diffraction (XRD, transmission electron microscope (TEM, X-ray photoelectron spectroscopy (XPS, and UV-vis adsorption spectra and then were taken as a new “photosensitizer” for photodynamic therapy (PDT. The photocatalytic inactivation of Fe-doped TiO2 on Leukemic HL60 cells was investigated using PDT reaction chamber based on LED light source, and the viability of HL60 cells was examined by Cell Counting Kit-8 (CCK-8 assay. The experimental results showed that the growth of leukemic HL60 cells was significantly inhibited by adding TiO2 nanoparticles, and the inactivation efficiency could be effectively enhanced by the surface modification of TiO2 nanoparticles with Fe doping. Furthermore, the optimized conditions were achieved at 5 wt% Fe/TiO2 at a final concentration of 200 μg/mL, in which up to 82.5% PDT efficiency for the HL60 cells can be obtained under the irradiation of 403 nm light (the power density is 5 mW/cm2 within 60 minutes.

  19. 15,16-Dihydrotanshinone I from the Functional Food Salvia miltiorrhiza Exhibits Anticancer Activity in Human HL-60 Leukemia Cells: in Vitro and in Vivo Studies.

    Science.gov (United States)

    Liu, Jun-Jen; Wu, Hsueh-Hsia; Chen, Tzu-Ho; Leung, Wan; Liang, Yu-Chih

    2015-08-17

    15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 μg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo; the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways.

  20. Ant 4,4, a polyamine-anthracene conjugate, induces cell death and recovery in human promyelogenous leukemia cells (HL-60).

    Science.gov (United States)

    Traquete, Rui; Ghani, Radiah A; Phanstiel, Otto; Wallace, Heather M

    2013-04-01

    One of the major problems in cancer therapy is the lack of specificity of chemotherapeutic agents towards cancer cells, resulting in adverse side effects. One means to counter this is to selectively deliver the drug to the cancer cell. Cancer cells accumulate increased concentrations of polyamines compared to normal cells, mainly through an increased uptake of preformed polyamines via the polyamine transport system (PTS). Furthermore, the non-stringent structural requirements of the PTS enable the transport of a range of polyamine-based molecules. Thus, the PTS can be used to transport compounds linked to polyamines selectively to cancer cells. In our laboratory, polyamine-anthracene conjugates have shown potent anti-tumour activity towards HL-60 cells. The aim of this study was to determine the cytotoxicity of Ant-4,4, a homospermidine-anthracene conjugate, and assess the long-term effects by determining whether cancer cells were able to recover from treatment. During exposure, Ant-4,4 was an effective growth-inhibitory agent in HL-60 cells decreasing viable cell number, protein and polyamine content. Evidence indicates concomitant cell-cycle arrest and increased apoptosis. Once the drug was removed, HL-60 cells recovered gradually over time. Increasing cell number, protein content and polyamine content, as well as diminished effects on cell-cycle and apoptotic stimuli were observed over time. These data suggest that, despite being an effective way of delivering anthracene, these polyamine conjugates do not exert long-lasting effects on HL-60 cells.

  1. Residence in biofilms allows Burkholderia cepacia complex (Bcc) bacteria to evade the antimicrobial activities of neutrophil-like dHL60 cells

    Science.gov (United States)

    Murphy, Mark P.; Caraher, Emma

    2015-01-01

    Bacteria of the Burkholderia cepacia complex (Bcc) persist in the airways of people with cystic fibrosis (CF) despite the continuous recruitment of neutrophils. Most members of Bcc are multidrug resistant and can form biofilms. As such, we sought to investigate whether biofilm formation plays a role in protecting Bcc bacteria from neutrophils. Using the neutrophil-like, differentiated cell line, dHL60, we have shown for the first time that Bcc biofilms are enhanced in the presence of these cells. Biofilm biomass was greater following culture in the presence of dHL60 cells than in their absence, likely the result of incorporating dHL60 cellular debris into the biofilm. Moreover, we have demonstrated that mature biofilms (cultured for up to 72 h) induced necrosis in the cells. Established biofilms also acted as a barrier to the migration of the cells and masked the bacteria from being recognized by the cells; dHL60 cells expressed less IL-8 mRNA and secreted significantly less IL-8 when cultured in the presence of biofilms, with respect to planktonic bacteria. Our findings provide evidence that biofilm formation can, at least partly, enable the persistence of Bcc bacteria in the CF airway and emphasize a requirement for anti-biofilm therapeutics. PMID:26371179

  2. Residence in biofilms allows Burkholderia cepacia complex (Bcc) bacteria to evade the antimicrobial activities of neutrophil-like dHL60 cells.

    Science.gov (United States)

    Murphy, Mark P; Caraher, Emma

    2015-11-01

    Bacteria of the Burkholderia cepacia complex (Bcc) persist in the airways of people with cystic fibrosis (CF) despite the continuous recruitment of neutrophils. Most members of Bcc are multidrug resistant and can form biofilms. As such, we sought to investigate whether biofilm formation plays a role in protecting Bcc bacteria from neutrophils. Using the neutrophil-like, differentiated cell line, dHL60, we have shown for the first time that Bcc biofilms are enhanced in the presence of these cells. Biofilm biomass was greater following culture in the presence of dHL60 cells than in their absence, likely the result of incorporating dHL60 cellular debris into the biofilm. Moreover, we have demonstrated that mature biofilms (cultured for up to 72 h) induced necrosis in the cells. Established biofilms also acted as a barrier to the migration of the cells and masked the bacteria from being recognized by the cells; dHL60 cells expressed less IL-8 mRNA and secreted significantly less IL-8 when cultured in the presence of biofilms, with respect to planktonic bacteria. Our findings provide evidence that biofilm formation can, at least partly, enable the persistence of Bcc bacteria in the CF airway and emphasize a requirement for anti-biofilm therapeutics.

  3. INDUCTION OF CELL PROLIFERATION AND APOPTOSIS IN HL60 AND HACAT CELLS BY ARSENIC, ARSENATE, AND ARSENIC-CONTAMINATED DRINKING WATER

    Science.gov (United States)

    Induction of cell proliferation and apoptosis in HL-60 and HaCaT cells by arsenite, arsenate and arsenic-contaminated drinking water. T-C. Zhang, M. Schmitt, J. L. Mumford National Research Council, Washington DC and U.S. Environmental Protection Agency, NHEERL, Research Triangle...

  4. Effects of anti-CXCR4 monoclonal antibody 12G5 on proliferation and apoptosis of human acute myelocytic leukemia cell line HL-60

    Institute of Scientific and Technical Information of China (English)

    WEI Li; KONG Pei-yan; SHI Zhan-zhong; ZENG Dong-feng; CHEN Xing-hua; CHANG Cheng; PENG Xian-gui; ZHANG Yi; LIU Hong

    2007-01-01

    Objective: To investigate the expression of CXCR4 on HL-60 cell line and the proliferation,apoptosis of HL-60 cell line cocultured with bone marrow stromal cells, so as to assess the possibility of 12G5, an anti-CXCR4 monoclonal antibody, in eradicating the minimal residual disease. Methods: The activity of SDF-1 was inhibited by 10 μg/ml 12G5. After treatment with 12G5, the status of adhesion was observed, and the adhesion rates, apoptosis and cell cycles were detected after 24 h of treatment. Cell growth rates were measured by trypan blue exclusion. Cell growth curve was plotted, and the expression of PCNA and apoptosis related protein including PCNA, Bcl-2 and Fas were detected with immunohistochemical technique. Results: (1) There was middling degree expression of CXCR4 on HL-60 membrane. From 0 h to 6 h, as the time of 12G5 incubation along, the expression of CXCR4 decreased gradually. (2)After treatment for 24 h, the adhesion rates in the experiment group and the control were (39.4±7.9)%and (51.4±5.9)%, respectively. (3)After treatment for 24 h, the percentage of HL-60 cells in G0/G1 phase were (55.21±4.9)%, and that in S phase and G2/M phase were (30.40±4.1)% and (14.39±5.2)%, respectively, with the corresponding proportions being (44.67±2.2)%, (45.30±3.7)%, and (10.03±2.6)% in the control. (4) The percentage of apoptotic HL-60 cells was (8.95±1.7)% in the experiment group, compared to (3.97±2.4)% in the control. (5)The survival rates of HL-60 cells decreased markedly at 48 h to 96 h, and the proliferation slowed down at this time duration. (6)The expression of PCNA and Bcl-2 down-regulated significantly, but the Fas protein expression was up-regulated. Conclusion: 12G5 could inhibit the capability of adhesion and proliferation of HL-60 cells and it can induce more cells to enter G0/G1 phase and promote apoptosis. It may be helpful by inhibiting the bioactivity of SDF-1 with 12G5 in the therapy of marrow residual disease.

  5. SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

    Science.gov (United States)

    Xiao, Jie; Yin, Songmei; Li, Yiqing; Xie, Shuangfeng; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan; Feng, Jianhong

    2009-08-01

    S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

  6. [Effect of 5-aza-CdR demethylation on expression of SHP-1 and C-kit genes in leukemia HL-60 cells].

    Science.gov (United States)

    Meng, Zhen; Li, Ying-Hua; Wang, Dong-Mei; Luo, Jian-Min

    2014-12-01

    This study was aimed to investigate the expression level of SHP-1 and C-kit genes in acute leukemia HL-60 cells and effect of 5-aza-CdR demethylation on expression of SHP-1 and C-kit genes. RT-PCR was used to detect the mRNA expression level of SHP-1 and C-kit mRNA in HL-60 cells of the drug-treated group and control group.The methylation specific PCR (MSP) was applied to measure the methylation status of SHP-1 and C-kit genes in HL-60 cells.The results showed that after being treated with 5-aza-CdR, the recovery of SHP-1 gene expression was observed in HL-60 cells in which SHP-1 mRNA originally was not expressed. Meanwhile, the high expression level of C-kit mRNA in HL-60 cells was decreased. When HL-60 cells were treated with 0, 0.5, 1.0, 2.0 µmol/L 5-aza-CdR, the demethylation effect was enhanced, the expression of SHP-1 mRNA displayed an ascending tendency, and the expression of C-kit mRNA showed an descending tendency in dose-dependent manner (P HL-60 cells and recovery of expression after treatment with 5-aza-CdR suggest that the hypermethylation of SHP-1 gene relates with pathogenesis of leukemia, and the abnormal increase of C-kit mRNA expression maybe exist in formation of leukemia. The effect of 5-aza-CdR on expression of SHP-1 and C-kit shows dose-dependency, the higher the 5-aza-CdR concentration, the higher the SHP-1 expression and the lower the C-kit expression, moreover, the effect of 5-aza-CdR shows time-dependency in specific concentration.The SHP-1 mRNA expression negatively correlates with C-kit mRNA expression, suggesting that the decrease or absence of SHP-1 expression in leukemia cells weakens the negative regulation on C-kit signaling pathway, thus plays a role in the formation of leukemia.

  7. Steroidal saponins from the aerial parts of Dracaena draco and their cytostatic activity on HL-60 cells.

    Science.gov (United States)

    Mimaki, Y; Kuroda, M; Ide, A; Kameyama, A; Yokosuka, A; Sashida, Y

    1999-03-01

    Chemical examination of the aerial parts of Dracaena draco has led to the isolation of a total of nine steroidal saponins, including five new ones. The structures of the new saponins were determined by spectral data and a few chemical transformations to be (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23,24-tetrol 1-O-{O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L -arabinopyranosyl} 24-O-beta-D-fucopyranoside, (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta, 23,24-tetrol 1-O-{O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L -arabinopyranoside}, (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23,24-tetrol 1-O-{O-(4-O- acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L-arabinopyransoide} , (23S)-spirosta-5,25(27)-diene-1 beta,3 beta,23-triol 1-O-{O-alpha-L- rhamnopyranosyl)-(1-->2)-alpha-L-arabinopyranoside} and (23S,24S)-spirosta-5,25(27)-diene-1 beta,3 beta,23-triol 1-O-{O-(4-O-acetyl-alpha-L-rhamnopyranosyl)-(1-->2)-alpha-L- arabinopyranoside}. The isolated saponins were evaluated for their cytostatic activity on leukemia HL-60 cells.

  8. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells.

    Science.gov (United States)

    Niu, Guomin; Yin, Songmei; Xie, Shuangfeng; Li, Yiqing; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavonol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

  9. Antiproliferative activity of O4-benzo[c]phenanthridine alkaloids against HCT-116 and HL-60 tumor cells.

    Science.gov (United States)

    Hatae, Noriyuki; Fujita, Erina; Shigenobu, Saori; Shimoyama, Sayumi; Ishihara, Yuhsuke; Kurata, Yuhki; Choshi, Tominari; Nishiyama, Takashi; Okada, Chiaki; Hibino, Satoshi

    2015-07-15

    The O4-benzo[c]phenanthridine alkaloids exhibit potent antiproliferative activity against cancer cells, which is derived from their ability to inhibit of topoisomerase I and II. It has been reported that in the alkaloids a cationic quaternary ammonium atom, which results in resonance effects between ring A and B, is necessary for increased antiproliferative activity. These findings indicate the role of their substituents at ring A on inhibition of tumor cell proliferation. In the present study, we systematically assessed the cytotoxic activities of naturally occurring alkaloids and their derivatives containing various ring A substituents against two tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. Among the cationic iminium alkaloids, which displayed more potent activity than the corresponding neutral derivatives, and the 7,8-oxygenated benzo[c]phenanthridine alkaloids, chelerythrine and NK109, exhibited stronger antiproliferative activity than the 8,9- and 9,10-oxygenated alkaloids. The activity of cationic iminium alkaloids could be correlated with the bond lengths of their ring A substituents and the electrostatic potentials of their ammonium molecules by DFT calculation.

  10. Differentially expressed nuclear proteins in human CCRF-CEM, HL-60, MEC-1 and Raji cells correlate with cellular properties.

    Science.gov (United States)

    Henrich, Silke; Crossett, Ben; Christopherson, Richard I

    2007-10-01

    The human cell lines CCRF-CEM (T-cell acute lymphocytic leukemia), HL-60 (acute myeloid leukemia), MEC-1 (B-cell chronic lymphocytic leukemia) and Raji (Burkitt's B-cell lymphoma) have been analysed for differences in their nuclear proteomes. Using 2-D DIGE, 55 nuclear proteins have been identified that are differentially expressed (p<0.025) between the four cell lines, including proteins associated with transcription, proliferation, DNA repair and apoptosis. Of these 55 proteins, 22 were over-expressed in just one cell line, and four were down-regulated in one cell line. Proteins uniquely over-expressed between myeloid and lymphoid cell lines include those that may have use as markers for diagnosis, disease progression and B-cell maturation and differentiation. Expression of various proliferation-associated nuclear proteins correlated with relative growth rates of the cell lines, giving these proteins potential diagnostic applications for distinction of chronic versus acute subtypes of haematological malignancies. Identification of these differentially expressed nuclear proteins should facilitate elucidation of the molecular mechanisms underlying leukocyte differentiation and transformation to leukemias and lymphomas. The nuclear expression profiles should enable classification of subtypes of leukemia, and identify potential nuclear protein targets for development of diagnostic and therapeutic strategies.

  11. In vitro hydroquinone-induced instauration of histone bivalent mark on human retroelements (LINE-1) in HL60 cells.

    Science.gov (United States)

    Mancini, Monica; Mandruzzato, Martina; Garzia, Alba C; Sahnane, Nora; Magnani, Elena; Macchi, Filippo; Oulad-Abdelghani, Mustapha; Oudet, Pierre; Bollati, Valentina; Fustinoni, Silvia; Furlan, Daniela; Bonapace, Ian M

    2016-12-13

    Benzene is extensively used in industry despite its leukemogenic activity, representing a significant occupational hazard. We investigated if long-term treatment with low-doses hydroquinone (HQ), a benzene metabolite, might be sufficient to alter in vitro the epigenetic signature underlining LINE-1 sequences, a poorly explored step in health risks associated with benzene exposure. In HL-60 cell line, exploring the epigenetic events occurring in chromatin, we found the transient instauration of the distinctive signature combining the repressive H3Lys27 tri-methylation mark and the activating H3Lys4 tri-methylation mark (H3K27me3/H3K4me3), indicating a tendency toward a poised chromatin conformation. These alterations are lost in time after short-term treatments, while the long-term setting, performed using a concentration within the levels of total HQ in peripheral blood of benzene-exposed workers, showed a gradual increase in H3K4me3. We observed the absence of statistically significant variations in DNA methylation and expression levels of LINE-1, despite a decrease in protein levels of UHRF1, DNA methyl-transferases and histone methyl-transferases. In conclusion, in vitro treatment with low-dose HQ determined the instauration of a reversible poised state of chromatin in LINE-1 sequences, suggesting that prolonged exposure could cause persistent epigenetic alterations.

  12. CHANGES OF POLYAMINE METABOLISM IN HL-60 CELLS DURING THE INDUCTION OF DIFFERENTIATION BY RETINOIC ACID AND DIMETHYLSULFOXIDE

    Institute of Scientific and Technical Information of China (English)

    缪金明; 潘瑞彭; 欧阳仁荣

    1992-01-01

    The polyamines putrescine, spermidine and spermine have been implicated in the regulation of cell proliferation and differentiation. In this study, the changes of intracellular polyamine contents and activity of ornithine decarboxylase, a rate-limiting enzyme in the polyamine synthetic pathway, were studied. The results showed that both retinoic acid (RA) and dimethylsulfoxide (DMSO) could elevate intracellular putrescine level by more than 2-fold over control value, then it declined gradually. In RA-treated cells, transient increase in spermidine and spermine levels was noted. In contrast, the spermidine and spermine levels in DMSO-treated cells declined to about 50% of the level of control cells at 96 h. The measurement of ornithine decarboxylase activity demonstrated that the increase of intracellular putrescine in RA and DMSO treated cells was due to the polyamine synthesis by inducing ornithine decarboxylase which reached 2 to 4-fold higher over basic level at 2 h, and above 6-fold at 16 h. These results suggest that the polyamine metabolism may be involved in RA and DMSO-induced granulocytic differentiation of HL-60 promyelocytic leukemia cells.

  13. Induction of apoptosis in the human promyelocytic leukemia cell line HL60 by falconensone A and its derivatives, new polyenes.

    Science.gov (United States)

    Takahashi, N; Kubo, Y; Iwahori, A; Kawai, K I; Fukui, T

    2000-06-01

    Falconensones A and B are a new type of yellow pigment with structural similarity to retinoic acid isolated from the mycelial extract of ascomycetous fungi, Emericella falconensis or Emericella fruticulosa. In the present study we show that falconensone A alone induced apoptosis of HL60 human leukemia cells, while falconensone B, the 4'-nor-methyl derivative of falconensone A, had much lower activity. The synthetic derivatives of falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime, were more potent than natural falconensone A and B as far as the induction of apoptosis was concerned. The induction of apoptosis by the falconensones correlated with their inhibition of cell growth. In addition, falconensones A and B, and falconensone A dioxime, increased the generation of intracellular reactive oxygen species, while falconensone A p-bromophenylhydrazone was inactive. These results suggest that falconensone A, falconensone A p-bromophenylhydrazone and falconensone A dioxime are potential new apoptosis-inducing agents. The enhanced generation of reactive oxygen species in cells may be involved in apoptosis induced by falconensone A and falconensone A dioxime, but not by falconensone A p-bromophenylhydrazone. It is also suggested that the methyl residue at the 4' position of the falconensone A cyclopentenone ring may be essential for the induction of apoptosis. Based on these results, falconensone A and its derivatives may be clinically useful in the treatment of some leukemias.

  14. Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Guomin Niu; Songmei Yin; Shuangfeng Xie; Yiqing Li; Danian Nie; Liping Ma; Xiuju Wang; Yudan Wu

    2011-01-01

    Quercetin is one of the naturally occurring dietary flavo-nol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.

  15. 槲皮素联合硼替佐米或雷利度胺对抑制HL-60细胞增殖的影响%Effects of quercetin in combination with bortezomib or lenalidomide on inhibition of proliferation of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    肖洁; 牛国敏; 尹松梅; 谢双峰; 李益清; 聂大年; 马丽萍; 王秀菊; 吴裕丹

    2014-01-01

    目的:前期研究证实槲皮素可有效抑制白血病细胞株HL-60细胞增殖,本研究旨在进一步筛选与槲皮素有协同抗白血病作用的药物。方法:不同浓度硼替佐米与HL-60细胞孵育48 h,槲皮素联合硼替佐米孵育HL-60细胞48 h。不同浓度雷利度胺与HL-60细胞孵育48 h,槲皮素联合雷利度胺孵育HL-60细胞48 h。CCK-8法检测细胞存活率。结果:硼替佐米可显著抑制HL-60细胞的增殖。槲皮素联合硼替佐米的IC50值是49.24μmol/L,比单用槲皮素下降13.44μmol/L;等辐射分析法显示两药有协同作用。低浓度雷利度胺(5、10、20、40、80μmol/L)的细胞存活率与对照组比较无统计学差异,高浓度雷利度胺(160、320μmol/L)细胞存活率显著降低;雷利度胺联合槲皮素的IC50值较单用槲皮素无明显下降;等辐射分析法显示两药无协同作用。结论:硼替佐米可显著抑制HL-60细胞的增殖,槲皮素与硼替佐米有协同抑制HL-60细胞增殖的作用。雷利度胺抑制HL-60细胞增殖作用较弱,与槲皮素在抑制HL-60细胞增殖方面无协同作用。%Objective Our preliminary study demonstrates that quercetin can inhibit the proliferation of HL-60 cells. This sudy aimed to find some drugs which could have synergistic effects with quercetin on apoptosis of HL-60 cells. Methods HL-60 cells were cultured with bortezomib at different concentrations (1, 2, 4, 8, 16, and 32μmol/L) alone or combined with quercetin at different concentrations for 48 h. HL-60 cells were cultured with lenalidomide at different concentrations (5, 10, 20, 40, 80, 160, and 320 μmol/L) alone or in combination with quercetin at different concentrations for 48 h. The CCK-8 assay was used to determine the effects on proliferation of HL-60 cells. Results Bortezomib significantly inhibited the proliferation of HL-60 cells (P 0.05). The results of cell viability of HL-60 cells treated by lenalidomide

  16. Quercetin induces mitochondrial-derived apoptosis via reactive oxygen species-mediated ERK activation in HL-60 leukemia cells and xenograft.

    Science.gov (United States)

    Lee, Wei-Jiunn; Hsiao, Michael; Chang, Junn-Liang; Yang, Shun-Fa; Tseng, Tsui-Hwa; Cheng, Chao-Wen; Chow, Jyh-Ming; Lin, Ke-Hsun; Lin, Yung-Wei; Liu, Chung-Chi; Lee, Liang-Ming; Chien, Ming-Hsien

    2015-07-01

    Quercetin is a plant-derived bioflavonoid that was recently shown to have multiple anticancer activities in various solid tumors. Here, novel molecular mechanisms through which quercetin exerts its anticancer effects in acute myeloid leukemia (AML) cells were investigated. Results from Western blot and flow cytometric assays revealed that quercetin significantly induced caspase-8, caspase-9, and caspase-3 activation, poly ADP-ribose polymerase (PARP) cleavage, and mitochondrial membrane depolarization in HL-60 AML cells. The induction of PARP cleavage by quercetin was also observed in other AML cell lines: THP-1, MV4-11, and U937. Moreover, treatment of HL-60 cells with quercetin induced sustained activation of extracellular signal-regulated kinase (ERK), and inhibition of ERK by an ERK inhibitor significantly abolished quercetin-induced cell apoptosis. MitoSOX red and 2',7'-dichlorofluorescin fluorescence, respectively, showed that mitochondrial superoxide and intracellular peroxide levels were higher in quercetin-treated HL-60 cells compared with the control group. Moreover, both N-acetylcysteine and the superoxide dismutase mimetic, MnTBAP, reversed quercetin-induced intracellular reactive oxygen species production, ERK activation, and subsequent cell death. The in vivo xenograft mice experiments revealed that quercetin significantly reduced tumor growth through inducing intratumoral oxidative stress while activating the ERK pathway and subsequent cell apoptosis in mice with HL-60 tumor xenografts. In conclusions, our results indicated that quercetin induced cell death of HL-60 cells in vitro and in vivo through induction of intracellular oxidative stress following activation of an ERK-mediated apoptosis pathway.

  17. Polysaccharopeptides derived from Coriolus versicolor potentiate the S-phase specific cytotoxicity of Camptothecin (CPT on human leukemia HL-60 cells

    Directory of Open Access Journals (Sweden)

    Jiang Pingping

    2010-04-01

    Full Text Available Abstract Background Polysaccharopeptide (PSP from Coriolus versicolor (Yunzhi is used as a supplementary cancer treatment in Asia. The present study aims to investigate whether PSP pre-treatment can increase the response of the human leukemia HL-60 cells to apoptosis induction by Camptothecin (CPT. Methods We used bivariate bromodeoxyuridine/propidium iodide (BrdUrd/PI flow cytometry analysis to measure the relative movement (RM of the BrdUrd positively labeled cells and DNA synthesis time (Ts on the HL-60 cell line. We used annexin V/PI flow cytometry analysis to quantify the viable, necrotic and apoptotic cells. The expression of cyclin E and cyclin B1 was determined with annexin V/PI flow cytometry and western blotting. Human peripheral blood mononuclear cells were used to test the cytotoxicity of PSP and CPT. Results PSP reduced cellular proliferation; inhibited cells progression through both S and G2 phase, reduced 3H-thymidine uptake and prolonged DNA synthesis time (Ts in HL-60 cells. PSP-pretreated cells enhanced the cytotoxicity of CPT. The sensitivity of cells to the cytotoxic effects of CPT was seen to be the highest in the S-phase and to a small extent of the G2 phase of the cell cycle. On the other hand, no cell death (measured by annexin V/PI was evident with the normal human peripheral blood mononuclear cells with treatment of either PSP or CPT. Conclusion The present study shows that PSP increases the sensitization of the HL-60 cells to undergo effective apoptotic cell death induced by CPT. The pattern of sensitivity of cancer cells is similar to that of HL-60 cells. PSP rapidly arrests and/or kills cells in S-phase and did not interfere with the anticancer action of CPT. PSP is a potential adjuvant to treat human leukemia as rapidly proliferating tumors is characterized by a high proportion of S-phase cells.

  18. Control of macrophage cell differentiation in human promyelocytic HL-60 leukemia cells by 1,25-dihydroxyvitamin D/sub 3/ and phorbol-12-myristate-13-acetate

    Energy Technology Data Exchange (ETDEWEB)

    Murao, S.; Gemmell, M.A.; Callaham, M.F.; Anderson, N.L.; Huberman, E.

    1983-10-01

    Human promyelocytic leukemia cells (HL-60) were induced to differentiate into macrophage-like cells in a dose (3 x 10/sup -10/ to 10/sup -7/ M) and time (1 to 6 days)-dependent manner by 1,25-dihydroxyvitamin D/sub 3/ and the tumor promoter, phorbol-12-myristate-13-acetate. Differentiation was determined by an increase in the percentage of morphologically mature cells, in lysozyme and nonspecific esterase activities, and in reactivity with the murine OKM1 monoclonal antibody. Two HL-60 cell variants, designated as R-80 and B-II, were also examined. R-80 cells, which are resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate, also exhibited resistance, although to a lesser degree, to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/. Te resistance to the action of the two compounds is presumably not due to similar binding sites for the two inducers, since 1,25-dihydroxyvitamin D/sub 3/ was unable to compete for the phorbol diester binding sites as measured by (/sup 3/H)phorbol-12,13-dibutyrate binding. B-II cells were resistant to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide. Two-dimensional electrophoretic analysis of HL-60 cell protein patterns indicated that treatment of the HL-60 cells with 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide caused the cells to express various monocyte-macrophage and granulocyte marker proteins. These results indicate that 1,25-dihydroxyvitamin D/sub 3/ induces in the HL-60 cells a phenotype that resembles, but is not identical to, that of peripheral monocytes-macrophages. 40 references, 3 figures, 1 table.

  19. Interaction between {alpha}5{beta}1 integrin and secreted fibronectin is involved in macrophage differentiation of human HL-60 myeloid leukemia cells.

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    Laouar, A.; Collart, F. R.; Chubb, C. B. H.; Xie, B.; Huberman, E.; Center for Mechanistic Biology and Biotechnology; anl-cmb

    1999-01-01

    We examined the role of fibronectin (FN) and FN-binding integrins in macrophage differentiation. Increased FN and {alpha}5{beta}1 integrin gene expression was observed in phorbol 12-myristate 13-acetate PMA-treated HL-60 cells and PMA- or macrophage-CSF-treated blood monocytes before the manifestation of macrophage markers. After treatment of HL-60 cells and monocytes, newly synthesized FN was released and deposited on the dishes. An HL-60 cell variant, HL-525, which is deficient in the protein kinase C{beta} (PKC-{beta}) and resistant to PMA-induced differentiation, failed to express FN after PMA treatment. Transfecting HL-525 cells with a PKC-{beta} expression plasmid restored PMA-induced FN gene expression and macrophage differentiation. Untreated HL-525 cells (which have a high level of the {alpha}5{beta}1 integrin) incubated on FN differentiated into macrophages. The percentage of cells having a macrophage phenotype induced by PMA in HL-60 cells, by FN in HL-525 cells, or by either PMA or macrophage-CSF in monocytes was reduced in the presence of mAbs to FN and {alpha}5{beta}1 integrin. The integrin-signaling nonreceptor tyrosine kinase, p72{sup Syk}, was activated in PMA-treated HL-60 and FN-treated HL-525 cells. We suggest that macrophage differentiation involves the activation of PKC-{beta} and expression of extracellular matrix proteins such as FN and the corresponding integrins, {alpha}5{beta}1 integrin in particular. The stimulated cells, through the integrins, attach to substrates by binding to the deposited FN. This attachment, in turn, may through integrin signaling activate nonreceptor tyrosine kinases, including p72{sup Syk}, and later lead to expression of other genes involved in evoking the macrophage phenotype.

  20. Up- or downregulation of tescalcin in HL-60 cells is associated with their differentiation to either granulocytic or macrophage-like lineage.

    Science.gov (United States)

    Levay, Konstantin; Slepak, Vladlen Z

    2010-04-15

    Tescalcin is a 25-kDa EF-hand Ca(2+)-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16-48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15-30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types.

  1. Uvangoletin induces mitochondria-mediated apoptosis in HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances.

    Science.gov (United States)

    Zheng, Zhuanzhen; Qiao, Zhenhua; Gong, Rong; Wang, Yalin; Zhang, Yiqun; Ma, Yanping; Zhang, Li; Lu, Yujin; Jiang, Bo; Li, Guoxia; Dong, Chunxia; Chen, Wenliang

    2016-02-01

    This study investigated the cytotoxic effect of uvangoletin on HL-60 cells, and the effects of uvangoletin on myelosuppression, leucopenia, gastrointestinal tract disturbances and the possible cytotoxic mechanisms by using CCK-8, flow cytometry, western blot, xenograft, cyclophosphamide-induced leucopenia, copper sulfate-induced emesis and ethanol-induced gastric mucosal lesions assays. The results of CCK-8, flow cytometry and western blot assays indicated that uvangoletin showed the cytotoxic effect on HL-60 cells and induced the apoptosis of HL-60 cells by downregulating the expression levels of anti-apoptotic proteins (Survivin, Bcl-xl and Bcl-2), upregulating the expression levels of pro-apoptotic proteins (Smac, Bax, Bad, c-caspase-3 and c-caspase-9), and promoting the release of cytochrome c from mitochondria to cytoplasm. Further, the results of xenograft assay suggested that uvangoletin inhibited the HL-60-induced tumor growth without adverse effect on body weight of nude mice in vivo by regulating the expression levels of above apoptotic proteins. The results indicated that the reductions of WBCs count and thighbone marrow granulocytes percentage in cyclophosphamide-induced leucopenia assay, the incubation period and number of emesis in copper sulfate-induced emesis assay and the gastric mucosal lesions in ethanol-induced gastric mucosal lesions assay were not exacerbated or reversed by uvangoletin. In conclusion, the research preliminarily indicated that uvangoletin induced apoptosis of HL-60 cells in vitro and in vivo without adverse reactions of myelosuppression, leucopenia and gastrointestinal tract disturbances, and the pro-apoptotic mechanisms may be related to mitochondria-mediated apoptotic pathway.

  2. Quercetin simultaneously induces G0 /G1 -phase arrest and caspase-mediated crosstalk between apoptosis and autophagy in human leukemia HL-60 cells.

    Science.gov (United States)

    Chang, Junn-Liang; Chow, Jyh-Ming; Chang, Jer-Hwa; Wen, Yu-Ching; Lin, Yung-Wei; Yang, Shun-Fa; Lee, Wei-Jiunn; Chien, Ming-Hsien

    2017-03-02

    Quercetin is a plant-derived bioflavonoid with high anticancer activity in various tumors. Herein, the molecular mechanisms by which quercetin exerts its anticancer effects against HL-60 acute myeloid leukemia (AML) cells were investigated. Results showed that quercetin suppressed cell proliferation in the HL-60 cell line in vitro and in vivo. Quercetin-induced G0 /G1 -phase arrest occurred when expressions of cyclin-dependent kinase (CDK)2/4 were inhibited and the CDK inhibitors, p16 and p21, were induced. Moreover, quercetin treatment not only activated proapoptotic signaling like poly (ADP ribose) polymerase (PARP)-1 cleavage and caspase activation but also triggered autophagy events as shown by the increased expression of light chain 3 (LC3)-II, decreased expression of p62, and formation of acidic vesicular organelles. Interestingly, it was found that use of the autophagy inhibitor, 3-methyladenine, significantly enhanced quercetin-mediated apoptotic cell death as analyzed by MTS and DNA fragmentation assays. Moreover, pretreatment of HL-60 cells with the pan-caspase inhibitor, Z-VAD-fmk, dramatically reversed quercetin-mediated apoptotic and autophagic cell death. Although apoptosis and autophagy are two independent cell death pathways, our findings indicated that quercetin can activate caspases to trigger these two pathways, and both pathways played contrary roles in quercetin-mediated HL-60 cell death. In conclusion, besides promoting apoptosis, quercetin also induced cytoprotective autophagy in HL-60 cells, and inhibition of autophagy may be a novel strategy to enhance the anticancer activity of quercetin in AML.

  3. Viral fusogenic membrane glycoprotein induces HL-60 cell death via inhibiting NF-κB activity%病毒融合基因包膜糖蛋白通过抑制NF-κB活性诱导HL-60细胞死亡

    Institute of Scientific and Technical Information of China (English)

    谭丽; 谭获; 李小龙

    2012-01-01

    目的 探讨猿猴白血病病毒融合基因包膜糖蛋白(GALV-FMG)诱导白血病HL-60细胞的死亡效应与NF-κB的关系.方法 将HL-60细胞分为pcDNA3.1(+)-GALV-FMG质粒转染(A)组、pcDNA3.1(+)空载体对照(B)组和空白对照(C)组.脂质体转染后,应用乳酸脱氢酶释放实验检测GALV-FMG引起HL-60细胞的死亡效应,免疫荧光和凝胶电迁移检验分析GALV-FMG在诱导HL-60细胞死亡中NF-κB的活性.结果 与B、C组相比,A组细胞中NF-κB的活性受到了明显的抑制,细胞死亡的发生显著增加(P<0.05).结论 GALV-FMG的表达可诱导HL-60细胞发生死亡;其机制可能与NF-κB的活性抑制有关.%Objective To investigate the relationship between HL-60 cell death induced by gibbon ape leukemia virus fusion gene envelope glycoprotein (GALV-FMG) and NF-kB activity. Methods HL-60 cells were divided into three groups of A[transfected with pcDNA3. l( + )-GALV-FMG],B[transfected with empty pcDNA3.1( + ) vector] and C(blank controls). After lipofectamine transfection, HL-60 cell death induced by GALV-FMG was detected by lactate dehydrogenase release test, and NF-kB activity was analyzed by immunofluorescence and electrophoretic mobility shift assay. Results Compared with groups of B and C, NF-kB activity was significantly inhibited and cell death was remarkably increased in group C(P<0. 05). Conclusion GALV-FMG expression can induce HL-60 cell death, which may be related with inhibition of NF-kB activity.

  4. Down-regulation of Mcl-1 by small interference RNA induces apoptosis and sensitizes HL-60 leukemia cells to etoposide.

    Science.gov (United States)

    Karami, Hadi; Baradaran, Behzad; Esfehani, Ali; Sakhinia, Masoud; Sakhinia, Ebrahim

    2014-01-01

    Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Myeloid cell leukemia-1 (Mcl-1), a death-inhibiting protein that regulates apoptosis, has been shown to be overexpressed in numerous malignancies. In addition, it has been demonstrated that the expression level of the Mcl-1 gene increases at the time of leukemic relapse following chemotherapy. The aim of this study was to target Mcl-1 by small interference RNA (siRNA) and analyze its effects on survival and chemosensitivity of acute myeloid leukemia cell line HL-60. siRNA transfection was performed with a liposome approach. The expression levels of mRNA and protein were measured by real-time quantitative PCR and Western blot analysis, respectively. Trypan blue assays were performed to evaluate tumor cell growth after siRNA transfection. The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Apoptosis was quantified using a DNA-histone ELISA assay. Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a time- dependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Surprisingly, pretreatment with siMcl-1 synergistically enhanced the cytotoxic effect of etoposide. Furthermore, Mcl-1 down-regulation significantly increased apoptosis sensitivity to etoposide. No significant biological effects were observed with negative control siRNA treatment. Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. Therefore, siMcl-1 may be a potent adjuvant in leukemia chemotherapy.

  5. BCL-xL/MCL-1 inhibition and RARγ antagonism work cooperatively in human HL60 leukemia cells.

    Science.gov (United States)

    Perri, Mariarita; Yap, Jeremy L; Yu, Jianshi; Cione, Erika; Fletcher, Steven; Kane, Maureen A

    2014-10-01

    The acute promyelocytic leukemia (APL) subtype of acute myeloid leukemia (AML) is characterized by chromosomal translocations that result in fusion proteins, including the promyelocytic leukemia-retinoic acid receptor, alpha fusion protein (PML-RARα). All-trans retinoic acid (atRA) treatment is the standard drug treatment for APL yielding cure rates > 80% by activating transcription and proteasomal degradation of retinoic acid receptor, alpha (RARα). Whereas combination therapy with As2O3 has increased survival further, patients that experience relapse and are refractory to atRA and/or As2O3 is a clinically significant problem. BCL-2 family proteins regulate apoptosis and over-expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL-2) family proteins has been associated with chemotherapeutic resistance in APL including impairment of the ability of atRA to induce growth arrest and differentiation. Here we investigated the novel BH3 domain mimetic, JY-1-106, which antagonizes the anti-apoptotic BCL-2 family members B-cell lymphoma-extra large (BCL-xL) and myeloid cell leukemia-1 (MCL-1) alone and in combination with retinoids including atRA, AM580 (RARα agonist), and SR11253 (RARγ antagonist). JY-1-106 reduced cell viability in HL-60 cells alone and in combination with retinoids. The combination of JY-1-106 and SR11253 had the greatest impact on cell viability by stimulating apoptosis. These studies indicate that dual BCL-xL/MCL-1 inhibitors and retinoids could work cooperatively in leukemia treatment.

  6. Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Judit Molnár

    2016-02-01

    Full Text Available Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1, 3β-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13-en-12,6α-olide (2, iso-seco-tanapartholide 3-O-methyl ether (3 and 4β,15-dihydro-3-dehydrozaluzanin C (4, were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica. When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC50 values in the range 3.6–13.5 μM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1–3 elicited increases in the hypodiploid (subG1 population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258–propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.

  7. Fractionation of the Hypericum perforatum L. extract: PMF, and PDT effects of the fractions against HL-60 leukemic cells

    Science.gov (United States)

    Tsontou, M.; Dimitriou, H.; Filippidis, G.; Tsimaris, I.; Kalmanti, M.; Skalkos, D.

    2007-02-01

    In the last three years we have prepared and studied the polar methanolic extract PMF, of the herb Hypericum perforatum L, and studied as a new, alternative photosensitizing substance for PDT. Hypericum perforatum L., as well as PMF, contains a number of naphthodianthrone derivatives (hypericins), such as hypericin and pseudohypericin, as its main photosensitizing constituents. PMF has been tested as a PDT agent in vitro in bladder cancer cells, leukemia cells, and in vivo in rat tumor bearing urinary bladder. In order to evaluate the contribution of the hypericins in the overall PDT action, and prepare a better photosensitizing extract than PMF, we have separated the extract in four main fractions (1,2,3,4), and tested their PDT effects against the HL-60 leukemic cells. The concentration of hypericins in the extracts was found 0.08% for fraction 1, 0.09% for fraction 2, 0.8% for fraction 3, and 2,8% for fraction 4. The PDT activity observed among the fractions was proportional to their hypericins concentration, thus increasing in the order of increasing number: fraction 4 > fraction 3 > fraction 2 > fraction 1. Fraction 4 proved to be the most powerful fraction. However, despite its relatively high hypericins concentration (2.8%), compared with the total extract PMF (1.37%), fraction 4 proved to be less active in the cell line tested. This result indicates that there are other photosensitizing constituents within the PMF extract which contribute significantly in the overall PDT action, and therefore the extract should be used as it is for further PDT studies, without any further purification.

  8. Purification of a 53kD pI 4. 8 cytosolic phosphoprotein from HL60

    Energy Technology Data Exchange (ETDEWEB)

    Biser, P.S.; Spearman, T.N.; Bruzzone, M.; Durham, J.P.

    1987-05-01

    In order to study the potential role of a 53kD pI 4.8 phosphoprotein in the differentiation of HL60 using monoclonal antibodies, a partial purification has been carried out. Cytosol from cells differentiated with 1 M retinoic acid was applied to a DEAE-cellulose column and eluted with a linear NaCl gradient. Fractions were screened by in vitro phosphorylation of aliquots using /sup 32/P ATP and highly purified protein kinase C, SDS-PAGE, and autoradiography. Fraction which showed autoradiographic bands of the correct molecular weight were further analyzed using 2-D electrophoresis involving isolectric focusing over a pH range of 4-6 followed by SDS-PAGE on a 10% slab gel. Autoradiograms of these gels showed the 53 kD pI 4.8 phosphoprotein to elute with a peak at 0.24 NaCl. This 53 kD pI 4.8 protein was identified as the 53kD pI 4.8 phosphoprotein whose synthesis and phosphorylation is induced by retinoic acid by DEAE chromatography of cytosol from cells labelled in vivo with /sup 32/PO/sub 4//sup -2/ followed by 2-D electrophoresis. Fractions containing the 53 kD pI 4.8 protein were concentrated and applied to a chromatofocusing column which was eluted with a gradient from pH 6 to 4. Analysis of fractions via in vitro phosphorylation and SDS PAGE showed the 53 kD pI 4.8 protein eluting with a peak at pH 4.8 as a silver-stained band well separated from contaminating proteins. Experiments are currently in progress to produce monoclonal antibodies to the 53 kD pI 4.8 protein using the partially purified antigen.

  9. Production of hydrogen peroxide is responsible for the induction of apoptosis by hydroxytyrosol on HL60 cells.

    Science.gov (United States)

    Fabiani, Roberto; Fuccelli, Raffaela; Pieravanti, Federica; De Bartolomeo, Angelo; Morozzi, Guido

    2009-07-01

    Hydroxytyrosol [3,4-dihydroxyphenylethanol (3,4-DHPEA)], a phenolic compound found exclusively in olive oil, exerts growth-suppressive and pro-apoptotic effects on different cancer cells. Although some molecular mechanisms involved in the pro-apoptotic activity of 3,4-DHPEA have been proposed, the initial stress signals responsible of this phenomenon are not known. Our aim was to assess the involvement of reactive oxygen species as mediators of apoptosis induced by 3,4-DHPEA on HL60 cells. Apoptosis was determined by analyzing the nuclear fragmentation by both fluorescence microscopy and flow cytometry. The externalization of phosphatidylserine was evidenced using an Annexin V-FITC kit. The concentration of H(2)O(2) in the culture medium was measured by the ferrous ion oxidation-xylenol orange method. The pro-apoptotic effect of 3,4-DHPEA (100 muM) was prevented by N-acetyl-cysteine, ascorbate, and alpha-tocopherol. Catalase suppressed the 3,4-DHPEA-induced apoptosis, while the Fe(II)-chelating reagent o-phenantroline showed no effect, suggesting the involvement of H(2)O(2 )but not of OH(*). Indeed, 3,4-DHPEA caused accumulation of H(2)O(2) in the culture medium. Tyrosol (p-hydroxyphenylethanol) and caffeic acid, compounds structurally similar to 3,4-DHPEA but not able to generate H(2)O(2), did not induce an appreciable apoptotic effect. This is the first study demonstrating that apoptosis induction by 3,4-DHPEA is mediated by the extracellular production of H(2)O(2).

  10. 2 (±)-7, 8, 3′, 4′, 5′-Pentamethoxyflavan induces G2/M phase arrest and apoptosis in human leukemia HL60 cells

    Institute of Scientific and Technical Information of China (English)

    TAI Wen-jiao; LI Yan-chun; LI Te; ZHANG Wei-ge; MA En-long

    2008-01-01

    Objective Flavans are a set of naturally occurring flavonoids possessing a 2-phenylchroman nucleus, which are widely distributed in the plant kingdom. A number of flavan compounds exhibit antitumor activities. In our previous report, a straightforward synthetic procedure for 2 (±)-7, 8, 3′, 4′, 5′-pentamethoxyflavan (PMF) was developed. To be more important, PMF showed growth inhibitory effect on various human tumor cell lines, especially against HL60 ceils. In the present study, we aim to investigate the molecular mechanisms of action of PMF in HL60 cells. This is the first report of the molecular mechanisms on anti-tumor effect of flavan compounds. Methods Trypan blue exclusion experiment was used for cell growth inhibition assay. Cell apoptosis, cell cycle distribution and the mitochondrial membrane potential (MMP) were assessed by flowcytometric analysis after AO/EB, PI and Rh123 flurescence staining, respectively. Cell cycle- and apoptosis-related proteins were detected using western blotting analysis. Results PMF (1-30 μM) inhibited the growth of HL60 cells in a time- and concentration-dependent manner. Antiproliferative effect of PMF on HL60 cells was associated with G2/M cell cycle arrest, which was mediated by regulating the expression of p21, Cdc25C and cyclin A proteins and inhibiting the phosphorylation of Cdc2 at Thr161. The prolonged PMF treatment also induced apoptosis of HL60 cells, which was characterized by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase, easpase-3, caspase-8 and caspase-9, changes of Bcl-2 and Bax expression and a decrease in the mitochondrial membrane potential (MMP). Furthermore, caspase-3 inhibitor, not caspase-8 inhibitor and caspase-9 inhibitor, completely blocked PMF-caused apoptosis. Conclusions PMF inhibited the growth of HL60 cells via induction of G2/M arrest and apoptosis. Blockade of cell cycle was associated with the downregulation of Cdc2 complex activity. Both death receptor and mitochondrial

  11. [Demethylation effect of inhibitor As2O3 on expression of SHP-1 and C-kit genes in leukemia HL-60 cells].

    Science.gov (United States)

    Meng, Zhen; Wang, Dong-Mei; Li, Ying-Hua; Liu, Xiao; Guo, Su-Qing; Luo, Jian-Min

    2013-06-01

    This study was aimed to investigate the expression level of SHP-1 and C-kit genes in acute leukemia HL-60 cells and effect of inhibitor As2O3 demethylation on SHP-1 and C-kit genes expression. RT-PCR was used to detect the expression level of SHP-1 and C-kit mRNA in drug-treated cell group and control group. The methylation specific PCR (MSP) was applied to measure the methylation status of SHP-1 gene in HL-60 cells. The results showed that after being treated with As2O3 the recovery of SHP-1 gene expression was observed in HL-60 cells in which SHP-1 mRNA originally did not expressed, meanwhile the expression level of C-kit mRNA in HL-60 cells with high expression decreased. When HL-60 cells were treated with As2O3 of 1.0, 2.5, 5.0 µmol/L, the demethylation effects was enhanced, the expression of SHP-1 mRNA displayed an ascending tendency, and expression of C-kit mRNA showed an descending tendency in dose-dependent manner (P HL-60 cells and recovery of expression after treatment with As2O3 suggest the hypermethylation of SHP-1 gene related with pathogenesis of leukemia, and the abnormal increase of C-kit mRNA expression maybe exist in formation of leukemia. The effect of As2O3 on expression of SHP-1 and C-kit shows dose-dependency, the higher the As2O3 concentration, the higher the SHP-1 expression and the lower the C-kit expression, moreover, the effect of As2O3 shows time-dependency in specific concentration. The SHP-1 mRNA expression negatively relates with C-kit mRNA expression, suggesting that the decrease or absence of SHP-1 expression in leukemia cells weakens the negative regulation on C-kit signaling pathway, thus plays a role in the formation of leukemia.

  12. In vitro anticancer property of a novel thalidomide analogue through inhibition of NF-κB activation in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Min LI; Wan SUN; Ya-ping YANG; Bo XU; Wen-yuan YI; Yan-xia MA; Zhong-jun LI; Jing-rong CUI

    2009-01-01

    Aim: To investigate the anticancer property and possible mechanism of action of a novel sugar-substituted thalidomide derivative (STA-35) on HL-60 cells in vitro. Methods: TNF-α-induced NF-κB activation was determined using a reporter gene assay. The MTT assay was used to mea-sure cytotoxicity of the compound. The appearance of apoptotic Sub-G1 cells was detected by flow cytometry analysis. PARP cleavage and protein expression of NF-κB p65 and its inhibitor IκB were viewed by Western blotting. Results: STA-35 (1-20 μmol/L) suppressed TNF-α-induced NF-κB activation in transfected cells (HEK293/pNiFty-SEAP) in a dose- (1-20 μmol/L) and time-dependent (0-48 h) manner. It was also shown that STA-35 exerted a dose-dependent inhibitory effect on HL-60 cell proliferation with an IC50 value of 9.05 μmol/L. In addition, STA-35 induced apoptosis in HL-60 cells, as indicated by the appearance of a Sub-G1 peak in the cell cycle distribution, as well as poly ADP-ribose polymerase (PARP) cleavage. Subsequently, both NF-κB p65 and its inhibitor IκB gradually accumulated in cytoplasmic extracts in a dose- and time-dependent manner, indicating the blockage of NF-κB translocation induced by TNF-α from the cytoplasm to the nucleus. Conclusion: A novel sugar-substituted thalidomide derivative, STA-35, is potent toward HL-60 cells in vitro and induces apoptosis by the suppression of NF-κB activation.

  13. Mangiferin induces cell cycle arrest at G2/M phase through ATR-Chk1 pathway in HL-60 leukemia cells.

    Science.gov (United States)

    Peng, Z G; Yao, Y B; Yang, J; Tang, Y L; Huang, X

    2015-05-12

    This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.

  14. 18α-Glycyrrhetinic Acid Induces Apoptosis of HL-60 Human Leukemia Cells through Caspases- and Mitochondria-Dependent Signaling Pathways.

    Science.gov (United States)

    Huang, Yi-Chang; Kuo, Chao-Lin; Lu, Kung-Wen; Lin, Jen-Jyh; Yang, Jiun-Long; Wu, Rick Sai-Chuen; Wu, Ping-Ping; Chung, Jing-Gung

    2016-07-01

    In this study we investigate the molecular mechanisms of caspases and mitochondria in the extrinsic and intrinsic signal apoptosis pathways in human leukemia HL-60 cells after in vitro exposure to 18α-glycyrrhetinic acid (18α-GA). Cells were exposed to 18α-GA at various concentrations for various time periods and were harvested for flow cytometry total viable cell and apoptotic cell death measurements. Cells treated with 18α-GA significantly inhibited cell proliferation and induced cell apoptosis in a dose-dependent manner, with an IC50 value of 100 μM at 48 h. The cell growth inhibition resulted in induction of apoptosis and decreased the mitochondria membrane potential (ΔΨm) and increased caspase-8, -9 and -3 activities. Furthermore, cytochrome c and AIF were released from mitochondria, as shown by western blotting and confirmed by confocal laser microscopy. Western blotting showed that 18α-GA increased the levels of pro-apoptotic proteins such as Bax and Bid and decreased the anti-apoptotic proteins such as Bcl-2 and Bcl-xl, furthermore, results also showed that 18α-GA increased Fas and Fas-L which are associated with surface death receptor in HL-60 cells. Based on those observations, the present study supports the hypothesis that 18α-GA-induced apoptosis in HL-60 cells involves the activation of the both extrinsic and intrinsic apoptotic pathways.

  15. Juglone, from Juglans mandshruica Maxim, inhibits growth and induces apoptosis in human leukemia cell HL-60 through a reactive oxygen species-dependent mechanism.

    Science.gov (United States)

    Xu, Hua Li; Yu, Xiao Feng; Qu, Shao Chun; Qu, Xiang Ru; Jiang, Yan Fang; Sui, Da Yuan

    2012-03-01

    Juglone, a major chemical constituent of Juglans mandshruica Maxim, is a promising anticancer agent that has shown a strong activity against cancer cells in vitro. Our previous study showed that juglone inhibited the proliferation of HL-60 cells with an IC50 value ∼8 μM. To further explore the proapoptotic mechanism of juglone, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by juglone in HL-60 cells. The generation of ROS was about 2 to 8-fold as compared to control cell after treatment with juglone (2, 4 and 8 μM) for 24 h. The glutathione (GSH) depletion was consistent with ROS generation after treatment with juglone. Reversal of apoptosis in antioxidants (NAC and catalase) pretreated cells indicated the involvement of ROS in juglone-induced apoptosis. The cleavage of PARP and procaspase-3 and -9, loss of mitochondrial membrane potential (△Ψm), and release of cytochrome c (Cyt c) and Smac induced by juglone were significantly blocked by NAC. NAC also prevented the inhibition the phosphorylation of Akt and mTOR proteins by juglone. Collectively, these results indicated that ROS played a significant role in the apoptosis induced by juglone in human leukemia cell HL-60.

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

    Directory of Open Access Journals (Sweden)

    LATIFAH SAIFUL YAZAN

    2009-01-01

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

  17. Influence of Radix Isatidis on the Endotoxin-induced Release of TNF-α and IL-8 from HL-60 Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The effects of active antiendotoxin chemical fraction isolated from Radix Isatidis (fraction D) on TNF-α and IL-8 secretion in HL-60 cells induced by lipopolysaccharide (LPS) were studied. The appropriate densities of cell suspension and fraction D solution were determined by MTT colorimetric method. Fraction D and LPS were added to HL-60 cell suspension with three different methods respectively. The contents of TNF-α and IL-8 in the cultured supernatant induced by LPS were detected by using ELISA method. The results showed that the absorbance (A) was directly proportional to the number of cells and the linearity was good in the range from 0.25 × 105 to 2 ×105 cell/mL cell suspension. The fraction D significantly inhibited the oversecretion of TNF-α and IL-8 in HL-60 cells induced by LPS at the concentration of 7. 812 mg/mL which had no cytotoxicity. It was indicated that the antiendotoxin mechanism of the active fraction from Radix Isatidis was contributed to the inhibition of the oversecretion of cytokines induced by LPS.

  18. Analysis of Spontaneous,gamma ray-and ethylnitrosourea-induced hprt mutants in HL-60 cells With multiplex PCR

    Institute of Scientific and Technical Information of China (English)

    Sheng-Xue Liu; Jia Cao; Hui An; Hua-Min Shun; Lu-Jun Yang; Yong Liu

    2003-01-01

    AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and 60Co γ-rays.METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and 60Co γ-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique.RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200μg/ml, P<0.01; in the group with dosage of 60Co γ-ray with 2-4 Gy, P<0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 μg/ml, P<0.05; in the group of 60Co γ-ray with 1-4 Gy, P<0.05) significantly. In the 13spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %,P<0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P<0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gammaray-induced mutants, 88.1% in ENU-induced mutants).CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or 60Co γ-ray.Although both ENU and γ-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.

  19. A new 2-aminosteroid induces cellular differentiation and upregulates the expression of MafB and Egr-1 genes respectively in HL-60 and K562 leukemia cells

    Institute of Scientific and Technical Information of China (English)

    HE Qun; LI Qiong; YUAN Lin-bo; HE Jun

    2005-01-01

    Background In previous work, we suggested that some 2-aminosteroids inhibited proliferation and induced differentiation of both human and murine leukemia cells. Here, we reported the actions of another new 2-aminosteroid designated as H89712 on human leukemia cells. Methods Cell colony counting and MTT assay were used to determine proliferation. Cell morphology, histochemical staining, UV detection and cytometry were used to determine differentiation. RT-PCR was used to detect gene expression. Standard statistical method was used to analyze data.Results H89712 inhibited proliferation of HL-60 leukemia cells and the inhibition percentage in MTT assay was 18% at the dose of 10-8 mol/L and 65% at the dose of 10-5 mol/L, respectively. The inhibition for HL-60 in colony assay was 23% at the dose of 10-8 mol/L and 96% at the dose of 10-5 mol/L, respectively. H89712 also induced HL-60 cells toward macrophage-like differentiation. It was verified by flow cytometry that the percentage of positive CD14 expression in differentiated HL-60 cells was about 9 times higher than that of the control at the dose of 10-8 mol/L and 20 times higher than that of the control at the dose of 10-5 mol/L respectively, and this action involved upregulation of MafB gene in HL-60 leukemia cells. On the other hand, H89712 inhibited proliferation of K562 leukemia cells and the inhibition of K562 leukemia cells in MTT assay was shown by 34% at the dose of 10-8 mol/L and 88% at the dose of 10-5 mol/L respectively. The inhibition of K562 leukemia cells in colony assay was 53% at the dose of 10-8 mol/L and 100% at the dose of 10-5 mol/L respectively. H89712 also induced K562 cells toward erythroid-like differentiation and it was verified by flow cytometry that the percentage of positive CD71 expression in differentiated K562 cells was about 9 times higher than that of the control at the dose of 10-8 mol/L and 16 times higher than that of the control at the dose of 10-5 mol/L respectively. This action

  20. Granulocyte colony-stimulating factor inhibits CXCR4/SDF-1α signaling and overcomes stromal-mediated drug resistance in the HL-60 cell line.

    Science.gov (United States)

    Sheng, Xianfu; Zhong, Hua; Wan, Haixia; Zhong, Jihua; Chen, Fangyuan

    2016-07-01

    Combining cytarabine, aclarubicin and granulocyte colony-stimulating factor (G-CSF) has demonstrated marked efficacy in the treatment of elderly and relapsed/refractory patients with acute myeloid leukemia (AML); however, the role of G-CSF remains poorly understood. The present study aimed to investigate the ability of G-CSF to overcome stromal-mediated drug resistance and the underlying molecular mechanism. Two types of co-culture models were established in the HS-5 human bone marrow/stromal and HL-60 human promyelocytic leukemia cell lines, in order to imitate the interactions between stromal and leukemia cells in vitro, which is mediated by the stromal cell-derived factor (SDF)-1α signaling axis. In the present study, HL-60 cells were attracted and adhered to HS-5 cells using migration assay and flow cytometry, respectively; however, these interactions were inhibited by treatment with G-CSF and/or the C-X-C chemokine receptor type 4 (CXCR4) antagonist, AMD3100. Co-culture with HS-5 cells, including direct and indirect contact, protected HL-60 cells against spontaneous apoptosis or drug-induced apoptosis; however, these protective effects were disrupted by treatment with G-CSF and/or AMD3100. Notably, G-CSF and/or AMD3100 did not alter cell viability or apoptosis when HL-60 cells were cultured with medium alone. In addition, G-CSF significantly reduced the expression levels of surface CXCR4 protein, total CXCR4 protein and CXCR4 mRNA, and significantly upregulated the expression of microRNA (miR)-146a. Conversely, AMD3100 significantly reduced surface CXCR4 expression levels, but not the total CXCR4, CXCR4 mRNA or miR-146a expression levels. The results of the present study suggested that interfering with the CXCR4/SDF-1α signaling axis via G-CSF inhibited the migration and adhesion of HL-60 cells to HS-5 cells and eliminated HS5 cell-mediated protective effects. Furthermore, G-CSF administration reduced CXCR4 expression levels by upregulating the expression of

  1. Cell differentiation and down regulation of nm23 gene expression in HL-60 cells induced by 1,25 dihydroxyvitamin D3%1,25-二羟基维生素D3诱导HL-60细胞分化并下调nm23基因的表达

    Institute of Scientific and Technical Information of China (English)

    朱宁希; 吕庆华; 周莹; 虞荣喜

    2001-01-01

    目的观察1,25-二羟基维生素D3[1,25(OH)2D3]对白血病细胞株HL-60的诱导分化作用并探讨其机制.方法通过MTT试验、细胞形态观察、表面标志分析和硝基四氮唑蓝(NBT)还原反应, 观察1,25(OH)2D3作用后HL-60细胞增殖的改变和分化情况;通过流式细胞仪和RT-PCR检测细胞周期和nm23基因表达的变化.结果 HL-60细胞经1,25(OH)2D3作用后增殖受抑制、向成熟单核细胞系分化、细胞被阻滞在G1期、nm23基因的表达下调.结论 1,25(OH)2D3对HL-60细胞株具有诱导分化作用,其作用机制可能与下调nm23基因的表达有关.

  2. 全反式维甲酸与紫杉醇、阿霉素联合诱导HL-60细胞凋亡的研究%Studies on the apoptosis using ATRA in combination with taxol and ADR in HL-60 cell lines

    Institute of Scientific and Technical Information of China (English)

    尤学芬; 丁润生; 谢玉娟; 姚登福; 陆德炎

    2007-01-01

    目的:探讨全反式维甲酸(ATRA)分别与紫杉醇(Taxol)、阿霉素(ADR)单独或联合孵育HL-60细胞株能否增强凋亡诱导作用,并进一步研究bcl-2基因及家族bax、bcl-x基因在此过程中的作用.方法:①用台盼兰拒染法观察细胞生长活力;②在光镜和电镜下观察细胞形态变化;③用流式细胞仪(FCM)检测药物孵育前后细胞凋亡率(AP)的变化;④用RT-PCR法检测药物孵育前后bcl-2基因及其家族bax、bcl-x基因的表达水平.结果:①ATRA能增强Taxol和ADR对HL-60细胞的生长抑制作用;②ATRA能增强HL-60细胞对Taxol和ADR诱导的细胞凋亡;③在ATRA与Taxol、ADR联合诱导的HL-60细胞凋亡中,bcl-2、bcl-xl基因表达下降,bax、bcl-xs基因表达增加.结论:ATRA能增加Taxol和ADR对HL-60细胞的生长抑制作用及凋亡诱导作用,bcl-2基因及家族bax、bcl-x基因参与了ATRA与Taxol、ADR联合诱导细胞凋亡的调控.

  3. Role of alpha class glutathione transferases (GSTs) in chemoprevention: GSTA1 and A4 overexpressing human leukemia (HL60) cells resist sulforaphane and curcumin induced toxicity.

    Science.gov (United States)

    Sharma, Rajendra; Ellis, Bryan; Sharma, Abha

    2011-04-01

    Alpha-class glutathione transferases (α-GSTs) have been shown to protect cells from the harmful effects of reactive oxygen species (ROS) induced lipid peroxidation (LPO) during oxidative stress caused by various physico-chemical agents. While GSTA1-1/A2-2 isozymes exhibit high activity towards lipid and fatty acid hydroperoxides through their selenium independent glutathione peroxidase (GPx) activity, the GSTA4-4 isozyme efficiently metabolizes the LPO product 4-hydroxynonenal (4-HNE) by conjugating it with glutathione (GSH). Because of the fact that ROS generated by the chemopreventive agents, sulforaphane (SFN) and curcumin (Cur), are implicated in the mechanisms of cancer cell killing, the present studies were designed to investigate the contribution of ROS induced LPO in the cytotoxic effects of these agents and the role of α-class GSTs in modulating their toxicity. Human erythroleukemic (HL60) cells were stably transfected with the cDNA encoding the hGSTA1-1 and mGsta4-4 isozymes. After analysing the expression and activities of the respective GST isozymes, the effects of SFN and Cur on the extent of LPO, cytotoxicity and apoptosis were compared in empty vector (VT), hGSTA1-1 and mGsta4-4 expressing HL60 cells. These studies demonstrate that when compared with SFN, Cur was relatively more cytotoxic to HL60 cells. The ectopic expression of hGSTA1-1 and mGsta4-4 isozymes provided resistance to SFN and Cur induced cytotoxicity and apoptosis through a significant suppression of LPO in these cells. Overall, the results suggest that the expression of α-class GSTs in cancer cells can modulate the therapeutic efficacy of chemopreventive agents.

  4. Sulforaphane-induced apoptosis in human leukemia HL-60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray.

    Science.gov (United States)

    Shang, Hung-Sheng; Shih, Yung-Luen; Lee, Ching-Hsiao; Hsueh, Shu-Ching; Liu, Jia-You; Liao, Nien-Chieh; Chen, Yung-Liang; Huang, Yi-Ping; Lu, Hsu-Feng; Chung, Jing-Gung

    2017-01-01

    Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN-induced cytotoxic effects and whether or not they went through cell-cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL-60 cells. Cell viability, cell-cycle distribution, sub-G1 (apoptosis), reactive oxygen species (ROS) and Ca(2+) production, levels of mitochondrial membrane potential (ΔΨm ), and caspase-3, -8, and -9 activities were assayed by flow cytometry. Apoptosis-associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub-G1 phase development. Furthermore, SFN increased ROS and Ca(2+) production and decreased the levels of ΔΨm and activated caspase-3, -8, and -9 activities in HL-60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas-L, caspase-8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl-x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase -8, -3, -4, -6, and -7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL-60 cells via Fas- and mitochondria-dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311-328, 2017.

  5. 瑞香狼毒诱导HL-60细胞凋亡和调节SGC-7901细胞bcl-2蛋白表达%Stellera chamaejasme induced apoptosis of HL-60 cells and regulated expression of bcl-2 protein in SGC-7901 cells

    Institute of Scientific and Technical Information of China (English)

    贾正平; 王彦广; 樊俊杰; 谢景文; 徐丽婷; 刘盛

    2001-01-01

    Object To explore the antitumor mechanism of Stellera chamaejasme Linn.(SC).Methods SC containing-serum(SCCS)was derived from mice pretreated with different doses of SC.Cultured human leukemia HL-60 and human gastric adenocarcinoma SGC-7901 cells were used.Inhibition of proliferation was measured using MTT assay.Morphological assessment of apoptosis was performed with fluorescence microscope.DNA fragmentation was assessed by agarose gel electrophoresis and flow cytometry.Expression of bcl-2 protein was measured with immunohistochemistry.Results Exposure of exponentially growing HL-60 cells to mice serum containing 10% SC(pretreated with SC3,6, and 12 g/kg)for 48h resulted in growth inhibition in a dose-dependent manner.Typical morphological changes of apoptosis and DNA fragmentation in HL-60 cells were induced."Apobodies'in the apoptotic cells were observed,'ladder"pattern of agarose gel electrophoresis of DNA from 11.7% to 57.4%.Treatment with SC containing serum decreased the percentage of SGC-7901 cell of bcl-2 protein positive expression from 78.3% to 32.9%.Conclusion SC could induce apoptosis of HL-60 cells and decrease the expression of bcl-2 protein of gastric adenocarcinoma SGC-7901 cells.%目的探索瑞香狼素(SC)抗肿瘤机制.方法以HL-60和SGC-7901为靶细胞,用MTT比色法测定细胞增殖抑制,荧光显微镜观察凋亡细胞的形态学改变,DNA电泳和流式细胞仪检测DNA断裂,免疫组化检测bcl-2蛋白表达.结果含SC药物血清处理细胞48 h后,HL-60细胞增殖呈剂量依赖性抑制,并表现出典型的凋亡细胞形态学改变及DNA断裂:即染色体聚集、核固缩、断裂及阶梯状DNA电泳条带,G1期前细胞从11.7%增至57.4%;而SGC-7901细胞bcl-2蛋白表达率从78.3%下降到32.9%.结论 SC可诱导肿瘤细胞凋亡,降低bcl-2蛋白表达.

  6. 冬凌草甲素联合丙戊酸钠对HL-60细胞的生长抑制和诱导凋亡作用研究%Growth inhibition and apoptosis induced by oridonin combined with valproic acid in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    邹慧娟; 姜国胜

    2011-01-01

    Objective To investigate the apoptosis-inducing effect of oridonin combined with valproic acid( VPA)on leukemic cell line HL-60,and study the feasibility of oridonin combined with VPA to be used in clinical practice. Methods Oridonin of 6-12 μmol/L combined with VPA of 0. 5-1 mmol/L were added in exponential growth HL-60 cells respectively. Cell count assays were used to measure the growth inhibitory effect of oridonin combined with VPA or alone. Flow cytometry was used to evaluate apoptosis with Annexin V and propidium iodide (PI) double staining. Results Combined use of oridonin and VPA could synergistically inactivate HL-60 cells,and inhibit the cell proliferation and induce apoptosis in a dose-and time-dependent manner (P < 0.05 ). Conclusion Oridonin has a synergistic effect combined with VPA. Oridonin has a promising prospect in clinical use of leukemia.%目的 研究中药单体冬凌草甲素(ORI)联合组蛋白去乙酰化酶抑制剂丙戊酸钠(VPA)诱导急性早幼粒白血病细胞株HL-60凋亡,探讨其应用于临床的可行性.方法 在对数生长期的HL-60细胞中分别加入6~12 la,mol/L的ORI和0.5~1 mmol/L的VPA,采用细胞计数法测定ORI和VPA单独和联合应用时对细胞的生长抑制,并用Annexin V/PI双标法流式细胞术检测细胞凋亡.结果 ORI联合VPA可协同降低HL-60细胞活力,抑制细胞增殖,诱导细胞凋亡,比单独用药凋亡作用更为显著(P<0.05).结论 ORI和VPA具有协同作用,能高效杀灭白血病细胞.ORI和VPA是一种有望应用于白血病临床治疗的新型生物制剂.

  7. Regulation of CD11b transcription by decreasing PRC2 and increased acH4 level during ATRA-induced HL-60 differentiation

    Institute of Scientific and Technical Information of China (English)

    Huarong Tang; Fangping Chen; Qian Tan; Sanqin Tan; Linxin Liu; Fan Zhang

    2009-01-01

    Polycomb repressive complex 2 (PRC2),which mediates trimethylation of lysine 27 on histone H3 (K27me3),plays an important role in many types of stem cell differentiation.Here,we try to reveal how PRC2,PRC2-mediated repressive histone marker H3K27me3,and active histone marker histone H4 acetylation (acH4) regulate the CD11b transcription during all-trans retinoic acid (ATRA)-induced HL-60 leukemia cell differentiation.By using quantitative real-time polymerase chain reaction (qPCR) and western blot analysis,we found that the mRNA and protein expression levels of two members of PRC2 were decreased during ATRA-induced HL-60 differentiation,respectively.When treated with ATRA for 72 h,the EZH2 and SUZ12 mRNA levels were decreased to 35% and 38% of the control group,respectively.At the same time,the granulocytic mature surface marker CD11b expression was increased significantly at mRNA level detected by qPCR and protein level detected by flow cytometry.By using chromatin immunoprecipita-tion assay,we compared the local changes in SUZ12 binding and PRC2-mediated H3K27me3 at the promo-ter of CD11b during ATRA-induced HL-60 differ-entiation.Both the levels of SUZ12 binding and PRC2-mediated H3K27me3 at the promoter of CD11b were decreased for 4.1 and 3.8 folds,respectively.And we also found the increase in the acH4 level up to 4 folds after 72 h of ATRA treatment.These results suggested that the histone modification including PRC2-mediated repressive histone marker H3K27me3 and active histone marker acH4 may involve in CD11b transcription during HL-60 leukemia cells reprogram-ming to terminal differentiation.

  8. 4-Acetyl-12,13-epoxyl-9-trichothecene-3,15-diol isolated from the fruiting bodies of Isariajaponica Yasuda induces apoptosis of human leukemia cells (HL-60).

    Science.gov (United States)

    Oh, G S; Hong, K H; Oh, H; Pae, H O; Kim, I K; Kim, N Y; Kwon, T O; Shin, M K; Chung, H T

    2001-07-01

    The fruiting bodies of Isaria fungi have been traditionally used in Korea to treat cancer. An apoptosis-inducing compound, 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol, was isolated from the methanol extract of fruiting bodies of Isaria japonica Yasuda by bioassay-guided fractionation. The apoptosis of the human leukemia cells (HL-60) by the compound was accessed by propidium iodide-staining flow cytometric analysis, and apoptosis-inducing activity at IC50 concentration (10 nmol/l) was further confirmed by a nuclear morphological change, a ladder pattern of internucleosomal DNA fragmentation, and an activation of caspase-3.

  9. Opposite effects of two trichothecene mycotoxins, deoxynivalenol and nivalenol, on the levels of macrophage inflammatory protein (MIP)-1α and MIP-1β in HL60 cells.

    Science.gov (United States)

    Nagashima, Hitoshi; Nakagawa, Hiroyuki; Kushiro, Masayo

    2012-11-01

    To elucidate the mechanisms underlying the toxicities of the trichothecene mycotoxins deoxynivalenol and nivalenol, their effects on the secretion of anti-hematopoietic chemokines, macrophage inflammatory protein-1α (MIP-1α) and MIP-1β in human promyelocytic leukemia cell line HL60 were investigated. Exposure to deoxynivalenol for 24h significantly induced the secretion of chemokines. The induction of these chemokines may account for the leukopenia after exposure to trichothecene mycotoxins. Treatment with nivalenol decreased the secretion of these chemokines. Our finding that deoxynivalenol induces the secretion of these chemokines, whereas nivalenol has the opposite effect, clearly indicates that the toxicity mechanisms of deoxynivalenol and nivalenol differ.

  10. Histamine increases cytosolic Ca2+ in HL-60 promyelocytes predominantly via H2 receptors with an unique agonist/antagonist profile and induces functional differentiation.

    Science.gov (United States)

    Seifert, R; Höer, A; Schwaner, I; Buschauer, A

    1992-08-01

    Histamine H1 receptors mediate activation of phospholipase C, with subsequent increases in cytosolic Ca2+ concentration ([Ca2+]i), and H2 receptors mediate accumulation of cAMP. HL-60 promyelocytes possess H2 receptors, but it is not known whether these cells also possess H1 receptors. We studied the effects of histamine on [Ca2+]i and the functional importance of histamine receptors in HL-60 promyelocytes. In these cells, histamine and dimaprit increased [Ca2+]i with EC50 values of 15 microM and 30 microM, respectively. Diphenhydramine inhibited the effect of histamine (100 microM) on [Ca2+]i up to 40%, with an IC50 of 100 nM. Famotidine and cimetidine diminished the effect of histamine (100 microM) up to 75%, with IC50 values of 85 nM and 300 nM, respectively. Diphenhydramine plus famotidine abolished histamine-induced rises in [Ca2+]i. Impromidine, with an IC50 of 100 nM, abolished the effect of histamine (100 microM) on [Ca2+]i. Diphenhydramine, famotidine, cimetidine, and impromidine showed marked noncompetitive antagonism with histamine. Histamine-induced increases in [Ca2+]i were largely due to influx of Ca2+ from the extracellular space. Ca2+ influx was inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). Histamine activated phospholipase C. Histamine induced expression of formyl peptide receptors, which effect was abolished by famotidine. In U-937 promonocytes and in the human erythroleukemia cell lines HEL and K-562, histamine did not induce rises in [Ca2+]i. Our data suggest the following. (i) In HL-60 promyelocytes, histamine increases [Ca2+]i predominantly via H2 receptors and to a lesser extent via H1 receptors. (ii) The agonist/antagonist profile of the H2 receptor-mediated increases in [Ca2+]i differs markedly from that for cAMP accumulation, suggesting the involvement of different H2 receptor subtypes. (iii) In HL-60 promyelocytes, histamine activates nonselective cation channels and

  11. Pellitorine, a Potential Anti-Cancer Lead Compound against HL60 and MCT-7 Cell Lines and Microbial Transformation of Piperine from Piper Nigrum

    Directory of Open Access Journals (Sweden)

    Gwendoline Cheng Lian Ee

    2010-04-01

    Full Text Available Pellitorine (1, which was isolated from the roots of Piper nigrum, showed strong cytotoxic activities against HL60 and MCT-7 cell lines. Microbial transformation of piperine (2 gave a new compound 5-[3,4-(methylenedioxyphenyl]-pent-2-ene piperidine (3. Two other alkaloids were also found from Piper nigrum. They are (E-1-[3’,4’-(methylenedioxycinnamoyl]piperidine (4 and 2,4-tetradecadienoic acid isobutyl amide (5. These compounds were isolated using chromatographic methods and their structures were elucidated using MS, IR and NMR techniques.

  12. Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation.

    Science.gov (United States)

    Gabig, T G; Crean, C D; Mantel, P L; Rosli, R

    1995-02-01

    Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47-phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates

  13. The newly synthesized 2-(3-hydroxy-5-methoxyphenyl)-6,7-methylenedioxyquinolin-4-one triggers cell apoptosis through induction of oxidative stress and upregulation of the p38 MAPK signaling pathway in HL-60 human leukemia cells.

    Science.gov (United States)

    Cheng, Yung-Yi; Yang, Jai-Sing; Tsai, Shih-Chang; Liaw, Chih-Chuang; Chung, Jing-Gung; Huang, Li-Jiau; Lee, Kuo-Hsiung; Lu, Chi-Cheng; Chien, Hsi-Cheng; Tsuzuki, Minoru; Kuo, Sheng-Chu

    2012-10-01

    The aim of the present study was to discover the signaling pathways associated with 2-(3-hydroxy-5-methoxy-phenyl)-6,7-methylenedioxyquinolin-4-one (YYK1)-induced apoptosis in HL-60 human leukemia cells. YYK1 induced cytotoxic effects, cell morphological changes, decreased the cell number and increased reactive oxygen species (ROS) production and loss of mitochondrial membrane potential (ΔΨm) in HL-60 cells. YYK1-induced apoptosis was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Results from colorimetric assays and western blot analysis indicated that activities of caspase-7/-3, caspase-8 and caspase-9 were increased in YYK1-treated HL-60 cells. Western blot analysis showed that the protein levels of extrinsic apoptotic proteins (Fas/CD95, FasL and FADD), intrinsic related proteins (cytochrome c, Apaf-1, AIF and Endo G), the ratio of Bax/Bcl-2 and phosphorylated p38 MAPK were increased in HL-60 cells after YYK1 treatment. Cell apoptosis was significantly reduced after pre-treatment with N-acetylcysteine (NAC; a ROS scavenger) or diphenyleneiodonium chloride (DPI; a NADPH oxidase inhibitor). Blockage of p38 MAPK signaling by SB202190 abolished YYK1-induced Fas/CD95 upregulation and apoptosis in HL-60 cells. We conclude that YYK1 induces both of extrinsic and intrinsic apoptotic pathways via ROS-mediated activation of p38 MAPK signaling in HL-60 human leukemia cells in vitro.

  14. 黄芩苷对高致瘤HL-60细胞裸鼠移植瘤模型的体内抑瘤作用和机制探讨%Effects of Baicalin on HL-60 Cell Xenografts in Nude Mice and Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    郑静; 胡建达; 黄毅; 陈英玉; 李静; 陈步远

    2012-01-01

    This study was aimed to investigate the effects of baicalin on HL-60 cell xenografts in nude mice in vivo and explore its mechanism. Xenograft tumor model of HL-60 cells in nude mice was established, which was divided randomly into 6 groups: negative control group (injection of 5% NaHCO3), 25,50 and 100 mg/kg baicalin groups, combination group (50 mg/kg baicalin+ 2 mg/kg VP16) and positive control group(VP16 4 mg/kg). The nude mice with HL-60 cell xenografts were treated with drugs via intraperitoneal injection daily. After treatment for 14 days average weigh and inhibitory rate of transplanted tumor stripped from 5 nude mice in each group were calculated, and the ultrastructure change of xenografts cells were tested by transmission electron microscopy. Histopathologic examination was used to observed the change of main organs in nude mice. The expression of signaling molecular PDK/Akt proteins extracted from xenografts was detected by Western blot. The effects of baicalin on overall survival time in nude mice with HL-60 cell xenografts were evaluated. The results showed that baicalin could inhibit the growth of transplanted tumors in dose-dependent manner. There were more necrotic and apoptotic cells in mice of baicalin-treated groups and combination group than that in mice of negative control group. Baicalin could inhibit the proliferation of HL-60 cells in vivo by down-regulating the PI3K/Akt/Mtor signal pathway, where the expressions of p-Akt, Mtor and p-Mtor proteins decreased compared with negative control group, and no significant difference of Akt expression was found between different groups. Compared with negative control group, the median survival time of mice in combination group was more prolongated (P <0.05). It is concluded that baicalin can inhibit growth and induce apoptosis of HL-60 cell xenografts in nude mice, and prolong median survival time of nude mice. The possible mechanisms may be related to inhibition of Akt activity and down

  15. Synthesis and Biological Activity of Diastereomeric and Geometric Analogs of Calcipotriol, PRI-2202 and PRI-2205, Against Human HL-60 Leukemia and MCF-7 Breast Cancer Cells.

    Science.gov (United States)

    Milczarek, Magdalena; Chodyński, Michał; Filip-Psurska, Beata; Martowicz, Agnieszka; Krupa, Małgorzata; Krajewski, Krzysztof; Kutner, Andrzej; Wietrzyk, Joanna

    2013-10-31

    Diastereomeric and geometric analogs of calcipotriol, PRI-2202 and PRI-2205, were synthesized as advanced intermediates from vitamin D C-22 benzothiazoyl sulfones and side-chain aldehydes using our convergent strategy. Calcitriol, calcipotriol (PRI-2201) and tacalcitol (PRI-2191) were used as the reference compounds. Among a series of tested analogs the diastereomeric analog PRI-2202 showed the strongest antiproliferative activity on the human breast cancer cell line MCF-7, whereas the geometric analog PRI-2205 was the weakest. Both analogs were less potent in antiproliferative activity against HL-60 cells compared to the reference compounds. The ability to potentiate antiproliferative effect of cisplatin or doxorubicin against HL-60 cells or that of tamoxifen against the MCF-7 cell line was observed at higher doses of PRI-2202 or PRI-2205 than those of the reference compounds. The proapoptotic activity of tamoxifen, expressed as the diminished mitochondrial membrane potential, as well as the increased phosphatidylserine expression, was partially attenuated by calcitriol, PRI-2191, PRI-2201 and PRI-2205. The treatment of the MCF-7 cells with tamoxifen alone resulted in an increase in VDR expression. Moreover, a further increase in VDR expression was observed when the analogs PRI-2201 or PRI-2205, but not PRI-2191, were used in combination with tamoxifen. This observation could partially explain the potentiation of the antiproliferative effect of tamoxifen by vitamin D analogs.

  16. Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells.

    Science.gov (United States)

    Lee, Wei-Jiunn; Chen, Yun-Ru; Tseng, Tsui-Hwa

    2011-02-01

    Quercetin, a naturally occurring flavonoid abundant in fruits and vegetables, has been demonstrated as a multipotent bioflavonoid with great potential for the prevention and treatment of cancer. Apoptosis is thought to be an important response to most chemotherapeutic agents in leukemia cells. However, the underlying mechanism of induction of apoptosis by quercetin involving epigenetic regulation is poorly understood. In the present study, by evaluation of fragmentation of DNA, poly (ADP-ribose) polymerase (PARP) and procaspases, we found that quercetin was able to induce apoptosis of human leukemia HL-60 cells in a dose-dependent manner. Quercetin triggered the extrinsic apoptosis pathway through activation of caspase-8 and induction of Bid cleavage, Bax conformation change and cytochrome c release. Furthermore, quercetin induced Fas ligand (FasL) expression involving activation of the extracellular signal-regulated kinase (ERK) and Jun N-terminus kinase (JNK) signaling pathways. In addition to activation of c-Jun, quercetin increased histone H3 acetylation which resulted in the promotion of the expression of FasL. Quercetin exhibited potential for the activation of histone acetyltransferase (HAT) and the inhibition of histone deacetyltransferase (HADC), both of which contributed to histone acetylation. However, only the activation effect on HAT was associated with the ERK and JNK pathway. These results demonstrated that quercetin induced FasL-related apoptosis by transactivation through activation of c-jun/AP-1 and promotion of histone H3 acetylation in HL-60 cells.

  17. Tempranillo-derived grape seed extract induces apoptotic cell death and cell growth arrest in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Espino, Javier; González-Gómez, David; Moreno, Daniel; Fernández-León, María F; Rodríguez, Ana B; Pariente, José A; Delgado-Adámez, Jonathan

    2013-12-01

    Although grape seed extract (GSE) has proven to be effective against various cancers, few studies have investigated the effects of GSE on human leukemia. In this study, we analysed the mechanisms involved in the apoptotic effects induced by GSE on human promyelocytic leukemia HL-60 cells. Thus, GSE treatment succeeded in activating caspase-3 (P < 0.05), the activation being dose-dependent and time-dependent. Activation of caspase-3 induced by GSE was accompanied by mitochondrial membrane depolarization (P < 0.05). Moreover, disruption of mitochondrial integrity caused by GSE treatment subsequently led to activation of caspase-9 (P < 0.05), and also produced a slight increase in ROS levels (P < 0.05). Cytotoxic effects elicited by GSE treatment ultimately resulted in extensive S-phase arrest (P < 0.05) and a substantial increase in the intrinsic rate of apoptosis (P < 0.05). Our findings suggest that the GSE induces apoptotic cell death and cell growth inhibition in human leukemic HL-60 cells, which seems to be dependent on mitochondrial damage. Therefore, the GSE obtained from Tempranillo cultivars could be an effective approach to restrain uncontrolled cell proliferation and survival in leukemia cells.

  18. Pyrrocidine A, a metabolite of endophytic fungi, has a potent apoptosis-inducing activity against HL60 cells through caspase activation via the Michael addition.

    Science.gov (United States)

    Uesugi, Shota; Fujisawa, Nozomi; Yoshida, Jun; Watanabe, Mitsuru; Dan, Shingo; Yamori, Takao; Shiono, Yoshihito; Kimura, Ken-ichi

    2016-03-01

    Pyrrocidine A is a known antimicrobial compound produced by endophytic fungi and has a unique 13-membered macrocyclic alkaloid structure with an α,β-unsaturated carbonyl group. We have previously reported that pyrrocidine A shows potent cytotoxicity against human acute promyelocytic leukemia HL60 cells, and the activity is 70-fold higher than that of pyrrocidine B which is the analog lacking the α,β-unsaturated carbonyl group. Pyrrocidine A induced nuclear condensation, DNA fragmentation and caspase activation in HL60 cells. Since the DNA fragmentation was suppressed by pretreatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-fmk), caspase-mediated apoptosis contributes to pyrrocidine A-induced cytotoxicity. JFCR39 human cancer cells panel indicated that the mechanism of action of pyrrocidine A is different from other clinical anticancer drugs, and this compound broadly inhibited the growth of various cancer cell lines. The apoptosis induction by pyrrocidine A was suppressed by both N-acetyl-l-cysteine (NAC) and glutathione, both of which are thiol-containing antioxidants. Furthermore, pyrrocidine A directly bound to N-acetyl-l-cysteine methyl ester (NACM) through the Michael-type addition at the α,β-unsaturated carbonyl group and was detected by HPLC and liquid chromatography-ESI-tandem MS (LC-ESI-MS/MS) analyses. This indicates that this moiety is crucial for the potent apoptosis-inducing activity of pyrrocidine A.

  19. Enhancement Effect of human acute myeloid leukemia HL-60 cell line supernatant on Angiogenesis of Human Umbilical Vein Endothelial Cell%人急性白血病细胞株HL-60细胞培养上清液对人脐静脉内皮细胞血管形成能力的影响

    Institute of Scientific and Technical Information of China (English)

    潘小兵; 陈建萍

    2011-01-01

    目的 研究人急性白血病细胞株(HL-60)上清液对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVECs)血管形成能力的影响.方法 以单独培养HUVECs为对照组,以HUVECs加HL-60细胞培养上清为实验组.采用MTT比色法分别在24h、48h、72h检测两组中HUVECs的吸光值;采用RT-PCR技术检测两组中HUVECs周期蛋白E(cyclinE )mRNA及血管内皮生长因子(VEGF)mRNA的表达情况.结果 实验组与对照组相比HUVECs增殖速度加快,72小时后差异有显著性意义(p〈0.05).72小时后,实验组中HUVECs CyclinE mRNA及VEGFmRNA表达明显增加,与对照组相比差异具有显著性意义(p〈0.01).结论 HL-60细胞培养上清液能够增强HUVECs的血管形成能力.

  20. Desmosdumotin-C A环衍生物TEP诱导HL-60细胞凋亡作用机制研究%The Effects and Mechanisms of an A ring Derivative of Desmosdumotin-C (TEP) induced Apoptosis in HL-60 Cells

    Institute of Scientific and Technical Information of China (English)

    郭宏举; 向卓; 常李荣; 史宁; 梁海; 郑凯音; 吴久鸿

    2016-01-01

    This study focused on the effects of TEP,an A ring derivative of desmosdumotin-C,on the human acute leukemia HL-60 cell apoptosis,and the underlying mechanisms.TEP-induced apoptosis and its effect on Fas,FasL,Bax,Bcl-2 expression were measured by flow cytometry,and the morphological changes of the apoptosis cells were further observed by transmission electron microscop.The results showed that 24 h after 40 μg/mL TEP treatment,the typical apoptotic morphological changes were induced in HL-60 cell.Meantime,40 μg/mL TEP significantly increased Fas,FasL,and Bax expression (P < 0.05),and decreased Bcl-2 expression (P < 0.05).All the results suggested that TEP effectively induced apoptosis in HL-60 cells,and the mechanism may be involved in upregulating Fas,FasL,Bax expression,and reducing Bcl-2 expression.%本研究主要探讨毛叶假鹰爪素C(Desmosdumotin C)A环衍生物TEP诱导人急性白血病HL-60细胞凋亡作用及机制.流式细胞技术检测TEP诱导细胞凋亡及其对凋亡细胞中Fas、FasL、Bax、Bcl-2表达率的影响,透射电镜观察凋亡细胞形态学改变.结果显示40μg/mL TEP作用细胞24h后,细胞可呈现典型的凋亡形态学变化;40 μg/mL TEP可明显提高凋亡细胞中Fas、FasL、Bax的表达(P<0.05),并可明显降低抗凋亡细胞Bcl-2的表达(P<0.05).以上实验结果表明毛叶假鹰爪素C(Desmosdumotin C,Des C)A环衍生物TEP可有效地诱导HL-60细胞凋亡,其作用机制可能与上调Fas、FasL、Bax表达以及下调Bcl-2表达有关.

  1. 鼠尾草酸联合1,25-二羟基维生素D3诱导HL-60细胞分化作用的研究%The differentiation of HL-60 cells induced by 1,25-dihydroxyvitamin D3 combined with carnosic acid

    Institute of Scientific and Technical Information of China (English)

    安慧萍; 任立红; 陈娟娟

    2007-01-01

    目的 研究鼠尾草酸与1,25-二羟基维生素D3[1,25(OH)2D3]联合应用对HL-60细胞分化的影响.方法 通过四唑氮蓝(MTT)法观察细胞生长,光学显微镜观察细胞形态,应用流式细胞仪测定细胞周期及成熟单核细胞分化标记CD14的表达,观察联合应用鼠尾草酸与低浓度1,25(OH)2D3对HL-60细胞的影响.结果 低浓度1,25(OH)2D3(1nmol/L)抑制HL-60细胞增殖、诱导细胞分化的作用与对照组比较无统计学意义(P>0.05).同浓度1,25(OH)2D3与10μmol/L鼠尾草酸联合处理HL-60细胞72h后,细胞形态具有成熟单核细胞特征、细胞增殖明显受抑制、细胞阻滞于G0/G1期、CD14的表达率为57.624%;其作用效应与对照组比较有显著性差异(P<0.01),与单独应用高浓度1,25(OH)2D3(100nmol/L)组比较作用相似,无统计学意义(P>0.05).结论 鼠尾草酸可增强1,25(OH)2D3对HL-60细胞的诱导分化、抑制增殖作用.

  2. L-securinine induces apoptosis in the human promyelocytic leukemia cell line HL-60 and influences the expression of genes involved in the PI3K/AKT/mTOR signaling pathway.

    Science.gov (United States)

    Han, Shuwen; Zhang, Gang; Li, Maidong; Chen, Dongyun; Wang, Ying; Ye, Wencai; Ji, Zhaoning

    2014-05-01

    The Securinega alkaloids are a class of natural products isolated from plants of the Euphorbiaceae family. L-securinine induces apoptosis in the human promyelocytic leukemia cell line HL-60 indicating its potential as an efficient natural antitumor drug with low toxicity. The aim of the present study was to investigate the apoptotic effects of L-securinine on HL-60 cells and to explore its potential underlying molecular mechanism(s) as an antitumor agent. HL-60 cells were cultured with L-securinine. The proliferation and changes in cell morphology were evaluated by cell counting Kit-8 (CCK-8) assay and electron microscopy, respectively. Induction of apoptosis and cell cycle progression were investigated by flow cytometry. The PI3K/AKT/mTOR pathway gene expression was measured by quantitative PCR (qPCR). L-securinine decreased the viability of HL-60 cells in a dose- and time-dependent manner, with IC50 values at 24, 48 and 72 h post-treatment of 47.88, 23.85 and 18.87 µmol/l, respectively. Numerous apoptotic bodies were observed in the HL-60 cells treated with 25 µmol/l L-securinine for 48 h. L-securinine at 12.5, 25 and 50 µmol/l increased the rate of apoptosis in HL-60 cells, and G1/S phase progression was retarded. Furthermore, L-securinine induced downregulation of PI3K, AKT and mTOR gene expression and upregulation of PTEN gene expression in a dose-dependent manner. In conclusion, L-securinine induces apoptosis and inhibition of cell cycle progression in HL-60 cells by modulation of the PI3K/AKT/mTOR pathway gene expression. These observations indicate the potential of L-securinine as an antitumor agent.

  3. Effects of co-treatment with puerariae radix flavones and arsenic trioxide on proliferation and apoptosis of Kasumi-1 cells and HL-60 cells%葛根总黄酮联合三氧化二砷对Kasumi-1和HL-60细胞增殖和凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    唐宇宏; 邵化敏; 朱红青; 姜鹏君; 季建敏; 沈群

    2011-01-01

    目的 探讨葛根总黄酮(PRF)联合三氧化二砷(ATO)对M2型急性髓系白血病细胞株Kasumi-1和HL-60细胞增殖和凋亡的影响.方法 以100 μg/mL PRF和1μmoL/L ATO联合处理Kasumi-1和HL-60细胞48 h(RRF+ ATO组),MTT法检测细胞增殖抑制率,Annexin V-FITC/PI双染法流式细胞术检测细胞早期凋亡率,Real-Time PCR检测凋亡抑制基因Bcl-2、Survivin mRNA表达,Western blotting检测细胞凋亡相关蛋白酶Caspase-3、-8、-9的表达.设立单用100μg/mL PRF的PRF处理组和空白对照组.结果 RRF+ ATO组Kasum-1细胞增殖抑制率、早期凋亡率以及Caspase-3、-9表达均显著高于PRF处理组(P<0.05),细胞中Bcl-2 mRNA表达的下调趋势明显.RRF+ ATO组HL-60细胞各项指标检测结果 与PRF处理组比较差异均无统计学意义(P>0.05).结论 PRF联合ATO能有效抑制Kasumi-1细胞增殖并诱导早期凋亡,而对HL-60细胞则无明显影响.%Objective To investigate the effects of co-treatment with puerariae radix flavones (PRF) and arsenic trioxide ( ATO) on proliferation and apoptosis of Kasumi-1 cells and HL-60 cells of acute myeloid leukemia Ml. Methods Kasumi-1 cells and HL-60 cells were treated with 100 jig/mL PRF and 1 funol/L ATO for 48 h (RRF + ATO group). MTT assay was employed to measure the inhibition rate of cell proliferation, Annexin V-FITC/PI double staining was used to determine the early apoptosis rate by flow cytometry, the expression of Bcl-2 and Survivin mRNA was detected by Real-Time PCR, and the expression of Caspase-3, Caspase-8 and Caspase-9 protein was detected by Western blotting. Cells only treated with 100 (ig/mL PRF were served as PRF group, and blank control group was also established. Results The inhibition rate of cell proliferation, early apoptosis rate and expression of Caspase-3 and Caspase-9 protein of Kasumi-1 cells in RRF + ATO group were significantly higher than those in PRF group (P 0.05). Conclusion Co-treatment with PRF and ATO can effectively

  4. Enhancement of differentiation induction of HL-60 cells by 1,25-dihydroxyvitaminD3 in combination with carnosic acid%鼠尾草酸增加1,25(OH)2D3诱导HL-60细胞分化及其机制研究

    Institute of Scientific and Technical Information of China (English)

    任立红; 陈娟娟; 安慧萍

    2008-01-01

    目的 已证实1,25(OH)2D3可以诱导HL-60细胞向成熟单核细胞分化,但其不足之处是高钙血症和形成耐药株.该实验应用鼠尾草酸(CA)联合1,25(OH)2D3研究对HL-60细胞分化的影响及引起细胞内活性氧(ROS)水平和细胞内钙离子浓度的改变.方法 应用鼠尾草酸,1,25(OH)2D3单独和联合应用处理HL-60细胞,共分为5组:空白对照组;低浓度1,25(OH)2D3(1 nmol/L)组、鼠尾草酸组;联合处理组(10 μmol/L鼠尾草酸和1 nmol/L 1,25(OH)2D3);高浓度1,25(OH)2D3(100 nmol/L)组.每24 h监测1次,通过四唑氮蓝(MTT)法观察细胞生长,共72 h;收集处理后72 h的HL-60细胞,通过光学显微镜观察细胞形态,用流式细胞仪(FCM)检测不同处理组细胞周期及单核细胞分化标记CD14的表达,ROS和细胞内钙离子浓度.结果 72 h后,联合处理组HL-60细胞与空白对照组相比,细胞增殖明显受抑制(Ab=0.560±0.020;P0.05),但与高浓度1,25(OH)2D3组相比细胞内钙离子水平明显下降(P<0.01).结论 鼠尾草酸可增强1,25(OH)2D3对HL-60细胞的诱导分化、增强抑制HL-60细胞增殖作用,细胞阻滞于G0/G1期,细胞内活性氧水平降低,这可能与细胞的诱导分化机制有关,且这一联合作用不增高细胞内钙离子水平.

  5. Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells.

    Directory of Open Access Journals (Sweden)

    Islam Husain

    Full Text Available Asparaginase is an important antileukemic agent extensively used worldwide but the intrinsic glutaminase activity of this enzymatic drug is responsible for serious life threatening side effects. Hence, glutaminase free asparaginase is much needed for upgradation of therapeutic index of asparaginase therapy. In the present study, glutaminase free asparaginase produced from Enterobacter cloacae was purified to apparent homogeneity. The purified enzyme was found to be homodimer of approximately 106 kDa with monomeric size of approximately 52 kDa and pI 4.5. Purified enzyme showed optimum activity between pH 7-8 and temperature 35-40°C, which is close to the internal environment of human body. Monovalent cations such as Na+ and K+ enhanced asparaginase activity whereas divalent and trivalent cations, Ca2+, Mg2+, Zn2+, Mn2+, and Fe3+ inhibited the enzyme activity. Kinetic parameters Km, Vmax and Kcat of purified enzyme were found to be 1.58×10-3 M, 2.22 IU μg-1 and 5.3 × 104 S-1, respectively. Purified enzyme showed prolonged in vitro serum (T1/2 = ~ 39 h and trypsin (T1/2 = ~ 32 min half life, which is therapeutically remarkable feature. The cytotoxic activity of enzyme was examined against a panel of human cancer cell lines, HL-60, MOLT-4, MDA-MB-231 and T47D, and highest cytotoxicity observed against HL-60 cells (IC50 ~ 3.1 IU ml-1, which was comparable to commercial asparaginase. Cell and nuclear morphological studies of HL-60 cells showed that on treatment with purified asparaginase symptoms of apoptosis were increased in dose dependent manner. Cell cycle progression analysis indicates that enzyme induces apoptosis by cell cycle arrest in G0/G1 phase. Mitochondrial membrane potential loss showed that enzyme also triggers the mitochondrial pathway of apoptosis. Furthermore, the enzyme was found to be nontoxic for human noncancerous cells FR-2 and nonhemolytic for human erythrocytes.

  6. Histamine increases cytosolic Ca2+ in dibutyryl-cAMP-differentiated HL-60 cells via H1 receptors and is an incomplete secretagogue.

    Science.gov (United States)

    Seifert, R; Höer, A; Offermanns, S; Buschauer, A; Schunack, W

    1992-08-01

    Human neutrophils and dibutyryl-cAMP (Bt2cAMP)-differentiated HL-60 cells possess receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), which mediate activation of phospholipase C, with subsequent increase in cytosolic Ca2+ concentration ([Ca2+]i) and activation of specific cell functions. In many cell types, histamine, via H1 receptors, activates phospholipase C, but it is unknown whether neutrophilic cells possess functional H1 receptors. We compared the effects of histamine with those of fMet-Leu-Phe on activation of these cells. In Bt2cAMP-differentiated HL-60 cells, substances increased [Ca2+]i in the effectiveness order fMet-Leu-Phe greater than histamine greater than betahistine. Pertussis toxin diminished fMet-Leu-Phe-induced rises in [Ca2+]i to a greater extent than those induced by histamine. H1 but not H2 antagonists inhibited histamine- and betahistine-induced rises in [Ca2+]i. fMet-Leu-Phe and histamine activated phospholipase C and increased [Ca2+]i through release of Ca2+ from intracellular stores and sustained influx of Ca2+ from the extracellular space. The substances also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). The stimulatory effects of histamine on [Ca2+]i were more sensitive to inhibition by 4 beta-phorbol 12-myristate 13-acetate than were those of fMet-Leu-Phe. Unlike fMet-Leu-Phe, histamine did not activate superoxide anion formation, release of beta-glucuronidase, and tyrosine phosphorylation. In neutrophils, histamine and betahistine did not induce rises in [Ca2+]i. Our data show that (i) in Bt2cAMP-differentiated HL-60 cells, histamine increases [Ca2+]i via H1 receptors coupled to pertussis toxin-sensitive and possibly, pertussis toxin-insensitive heterotrimeric regulatory guanine nucleotide-binding proteins, (ii) histamine activates nonselective cation channels, and (iii

  7. Rab27a is essential for the formation of neutrophil extracellular traps (NETs in neutrophil-like differentiated HL60 cells.

    Directory of Open Access Journals (Sweden)

    Tatsumi Kawakami

    Full Text Available Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs. NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an

  8. UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lu, M.L.; Sato, Mitsuhiro; Cao, Boliang; Richie, J.P. [Harvard Medical School, Boston, MA (United States)

    1996-08-20

    UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirements for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-{alpha}-and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis. 40 refs., 5 figs.

  9. Berberine and a Berberis lycium extract inactivate Cdc25A and induce {alpha}-tubulin acetylation that correlate with HL-60 cell cycle inhibition and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Musa [Department of Plant Sciences, Quaid-i-Azam University Islamabad (Pakistan); Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Giessrigl, Benedikt; Vonach, Caroline; Madlener, Sibylle [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Prinz, Sonja [Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Herbaceck, Irene; Hoelzl, Christine [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Bauer, Sabine; Viola, Katharina [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria); Mikulits, Wolfgang [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Quereshi, Rizwana Aleem [Department of Plant Sciences, Quaid-i-Azam University Islamabad (Pakistan); Knasmueller, Siegfried; Grusch, Michael [Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, Borschkegasse 8a (Austria); Kopp, Brigitte [Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Althanstrasse 14 (Austria); Krupitza, Georg, E-mail: georg.krupitza@meduniwien.ac.at [Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna (Austria)

    2010-01-05

    Berberis lycium Royle (Berberidacea) from Pakistan and its alkaloids berberine and palmatine have been reported to possess beneficial pharmacological properties. In the present study, the anti-neoplastic activities of different B. lycium root extracts and the major constituting alkaloids, berberine and palmatine were investigated in p53-deficient HL-60 cells. The strongest growth inhibitory and pro-apoptotic effects were found in the n-butanol (BuOH) extract followed by the ethyl acetate (EtOAc)-, and the water (H{sub 2}O) extract. The chemical composition of the BuOH extract was analyzed by TLC and quantified by HPLC. 11.1 {mu}g BuOH extract (that was gained from 1 mg dried root) contained 2.0 {mu}g berberine and 0.3 {mu}g/ml palmatine. 1.2 {mu}g/ml berberine inhibited cell proliferation significantly, while 0.5 {mu}g/ml palmatine had no effect. Berberine and the BuOH extract caused accumulation of HL-60 cells in S-phase. This was preceded by a strong activation of Chk2, phosphorylation and degradation of Cdc25A, and the subsequent inactivation of Cdc2 (CDK1). Furthermore, berberine and the extract inhibited the expression of the proto-oncogene cyclin D1. Berberine and the BuOH extract induced the acetylation of {alpha}-tubulin and this correlated with the induction of apoptosis. The data demonstrate that berberine is a potent anti-neoplastic compound that acts via anti-proliferative and pro-apoptotic mechanisms independent of genotoxicity.

  10. Arginase purified from endophytic Pseudomonas aeruginosa IH2: Induce apoptosis through both cell cycle arrest and MMP loss in human leukemic HL-60 cells.

    Science.gov (United States)

    Husain, Islam; Bala, Kiran; Wani, Abubakar; Makhdoomi, Ubaid; Malik, Fayaz; Sharma, Anjana

    2017-08-25

    Arginase is a therapeutic enzyme for arginine-auxotrophic cancers but their low anticancer activity, less proteolytic tolerance and shorter serum half-life are the major shortcomings. In this study, arginase from Pseudomonas aeruginosa IH2 was purified to homogeneity and estimated as 75 kDa on native-PAGE and 37 kDa on SDS-PAGE. Arginase showed optimum activity at pH 8 and temperature 35 °C. Mn(2+) and Mg(2+) ions enhanced arginase activity while, Li(+), Cu(2+), and Al(3+) ions reduced arginase activity. In-vitro serum half-life of arginase was 36 h and proteolytic half-life against trypsin and proteinase-K was 25 and 29 min, respectively. Anticancer activity of arginase was evaluated against colon, breast, leukemia, and prostate cancer cell lines and lowest IC50 (0.8 IU ml(-1)) was found against leukemia cell line HL-60. Microscopic studies and flow cytometric analysis of Annexin V/PI staining of HL-60 cells revealed that arginase induced apoptosis in dose-dependent manner. Cell cycle analysis suggested that arginase induced cell cycle arrest in G0/G1 phase. The increasing level of MMP loss, ROS generation and decreasing level of SOD, CAT, GPx and GSH suggested that arginase treatment triggered dysfunctioning of mitochondria. The cleavage of caspase-3, PARP-1, activations of caspase-8, 9 and high expression of proapoptotic protein Bax, low expression of anti-apoptotic protein Bcl-2 indicated that arginase treatment activates mitochondrial pathway of apoptosis. Purified arginase did not exert cytotoxic effects on human noncancer cells. Our study strongly supports that arginase could be used as potent anticancer agent but further studies are required which are underway in our lab. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Cellular uptake of {sup 99m}TcN-NOET in human leukaemic HL-60 cells is related to calcium channel activation and cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Guillermet, Stephanie; Vuillez, Jean-Philippe; Caravel, Jean-Pierre; Marti-Batlle, Daniele; Fagret, Daniel [Universite de Grenoble, Radiopharmaceutiques Biocliniques, La Tronche (France); Fontaine, Eric [Universite de Grenoble, Laboratoire de Bioenergetique Fondamentale et Appliquee, Grenoble (France); Pasqualini, Roberto [Cis Bio International Schering SA, Gif-sur-Yvette (France)

    2006-01-01

    A major goal of nuclear oncology is the development of new radiolabelled tracers as proliferation markers. Intracellular calcium waves play a fundamental role in the course of the cell cycle. These waves occur in non-excitable tumour cells via store-operated calcium channels (SOCCs). Bis(N-ethoxy, N-ethyldithiocarbamato) nitrido technetium (V)-99m ({sup 99m}TcN-NOET) has been shown to interact with L-type voltage-operated calcium channels (VOCCs) in cultured cardiomyocytes. Considering the analogy between VOCCs and SOCCs, we sought to determine whether {sup 99m}TcN-NOET also binds to activated SOCCs in tumour cells in order to clarify the potential value of this tracer as a proliferation marker. Uptake kinetics of {sup 99m}TcN-NOET were measured in human leukaemic HL-60 cells over 60 min and the effect of several calcium channel modulators on 1-min tracer uptake was studied. The uptake kinetics of {sup 99m}TcN-NOET were compared both with the variations of cytosolic free calcium concentration measured by indo-1/AM and with the variations in the SG{sub 2}M cellular proliferation index. All calcium channel inhibitors significantly decreased the cellular uptake of {sup 99m}TcN-NOET whereas the activator thapsigargin induced a significant 10% increase. In parallel, SOCC activation by thapsigargin, as measured using the indo-1/AM probe, was inhibited by nicardipine. These results indicate that the uptake of {sup 99m}TcN-NOET is related to the activation of SOCCs. Finally, a correlation was observed between the tracer uptake and variations in the proliferation index SG{sub 2}M. The uptake of {sup 99m}TcN-NOET seems to be related to SOCC activation and to cell proliferation in HL-60 cells. These results indicate that {sup 99m}TcN-NOET might be a marker of cell proliferation. (orig.)

  12. Expression of GPI-80, a beta2-integrin-associated glycosylphosphatidylinositol-anchored protein, requires neutrophil differentiation with dimethyl sulfoxide in HL-60 cells.

    Science.gov (United States)

    Takeda, Yuji; Fu, Junfen; Suzuki, Kichiya; Sendo, Dai; Nitto, Takeaki; Sendo, Fujiro; Araki, Yoshihiko

    2003-06-10

    GPI-80 is a member of the amidohydrolase family that has been proposed as a potential regulator of beta2-integrin-dependent leukocyte adhesion. GPI-80 is expressed mainly in human neutrophils. Our previous studies suggested that GPI-80 expression might be associated with myeloid differentiation. To verify this, we examined whether GPI-80 is expressed on the human promyelocytic leukemia cell line HL-60 following treatment with differentiation inducers. GPI-80 expression was induced in cells treated with dimethyl sulfoxide (DMSO) to stimulate differentiation down the neutrophil pathway. On the other hand, all-trans-retinoic acid (ATRA), another neutrophil-inducing reagent, induced no clear GPI-80 expression. Potent monocyte-inducing reagents such as 1alpha,25-dihydroxyvitamin D(3) or phorbol 12-myristate 13-acetate also had no significant effect on the protein expression. GPI-80-positive cells were found in the well-differentiated CD11b-positive and transferrin-receptor-negative cell population. Granulocyte colony-stimulating factor, which augments neutrophil differentiation of HL-60 cells, up-regulated GPI-80 expression in the presence of DMSO. Granulocyte/macrophage colony-stimulating factor, which is known to suppress the neutrophil maturation of cells, inhibited expression. Adhesion of DMSO-induced cells was regulated by anti-GPI-80 monoclonal antibody, similar to the regulation observed in neutrophils. These results suggest that use of DMSO to induce neutrophil differentiation provides suitable conditions for GPI-80 expression, and that this culture system may be a helpful model for further study of the regulation of GPI-80 expression during myeloid differentiation.

  13. Biodegradation of NSO-compounds under different redox-conditions

    DEFF Research Database (Denmark)

    Dyreborg, S.; Arvin, E.; Broholm, K.

    1997-01-01

    Laboratory experiments were carried out to investigate the potential of groundwater microorganisms to degrade selected heterocyclic aromatic compounds containing nitrogen, sulphur, or oxygen (NSO-compounds) under four redox-conditions over a period of 846 days. Eight compounds (pyrrole, 1-methylp...

  14. Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of HL60 and K562 cells to daunorubicin%Bcl-2反义核酸增强HL-60、K562细胞对柔红霉素敏感性的研究

    Institute of Scientific and Technical Information of China (English)

    雷小勇; 张洹; 何冬梅

    2003-01-01

    目的:研究bcl-2反义寡核苷酸作用后,白血病细胞株HL60、K562细胞对柔红霉素(DNR)敏感性的影响.方法:MTT法测HL60、K562细胞中DNR的半数抑制率(IC50);免疫荧光标记观测细胞Bcl-2蛋白水平;用Hoechest33258和碘化丙锭双染色法及流式细胞仪检测细胞凋亡.结果:靶向bcl-2 mRNA蛋白编码区反义寡核苷酸(AS-ODN1)和靶向翻译起始区的反义寡核苷酸(AS-ODN2)分别与DNR联合作用于HL60细胞后DNR的IC50值分别为0.124±0.011、0.149±0.012,分别与不加寡核苷酸组DNR的IC50值(0.173±0.021)或无义寡核苷酸联用DNR的IC50值(0.180±0.023)相比有显著差异,P<0.05.AS-ODN1和AS-ODN2分别与DNR联合作用于K562细胞后DNR的IC50值分别为0.078±0.007、0.079±0.008,分别与不加寡核苷酸组DNR的IC50值(0.106±0.011)或无义寡核苷酸联用DNR的IC50值(0.107±0.012)相比有显著差异,P<0.05.AS-ODN1和AS-ODN2分别与DNR同时作用于HL60或K562细胞后抑制Bcl-2蛋白表达及诱导细胞凋亡率分别与单用DNR或无义寡核苷酸联用DNR相比有显著差异,P<0.05.与AS-ODN2比较,AS-ODN1提高HL60细胞对DNR的敏感性作用要强些(P<0.05).结论:靶向bcl-2 mRNA蛋白编码区反义寡核苷酸和靶向翻译起始区的反义寡核苷酸能增强HL60和K562细胞对DNR的敏感性.

  15. Safrole induces G0/G1 phase arrest via inhibition of cyclin E and provokes apoptosis through endoplasmic reticulum stress and mitochondrion-dependent pathways in human leukemia HL-60 cells.

    Science.gov (United States)

    Yu, Chun-Shu; Huang, An-Cheng; Yang, Jai-Sing; Yu, Chien-Chih; Lin, Chin-Chung; Chung, Hsiung-Kwang; Huang, Yi-Ping; Chueh, Fu-Shin; Chung, Jing-Gung

    2012-05-01

    Safrole, a component of Piper betle inflorescence, is a carcinogen which has been demonstrated to induce apoptosis on human oral cancer HSC-3 cells in vitro and to inhibit HSC-3 cells in xenograft tumor cells in vivo. In our previous study, safrole promoted phagocytosis by macrophages and natural killer cell cytotoxicity in normal BALB/c mice. The cytotoxic effects of safrole on HL-60 cells were investigated by using flow cytometric analysis, comet assay, 4',6-diamidino-2-phenylindole (DAPI) staining, western blotting and confocal laser microscopy. The obtained results indicate that safrole induced a cytotoxic response through reducing the percentage of viable cells and induction of apoptosis in HL-60 cells in a dose-dependent manner. DAPI staining and comet assay also showed that safrole induced apoptosis (chromatin condensation) and DNA damage in HL-60 cells. The flow cytometric assay showed that safrole increased the production of reactive oxygen species (ROS) and Ca(2+) and reduced the mitochondrial membrane potential in HL-60 cells. Safrole enhanced the levels of the pro-apoptotic protein BAX, inhibited those of the anti-apoptotic protein BCL-2 and promoted the levels of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in HL-60 cells. Furthermore, safrole promoted the expression of glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and of activating transcription factor 6α (ATF-6α). Based on these findings, we suggest that safrole-induced apoptosis in HL-60 cells is mediated through the ER stress and intrinsic signaling pathways.

  16. Ethyl acetate fraction of Garcina epunctata induces apoptosis in human promyelocytic cells (HL-60) through the ROS generation and G0/G1 cell cycle arrest: a bioassay-guided approach.

    Science.gov (United States)

    Constant Anatole, Pieme; Guru, Santoh Kumar; Bathelemy, Ngamegni; Jeanne, Ngogang; Bhushan, Shashi; Murayama, Tetsuya; Saxena, Ajit Kumar

    2013-11-01

    Number of deaths due to cancer diseases is increasing in the world. There is an urgent need to develop alternative therapeutic measures against the disease. Our study reports the cytotoxicity activity of Garcina epunctata (gutifferae) in human promyelocytic leukemia cells (HL-60) and prostate cancer cells (PC-3) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Changes in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and morphological changes associated with apoptosis were examined by flow cytometry and Hoescht staining respectively. The results of in vitro antiproliferative screening of fractions and extract from G. epunctata indicated that three fractions inhibited the viability of PC-3 cells with IC₅₀ varied from 50 to 88 μ/ml while two fractions inhibited the proliferation of HL-60 cells with IC₅₀ range between 47.5 and 12 μg/ml. Among the entire fraction tested, Hex-EtOAc (75:25) showed cytotoxic effects on the two cell lines and EtOAc fraction was most active only HL-60 cells (12 μg/ml). Treatment of HL-60 cells with G. epunctata (20, 50, 100 μg/ml) for 24 h led to a significant dose-dependent increase in the percentage of cells in sub-G1 phase by analysis of the content of DNA in cells, and a number of apoptotic bodies containing nuclear fragments were observed in cells treated with 100 μg/ml. The EtOAc fraction of G. epunctata treatment significantly arrested HL-60 cells at the G0/G1 phase (pHL-60 cells, leading to cell cycle arrest and programmed cell death, which was confirmed to occur through the mitochondrial pathway.

  17. The anticancer potential of flavonoids isolated from the stem bark of Erythrina suberosa through induction of apoptosis and inhibition of STAT signaling pathway in human leukemia HL-60 cells.

    Science.gov (United States)

    Kumar, Sunil; Pathania, Anup Singh; Saxena, A K; Vishwakarma, R A; Ali, Asif; Bhushan, Shashi

    2013-09-25

    Erythrina suberosa is an ornamental tall tree found in India, Pakistan, Nepal, Bhutan, Burma, Thailand and Vietnam. We have isolated four known distinct metabolites designated as α-Hydroxyerysotrine, 4'-Methoxy licoflavanone (MLF), Alpinumisoflavone (AIF) and Wighteone. Among the four isolated metabolites the two flavonoids, MLF and AIF were found to be the most potent cytotoxic agent with IC50 of ∼20μM in human leukemia HL-60 cells. We are reporting first time the anticancer and apoptotic potential of MLF and AIF in HL-60 cells. Both MLF and AIF inhibited HL-60 cell proliferation and induce apoptosis as measured by several biological endpoints. MLF and AIF induce apoptosis bodies formation, enhanced annexinV-FITC binding of the cells, increased sub-G0 cell fraction, loss of mitochondrial membrane potential (Δψm), release of cytochrome c, Bax, activation of caspase-9, caspase-3 and PARP (poly ADP Ribose polymers) cleavage in HL-60 cells. MLF and AIF also increase the expression of apical death receptor, Fas, with inhibition of anti-apoptotic protein Bid. All the above parameters revealed that these two flavonoids induce apoptosis through both extrinsic and intrinsic apoptotic pathways in HL-60 cells. In spite of apoptosis, these two flavonoids significantly inhibit nuclear transcription factor NF-κB and STAT (Signal Transducer and Activator of Transcription) signaling pathway, which are highly expressed in leukemia. The present study provide an insight of molecular mechanism of cell death induced by MLF and AIF in HL-60 cells which may be useful in managing and treating leukemia.

  18. Inhibitory effect of compound 6F isolated from Pteris semipinnata L. on the activity of protein kinase C in HL-60 cells%半边旗提取物6F抑制HL-60细胞蛋白激酶C活性

    Institute of Scientific and Technical Information of China (English)

    何承伟; 梁念慈; 莫丽儿; 李金华; 张晓

    2003-01-01

    目的探讨半边旗提取物6F的细胞毒作用及诱导DNA片段化与蛋白激酶C(PKC)信号转导途径的关系,检测6F对PKC活性的影响.方法受试对象为HL-60细胞,超速离心法获得的胞液(可溶部分)及颗粒(不可溶部分,包括细胞膜系统及胞核)部分用作PKC活性测定.经0.4 g@L-1磷脂酰丝氨酸,0.04g@L-1甘油二油酸酯激动剂作用酶粗提物后,用液体闪烁计数仪计数[γ-32P]ATP参入外源底物的量以测定PKC活性.MTT法测定HL-60细胞的活力,二苯胺法测定6F诱导DNA片段化程度.结果在所测试的浓度范围内(0.5~312 μmol@L-1),化合物6F显著抑制胞液及颗粒部分PKC活性,最大抑制率达88.6%,呈浓度依赖关系(胞液部分r=0.781,P<0.05,颗粒部分r=0.931,P<0.01).6F诱导HL-60细胞DNA片段化及对细胞的毒性作用可被具有致癌作用的PKC激活剂肉豆蔻酸酯(PMA,浓度为65 nmol@L-1)拮抗,抑制率分别是30%和44%(P<0.01).PMA单独用使HL-60细胞线粒体将MTT还原为甲臜的能力增强14%(P<0.01),即增强细胞活力.结论化合物6F是PKC的抑制剂.6F对HL-60细胞DNA片段化的诱导作用及其细胞毒作用至少可部分归因于其对PKC活性的抑制作用.%AIM To investigate whether compotmd 6F isolated from Pteris semipinnata L inhibits the activity of protein kinase C (PKC) and whether the DNA fragment induction and cytotoxicity of 6F on HL-60 cells relate to PKC signaling pathway. METHODS HL-60 cells were used as in vitro model and its cytosol (soluble sample)and particle( insoluble sample including membrane system and nuclei) fractions obtained by ultracentrifugation were used as samples for PKC assay. PKC activity was measured by incorporation of [ γ-32p]ATP into exogenous substrate after stimulated by phosphatidylserine and diolein.Diphenylamine assay and MTT staining methods were applied for DNA fragmentation detection and cytotoxicity assay, respectively. RESUL TS PKC activities in cytosol and particle

  19. 扶正祛邪含药血清对白血病HL60/VCR细胞Bcl-2表达水平的影响%Effects and reversal mechanism of Fuzheng Quxie Prescription serum to the apoptosis gene (Bcl-2) of the human leukemia HL60/VCR cells

    Institute of Scientific and Technical Information of China (English)

    李秀军; 严鲁萍; 姚宇红

    2013-01-01

    目的:观察扶正祛邪中药复方含药血清对长春新碱诱导的急性早幼粒白血病耐药细胞株H L60/VCR0细胞耐药基因Bcl-2表达水平的影响.方法:采用流式细胞术检测法,选择HL60/VCR细胞为靶细胞,观察不同浓度扶正祛邪含药血清对其耐药基因Bcl-2表达的影响.结果:扶正祛邪含药血清对HL60/VCR细胞内凋亡抑制基因Bcl-2的表达有明显抑制作用,其中含药血清高、中、低剂量组荧光强度依次为401.67±0.86、453.69±0.40、516.66±0.40.结论:扶正祛邪复方中药抗白血病多药耐药的作用可能与下调Bcl-2的表达有关.

  20. Role of the p70 S6 kinase cascade in neutrophilic differentiation and proliferation of HL-60 cells-a study of transferrin receptor-positive and -negative cells obtained from dimethyl sulfoxide- or retinoic acid-treated HL-60 cells.

    Science.gov (United States)

    Kanayasu-Toyoda, Toshie; Yamaguchi, Teruhide; Oshizawa, Tadashi; Kogi, Mieko; Uchida, Eriko; Hayakawa, Takao

    2002-09-01

    Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.

  1. Induction of apoptosis by 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol from Isaria japonica Yasuda through intracellular reactive oxygen species formation and caspase-3 activation in human leukemia HL-60 cells.

    Science.gov (United States)

    Pae, H O; Oh, G S; Choi, B M; Seo, E A; Oh, H; Shin, M K; Kim, T H; Kwon, T O; Chung, H T

    2003-02-01

    Recently we have reported that the trichothecene mycotoxin 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (AETD) from the fruiting bodies of Isaria japonica Yasuda is a potent inducer of apoptosis in human promyelocytic HL-60 cells. The present study aims to characterize the molecular events leading to AETD-induced apoptosis in HL-60 cells. The percentage of apoptotic cells (annexin-V-positive cell population) increased dose- and time-dependently after AETD exposure. Apoptosis of HL-60 cells by AETD was associated with the formation of intracellular reactive oxygen species (ROS), the depletion of intracellular glutathione (GSH) and the activation of caspase-3. Pretreating the cells with the antioxidant N-acetyl-L-cystein (NAC) and the caspase-3 inhibitor Z-DEVD-fmk abrogated AETD-induced apoptosis and caspase-3 activation. NAC blocked intracellular ROS formation and GSH depletion, but Z-DEVD-fmk did not. These results indicate that AETD induces apoptosis in HL-60 cells by causing intracellular ROS formation and GSH depletion followed by the downstream event of caspase-3 activation.

  2. Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells

    DEFF Research Database (Denmark)

    Winzen, R; Wallach, D; Engelmann, H;

    1992-01-01

    Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol d...

  3. Stimulation of proliferation of HL60 cells by low concentrations of 12-O-tetradecanoylphorbol-13-acetate and its relationship to the mitogenic effects of insulin

    Energy Technology Data Exchange (ETDEWEB)

    Trayner, I.D.; Clemens, M.J. (St. George' s Hospital Medical School, London (United Kingdom))

    1992-03-01

    The effects of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the growth and differentiation of cultured human acute promyelocytic leukemia (HL60) cells have been studied using cells growing in a fully defined medium consisting of RPMI 1640 supplemented with selenium dioxide, insulin, and either transferrin or ferric citrate. High concentrations of TPA cause the expected inhibition of proliferation and induction of macrophage-like differentiation. In contrast, in cells deprived in insulin, which continue to grow at a slow rate, lower concentrations of TPA stimulate proliferation without inducing differentiation. The ability of higher concentrations of TPA to induce differentiation is independent of the presence of insulin. Low-TPA also stimulates the short-term incorporation of thymidine by three- to fourfold, as compared to a seven fold stimulation by insulin. The proliferative response to low TPA concentrations provides a useful model for dissecting the signaling pathways that control cell proliferation following stimulation by insulin and activators of protein kinase C.

  4. Sequential Enrichment with Titania-coated Magnetic Mesoporous Hollow Silica Microspheres and Zirconium Arsenate-modified Magnetic Nanoparticles for the Study of Phosphoproteome of HL60 Cells

    Science.gov (United States)

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-01-01

    As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively. PMID:25262027

  5. Cytotoxic and DNA-damaging effects of methyl tert-butyl ether and its metabolites on HL-60 cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Tang, G.H. [Xian Medical Univ. (China); Shen, Y.; Shen, H.M. [National Univ. of Singapore (Singapore)] [and others

    1996-12-31

    Methyl tert-butyl ether (MTBE) is a widely used oxygenate in unleaded gasoline; however, few studies have been conducted on the toxicity of this compound. This study evaluates the cytotoxic and DNA-damaging effects of MTBE and its metabolites in a human haemopoietic cell line, HL-60. The metabolites of MTBE studied include tertiary butyl alcohol (TBA), {alpha}-hydroxyisobutyric acid (HIBA), and formaldehyde. Comet assay is used to assess DNA damage, and the cytotoxicity is investigated by lactate dehydrogenease (LDH) release. The results show no significant cytotoxic effects of MTBE, TBA, and HIBA over a concentration ranging from 1 to 30 mM. Formaldehyde, in contrast, causes a substantial LDH release at a concentration of 5 {mu}M. Hydrogen peroxide, a known oxidative agent, at concentrations ranging from 10 to 100 {mu}M, produces a significant dose-related increase in DNA damage, whereas a much higher concentration of MTBE (1 to 30 mM) is required to produce a similar observation. The genotoxic effects of TBA and HIBA appear to be identical to that of MTBE. Conversely, DNA damage is observed for formaldehyde at a relatively low concentration range (5 to 100 {mu}M). These findings suggest that MTBE and its metabolites, except formaldehyde, have relatively low cytotoxic and genotoxic effects. 16 refs., 4 figs.

  6. Growth inhibition and apoptosis induction of Scutellaria luteo-coerulea Bornm. & Sint. on leukemia cancer cell lines K562 and HL-60

    Directory of Open Access Journals (Sweden)

    Mahsa Motaez

    2015-10-01

    Full Text Available Objective: Scutellaria (Lamiaceae has been implicated for medicinal purposes both in modern and traditional medicine. Some species of the genus Scutellaria has extensively been studied for anticancer activity. Scutellaria luteo-coerulea (S. luteo-coerulea is one of the Iranian species of the genus Scutellaria. Materials and Methods: In the present study, cytotoxic and apoptogenic properties of CH2Cl2, EtOAc, n-BuOH, and H2O fractions of S. luteo-coerulea were investigated on K562. Moreover, HL-60. DNA fragmentation in apoptotic cells were determined by propidium iodide (PI staining (sub-G1 peak. Results: Scutellaria luteo-coerulea inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions of S. luteo-coerulea, the CH2Cl2 fraction was found to be the most cytotoxic one among others. Sub-G1 peak in flow cytometry histogram of treated cells suggested the induction of apoptosis in S. luteo-coerulea. Conclusion: Scutellaria luteo-coerulea could be a novel candidate for further analytical elucidation in respect to fine major components responsible for the cytotoxic effect of the plant also clinical evaluations.

  7. Myeloid differentiation and retinoblastoma phosphorylation changes in HL-60 cells induced by retinoic acid receptor- and retinoid X receptor-selective retinoic acid analogs.

    Science.gov (United States)

    Brooks, S C; Kazmer, S; Levin, A A; Yen, A

    1996-01-01

    The ability of subtypes of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) singly and in combination to elicit myeloid differentiation, G1/0-specific growth arrest, and retinoblastoma (RB) tumor suppressor protein dephosphorylation was determined in the human myeloblastic leukemia cell line HL-60 using subtype-selective retinoic acid (RA) analogs. RA analogs that selectively bind only to RARs (Am580 and/or TTNPB) or to RXRs (Ro 25-6603, SR11237, and/or SR11234) did not elicit the above-mentioned three cellular responses. In contrast, simultaneous treatment with both an RAR-selective ligand (Am580 or TTNPB) and an RXR-selective ligand (Ro 25-6603, SR11237, or SR11234) induced all three cellular processes. An RAR alpha-selective ligand used with an RXR-selective ligand generated the same responses as did all-trans RA or 9-cis RA, which affect both families of receptors, suggesting an important role for RAR alpha among RAR subtypes in eliciting cellular response. Consistent with this finding, the RAR alpha antagonist, Ro 41-5253, reduced the level of the cellular responses elicited by treatment with an RAR alpha-selective ligand plus RXR-selective ligand. The coupling of the shift of RB to its hypophosphorylated form with G1/0 arrest and differentiation in response to ligands is consistent with a possible role of RB as a downstream target or effector of RAR alpha and RXR in combination.

  8. Neoplastic transformation of BALB/3T3 cells and cell cycle of HL-60 cells are inhibited by mango (Mangifera indica L.) juice and mango juice extracts.

    Science.gov (United States)

    Percival, Susan S; Talcott, Stephen T; Chin, Sherry T; Mallak, Anne C; Lounds-Singleton, Angela; Pettit-Moore, Jennifer

    2006-05-01

    The mango, Mangifera indica L., is a fruit with high levels of phytochemicals, suggesting that it might have chemopreventative properties. In this study, whole mango juice and juice extracts were screened for antioxidant and anticancer activity. Antioxidant activity of the mango juice and juice extracts was measured by 3 standard in vitro methods. The results of the 3 methods were in general agreement, although different radicals were measured in each. Anticancer activity was measured by examining the effect on cell cycle kinetics and the ability to inhibit chemically induced neoplastic transformation of mammalian cell lines. Incubation of HL-60 cells with whole mango juice and mango juice fractions resulted in an inhibition of the cell cycle in the G(0)/G(1) phase. A fraction of the eluted mango juice with low peroxyl radical scavenging ability was most effective in arresting cells in the G(0)/G(1) phase. Whole mango juice was effective in reducing the number of transformed foci in the neoplastic transformation assay in a dose-dependent manner. These techniques provide valuable screening tools for health benefits derived from mango phytochemicals.

  9. 27. Molecular spectra of acrylamide-induced mutation at hprt locus in human promyelocytic leukemia HL-60 and NB4. cell lines

    Institute of Scientific and Technical Information of China (English)

    刘胜学; 曹佳; 杨梦苏; 方志俊; 安辉

    2001-01-01

    The genotoxicity of acrylamide was investigated by methods of single cell clone culturing, two-way screening count, multiplex PCR amplification and electrophoresis technique. Acrylamide only showed clear mutagenesis until dose raised 700 mg*L-1 in HL-60 cells. The most frequent spontaneous mutation were point mutation (≥90.0%) and acrylamide-induced mutation mainly included partial deletion and point mutation (respectively 40.0%~66.7%/33.3%~60.0%). Total gene deletion wasn't discovered in both of cells. There were deletion mutation in all exons of hprt gene(except 7/8 exon), and toward the 3' end of the hprt gene. The most frequent acrylamide-induced mutation were point mutation and single exon deletion (93.3%/86.1%). There were not clear difference in both of cells. The results suggest that the spectra of spontaneous and acrylamide-induced mutants were different. and the smaller changes in genetic structure have something to do with mechanism.

  10. Sequential enrichment with titania-coated magnetic mesoporous hollow silica microspheres and zirconium arsenate-modified magnetic nanoparticles for the study of phosphoproteome of HL60 cells.

    Science.gov (United States)

    Yu, Qiong-Wei; Li, Xiao-Shui; Xiao, Yongsheng; Guo, Lei; Zhang, Fan; Cai, Qian; Feng, Yu-Qi; Yuan, Bi-Feng; Wang, Yinsheng

    2014-10-24

    As one of the most important types of post-translational modifications, reversible phosphorylation of proteins plays crucial roles in a large number of biological processes. However, owing to the relatively low abundance and dynamic nature of phosphorylation and the presence of the unphosphorylated peptides in large excess, phosphopeptide enrichment is indispensable in large-scale phosphoproteomic analysis. Metal oxides including titanium dioxide have become prominent affinity materials to enrich phosphopeptides prior to their analysis using liquid chromatography-mass spectrometry (LC-MS). In the current study, we established a novel strategy, which encompassed strong cation exchange chromatography, sequential enrichment of phosphopeptides using titania-coated magnetic mesoporous hollow silica microspheres (TiO2/MHMSS) and zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2), and LC-MS/MS analysis, for the proteome-wide identification of phosphosites of proteins in HL60 cells. In total, we were able to identify 11,579 unique phosphorylation sites in 3432 unique proteins. Additionally, our results suggested that TiO2/MHMSS and ZrAs-Fe3O4@SiO2 are complementary in phosphopeptide enrichment, where the two types of materials displayed preferential binding of peptides carrying multiple and single phosphorylation sites, respectively.

  11. Apoptosis of HL-60 human leukemia cells induced by Asiatic acid through modulation of B-cell lymphoma 2 family proteins and the mitogen-activated protein kinase signaling pathway.

    Science.gov (United States)

    Wu, Qiuling; Lv, Tingting; Chen, Yan; Wen, Lu; Zhang, Junli; Jiang, Xudong; Liu, Fang

    2015-07-01

    The toxicities of conventional chemotherapeutic agents to normal cells restrict their dosage and clinical efficacy in acute leukemia; therefore, it is important to develop novel chemotherapeutics, including natural products, which selectively target cancer-specific pathways. The present study aimed to explore the effect of the chemopreventive agent asiatic acid (AA) on the proliferation and apoptotic rate of the leukemia cell line HL-60 and investigated the mechanisms underlying its anti-tumor activity. The effect of AA on the proliferation of HL-60 cells was evaluated using the MTT assay. Annexin V-fluorescein isothiocyanate/propidium iodide double staining followed by flow cytometric analysis as well as Hoechst 33258 staining were used to analyze the apoptotic rate of the cells. Furthermore, changes of survivin, B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 expressions were detected by western blot analysis. AA blocked the growth of HL-60 cells in a dose- and time-dependent manner. The IC50-value of AA on HL-60 cells was 46.67 ± 5.08 µmol/l for 24 h. AA induced apoptosis in a dose-dependent manner, which was inhibited in the presence of Z-DEVD-FMK, a specific inhibitor of caspase. The anti-apoptotic proteins Bcl-2, Mcl-1 and survivin were downregulated by AA in a dose-dependent manner. Concurrently, AA inhibited ERK and p38 phosphorylation in a dose-dependent manner, while JNK phosphorylation was not affected. In conclusion, the present study indicated that the p38 and ERK pathways, as well as modulation of Bcl-2 family and survivin proteins were key regulators of apoptosis induced in HL-60 cells in response to AA.

  12. Programmed Cell Death Induced by (−)-8,9-Dehydroneopeltolide in Human Promyelocytic Leukemia HL-60 Cells under Energy Stress Conditions

    Science.gov (United States)

    Fuwa, Haruhiko; Sato, Mizuho; Sasaki, Makoto

    2014-01-01

    (+)-Neopeltolide is a marine macrolide natural product that exhibits potent antiproliferative activity against several human cancer cell lines. Previous study has established that this natural product primarily targets the complex III of the mitochondrial electron transport chain. However, the biochemical mode-of-actions of neopeltolide have not been investigated in detail. Here we report that (−)-8,9-dehydroneopeltolide (8,9-DNP), a more accessible synthetic analogue, shows potent cytotoxicity against human promyelocytic leukemia HL-60 cells preferentially under energy stress conditions. Nuclear morphology analysis, as well as DNA ladder assay, indicated that 8,9-DNP induced significant nuclear condensation/fragmentation and DNA fragmentation, and these events could be suppressed by preincubating the cells with a pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD). Immunoblot analysis demonstrated the release of cytochrome c from the mitochondria and the cleavage of full-length caspase-3 and poly(ADP-ribose) polymerase (PARP). These results indicated that 8,9-DNP induced caspase-dependent apoptotic programmed cell death under energy stress conditions. It was also found that 8,9-DNP induced non-apoptotic cell death in the presence/absence of zVAD under energy stress conditions. Immunoblot analysis showed the intracytosolic release of apoptosis-inducing factor (AIF), although it did not further translocate to the nucleus. It appears most likely that, in the presence of zVAD, 8,9-DNP triggered necrotic cell death as a result of severe intracellular ATP depletion. PMID:25419998

  13. Programmed Cell Death Induced by (−-8,9-Dehydroneopeltolide in Human Promyelocytic Leukemia HL-60 Cells under Energy Stress Conditions

    Directory of Open Access Journals (Sweden)

    Haruhiko Fuwa

    2014-11-01

    Full Text Available (+-Neopeltolide is a marine macrolide natural product that exhibits potent antiproliferative activity against several human cancer cell lines. Previous study has established that this natural product primarily targets the complex III of the mitochondrial electron transport chain. However, the biochemical mode-of-actions of neopeltolide have not been investigated in detail. Here we report that (−-8,9-dehydroneopeltolide (8,9-DNP, a more accessible synthetic analogue, shows potent cytotoxicity against human promyelocytic leukemia HL-60 cells preferentially under energy stress conditions. Nuclear morphology analysis, as well as DNA ladder assay, indicated that 8,9-DNP induced significant nuclear condensation/fragmentation and DNA fragmentation, and these events could be suppressed by preincubating the cells with a pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe-fluoromethylketone (zVAD. Immunoblot analysis demonstrated the release of cytochrome c from the mitochondria and the cleavage of full-length caspase-3 and poly(ADP-ribose polymerase (PARP. These results indicated that 8,9-DNP induced caspase-dependent apoptotic programmed cell death under energy stress conditions. It was also found that 8,9-DNP induced non-apoptotic cell death in the presence/absence of zVAD under energy stress conditions. Immunoblot analysis showed the intracytosolic release of apoptosis-inducing factor (AIF, although it did not further translocate to the nucleus. It appears most likely that, in the presence of zVAD, 8,9-DNP triggered necrotic cell death as a result of severe intracellular ATP depletion.

  14. Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

    2012-06-22

    The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

  15. Toxicological effects of three types of silver nanoparticles and their salt precursors acting on human U-937 and HL-60 cells.

    Science.gov (United States)

    Barbasz, Anna; Oćwieja, Magdalena; Walas, Stanisław

    2017-01-01

    The growing popularity of nanomaterials requires a systematic study of their effects on the human body. Silver nanoparticles (AgNPs), due to their antiseptic properties, are used in almost every area of life. The purpose of the study was to examine whether the precursor used for the synthesis of nanoparticles affects their bio-influence and modifies their impact on cells of the human immune system. To compare the effects of precursor silver salts (AgNO3, CH3COOAg and AgClO4) and corresponding nanoparticles (TAN TAA and TAC) cytotoxicity study was conducted on two cell lines U-937 and HL-60. For both cell lines, silver salts are more toxic than the corresponding nanoparticles. Cell viability after treatment with the two forms of silver (salt/particle) is dependent on silver dose and degree of cells differentiation. Addition of the silver salt of doses greater than 5 mg/L results in decreased cell viability by over 60%, whereas nanoparticles' addition reduces cell viability on average by 30%. On the basis of the determined LD50 values it can be stated that for the tested cells the most toxic are AgClO4 and TAC. Production of nitric oxide, which is a mediator of inflammation, is the greatest after treatment of the cells by TAC. Different interactions of studied nanoparticles with albumin has been found and it was shown that addition of albumin to the cells treated by nanoparticles reduces their toxic effects. Obtained by us highly purified, mono-disperse AgNPs exhibit diverse effects relative to the biological systems, depending on the precursor salt used.

  16. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yoon Shin; Lim, Goh-Woon [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung [Department of Microbiology, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Yoo, Eun-Sun [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Chan Ra, Jeong [Stem Cell Research Center, RNL BIO, Seoul 153-768 (Korea, Republic of); Ryu, Kyung-Ha, E-mail: ykh@ewha.ac.kr [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  17. A new daunomycin-peptide conjugate: synthesis, characterization and the effect on the protein expression profile of HL-60 cells in vitro.

    Science.gov (United States)

    Orbán, Erika; Manea, Marilena; Marquadt, Andreas; Bánóczi, Zoltán; Csík, Gabriella; Fellinger, Erzsébet; Bosze, Szilvia; Hudecz, Ferenc

    2011-10-19

    Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

  18. HMI Synoptic Maps Produced by NSO/NISP

    CERN Document Server

    Hughes, Anna L H; Marble, Andrew R; Oien, Niles A; Petrie, Gordon; Pevtsov, Alexei A

    2016-01-01

    Recently, the National Solar Observatory (NSO) Solar-atmosphere Pipeline Working Group has undertaken the production of synoptic maps from Helioseismic and Magnetic Imager (HMI) magnetograms. A set of maps has been processed spanning the data available for 2010-2015 using twice daily images (taken at UT midnight and noon) and running them through the same algorithms used to produce SOLIS/VSM 6302l mean-magnetic and spatial-variance maps. The contents of this document provide an overview of what these maps look like, and the processing steps used to generate them from the original HMI input data.

  19. Regulated expression of the MRP8 and MRP14 genes in human promyelocytic leukemic HL-60 cell treated with the differentiation-inducing agents mycophenolic acid and 1{alpha},25-Dihydroxyvitamin D{sub 3}

    Energy Technology Data Exchange (ETDEWEB)

    Warner-Bartnicki, A.L.; Murao, S.; Collart, F.R.; Huberman, E.

    1992-12-31

    The calcium-binding proteins MRP8 and MEP14 are present in mature monomyelocytic cells and are induced during differentiation. Previous studies have demonstrated that the proteins may mediate the growth arrest in differentiating HL-60 cells. We determined the levels of a protein complex (PC) containing MRP8 and MRP14 and investigated the mechanism by which the genes encoding these proteins are regulated in HL-60 cells treated with the differentiation-inducing agent mycophenorc acid (MPA)While the PC was barely detectable in untreated cells, MPA treatment resulted in elevated levels of the PC which were maximal at 3-4 d, and were found to directly parallel gains in the steady-state levels of MRP8 and MRP14 MRNA. Transcription studies with the use of nuclear run-on experiments revealed increased transcription initiation at the MRP8 and MRP14 promoters after MPA treatment. 1{alpha},25-Dihydroxyvitamin D{sub 3}, which induces HL-60 cell differentiation by another mechanism, was also found to increase transcription initiation at the MRP8 and MRP14 promoters. Our results suggest that this initiation is the major control of maturation agent-mediated increases in MRP8 and MRPl4 gene expression, and support a role for the PC in terminal differentiation of human monomyelocytic cells.

  20. Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: implications for autologous bone marrow purging

    Energy Technology Data Exchange (ETDEWEB)

    Gulliya, K.S.; Pervaiz, S.

    1989-03-01

    Laser photoradiation therapy was tested in an in vitro model for its efficacy in the elimination of non-Hodgkin's lymphoma cells. Results show that at 31.2 J/cm2 of laser light in the presence of 20 micrograms/mL of merocyanine 540 (MC540) there was greater than 5 log reduction in Burkitt's lymphoma (Daudi) cells. Similar tumor cell kill was obtained for leukemia (HL-60) cells at a laser light dose of 93.6 J/cm2. However, to obtain the same efficiency of killing for histiocytic lymphoma (U-937) cells, a higher dose of MC540 (25 micrograms/mL) was required. Clonogenic tumor stem cell colony formation was reduced by greater than 5 logs after laser photoradiation therapy. Under identical conditions for each cell line the percent survival for granulocyte-macrophage colony-forming units (CFU-GM, 45.9%, 40%, 17.5%), granulocyte/erythroid/macrophage/megakaryocyte (GEMM, 40.1%, 20.1%, 11.5%), colony-forming units (CFU-C, 16.2%, 9.1%, 1.8%), and erythroid burst-forming units (BFU-E, 33.4%, 17.8%, 3.9%) was significantly higher than the tumor cells. Mixing of gamma ray-irradiated normal marrow cells with tumor cells (1:1 and 10:1 ratio) did not interfere with the elimination of tumor cells. The effect of highly purified recombinant interferon alpha (rIFN) on laser photoradiation therapy of tumor cells was also investigated. In the presence of rIFN (30 to 3,000 U/mL), the viability of leukemic cells was observed to increase from 0% to 1.5% with a concurrent decrease in membrane polarization, suggesting an increase in fluidity of cell membrane in response to rIFN. However, at higher doses of rIFN (6,000 to 12,000 U/mL) this phenomenon was not observed. The viability of lymphoma cells remained unaffected at all doses of rIFN tested.

  1. An antisense oligodeoxynucleotide targeted against the type II sub. beta. regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

    Energy Technology Data Exchange (ETDEWEB)

    Tortora, G.; Clair, T.; Cho-Chung, Y.S. (National Institutes of Health, Bethesda, MD (USA))

    1990-01-01

    The type II{sub {beta}} regulatory subunit of cAMP-dependent protein kinase (RII{sub {beta}}) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII{sub {beta}} antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII{sub {beta}} antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII{sub {beta}} protein. Exposure to RII{sub {beta}} sense, RI{sub {alpha}} and RII{sub {alpha}} antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII{sub {beta}} regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells.

  2. Effect of an Intermediate-Frequency Magnetic Field of 23 kHz at 2 mT on Chemotaxis and Phagocytosis in Neutrophil-Like Differentiated Human HL-60 Cells

    Directory of Open Access Journals (Sweden)

    Shin Koyama

    2014-09-01

    Full Text Available Public concerns about potential health risks of intermediate-frequency (IF electromagnetic fields are increasing, especially as the use of induction-heating cooktops has spread extensively in Japan and Europe. In order to investigate the properties of IF electromagnetic fields, we examined the effect of exposure to a 23-kHz IF magnetic field of 2 mT for 2, 3, or 4 h on neutrophil chemotaxis and phagocytosis using differentiated human HL-60 cells. Compared with sham exposure, exposure to the IF magnetic field had no effect on neutrophil chemotaxis or phagocytosis. Previous studies demonstrated that exposure to a 23-kHz IF magnetic field of 2 mT (about 74-times the maximum value recommended by the International Commission for Nonionizing Radiation Protection guidelines may affect the first-line immune responses in humans. To our knowledge, this is the first study to evaluate the effects of IF magnetic fields on cellular immune responses. We found that exposure to an IF magnetic field of 2 mT has minimal if any effect on either the chemotaxis or phagocytic activity of neutrophil-like human HL-60 cells.

  3. Single pre-treatment with hypericin, a St. John's wort secondary metabolite, attenuates cisplatin- and mitoxantrone-induced cell death in A2780, A2780cis and HL-60 cells.

    Science.gov (United States)

    Jendželovská, Zuzana; Jendželovský, Rastislav; Hiľovská, Lucia; Kovaľ, Ján; Mikeš, Jaromír; Fedoročko, Peter

    2014-10-01

    St. John's wort (SJW, Hypericum perforatum L.) is a commonly used natural antidepressant responsible for the altered toxicity of some anticancer agents. These interactions have been primarily attributed to the hyperforin-mediated induction of some pharmacokinetic mechanisms. However, as previously demonstrated by our group, hypericin induces the expression of two ABC transporters: multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). Because cisplatin (CDDP) and mitoxantrone (MTX) are potential substrates of ABC transporters, we investigated the effect of 24h hypericin pre-treatment on the cytotoxicity of CDDP and MTX in human cancer cell lines. CDDP-sensitive and -resistant ovarian adenocarcinoma cell lines A2780/A2780cis, together with HL-60 promyelocytic leukemia cells and ABCG2-over-expressing cBCRP subclone, were used in our experiments. We present CDDP cytotoxicity attenuated by hypericin pre-treatment in both A2780 and A2780cis cells and MTX cytotoxicity in HL-60 cells. In contrast, hypericin potentiated MTX-induced death in cBCRP cells. Interestingly, hypericin did not restore cell proliferation in rescued cells. Nevertheless, hypericin did increase the expression of MRP1 transporter in A2780 and A2780cis cells indicating the impact of hypericin on certain resistance mechanisms. Additionally, our results indicate that hypericin may be the potential substrate of BCRP transporter. In conclusion, for the first time, we report the ability of hypericin to affect the onset and/or progress of CDDP- and MTX-induced cell death, despite strong cell cycle arrest. Thus, hypericin represents another SJW metabolite that might be able to affect the effectiveness of anti-cancer drugs and that could interact with ABC transporters, particularly with BCRP.

  4. Effects of human promyelocytic leukemia HL-60 cel supernatant on differetiation of mononulear cel s from human umbilical cord blood%急性早幼粒白血病细胞培养上清液对人脐带血单个核细胞分化的影响

    Institute of Scientific and Technical Information of China (English)

    郑毅; 方宁; 陈代雄

    2013-01-01

    目的白血病细胞能够通过自分泌方式分泌细胞因子而作用于自身,使细胞大量的增殖而不分化;白血病细胞所分泌的细胞因子也能作用于人脐带血(UCB)单个核细胞(MNCs)中的CD34+/CD133+细胞亚群使之增殖而抑制其分化。关于急性早幼粒白血病细胞(HL-60细胞)培养上清液能否使UCB- MNCs分化尚未见报道,本实验主要目的是观察HL-60细胞培养上清液对人UCB-MNCs分化的影响。方法实验分为①有HL-60细胞培养上清液+StemSpan® SFEM组;②StemSpan® SFEM组;③HL-60细胞培养上清液+高糖(HG)-DMEM组;④HG-DMEM组;⑤HL-60细胞培养上清液+低糖(LG)-DMEM组;⑥LG-DMEM组。收分离的人UCB-MNCs按2.5×106/瓶分别接种于6组培养液中,37℃,5%CO2,饱和湿度培养,于培养前和培养12天用流式细胞仪(FCM)分析表型CD34,CD90和CD166的变化。结果各实验组CD34,CD34/CD90和CD166阳性细胞百分比均较培养前升高。结论%Objective:Leukemic cel s can excrete cytokines by an autocrine way, which contribute to its large-scale proliferation, and the cytokines may also contribute to the proliferations of CD34+/CD133+ subpopulation of UCB-MNCs too. To the effect of human promyelocytic leukemia HL-60 cel supernatant on the differentiation of UCB-MNCs, there is not related report so far. The aim of this study is to investigate effects of HL-60 cel supernatant on differentiations of mononuclear cel s from human umbilical cord blood. Methods:Six various cultural system are listed as fol ows: ① HL-60 cel supernatant+ StemSpan–SFEM; ② StemSpan–SFEM; ③ HL-60 cel supernatant+ HG-DMEM; ④ HG-DMEM; ⑤HL-60 cel supernatant LG-DMEM; ⑥ LG-DMEM. 2.50×106 of UCB-MNCs per bottle with the above each cultural system is incubated at 37℃ in a 5%CO2, saturation humidity of incubator, the changes of phenotype of CD34,CD90 and CD166 analyzed by FCM after 12 days. Results: Positive cel s with CD34+, CD166+ and CD34+/CD

  5. GABAA receptor plasticity in Jurkat T cells.

    Science.gov (United States)

    Dionisio, Leonardo; Arias, Verónica; Bouzat, Cecilia; Esandi, María del Carmen

    2013-12-01

    GABAA receptors (GABAAR) mediate inhibitory neurotransmission in the human brain. Neurons modify subunit expression, cellular distribution and function of GABAAR in response to different stimuli, a process named plasticity. Human lymphocytes have a functional neuronal-like GABAergic system with GABAAR acting as inhibitors of proliferation. We here explore if receptor plasticity occurs in lymphocytes. To this end, we analyzed human T lymphocyte Jurkat cells exposed to different physiological stimuli shown to mediate plasticity in neurons: GABA, progesterone and insulin. The exposure to 100 μM GABA differently affected the expression of GABAAR subunits measured at both the mRNA and protein level, showing an increase of α1, β3, and γ2 subunits but no changes in δ subunit. Exposure of Jurkat cells to different stimuli produced different changes in subunit expression: 0.1 μM progesterone decreased δ and 0.5 μM insulin increased β3 subunits. To identify the mechanisms underlying plasticity, we evaluated the Akt pathway, which is involved in the phosphorylation of β subunits and receptor translocation to the membrane. A significant increase of phosphorylated Akt and on the expression of β3 subunit in membrane occurred in cells exposed 15 h to GABA. To determine if plastic changes are translated into functional changes, we performed whole cell recordings. After 15 h GABA-exposure, a significantly higher percentage of cells responded to GABA application when compared to 0 and 40 h exposure, thus indicating that the detected plastic changes may have a role in GABA-modulated lymphocyte function. Our results reveal that lymphocyte GABAAR are modified by different stimuli similarly and by similar mechanisms to those in neurons. This property is of significance for the development of future therapies involving pharmacological modulation of the immune response. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  6. Identity, Prosocial Behavior, and Generative Concern in German and Cameroonian Nso Adolescents

    Science.gov (United States)

    Busch, Holger; Hofer, Jan

    2011-01-01

    Little is known about whether ego identity statuses have the same developmental concomitants in different cultural contexts. Thus, 159 German and 158 Cameroonian Nso adolescents aged 15-18 were recruited to test if associations between identity and generativity (i.e. the desire to create a positive legacy) and prosocial behavior (i.e. helpful and…

  7. Establishment of Jurkat tet-on cell line

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  8. Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

    Directory of Open Access Journals (Sweden)

    Alessia Arcaro

    2015-01-01

    Full Text Available Heat shock 60 kDa protein 1 (HSP60 is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM of 4-hydroxy-2-nonenal (HNE yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL or by copper-catalyzed oxidation (oxLDL, but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.

  9. Rubratoxin-B-induced secretion of chemokine ligands of cysteine-cysteine motif chemokine receptor 5 (CCR5) and its dependence on heat shock protein 90 in HL60 cells.

    Science.gov (United States)

    Nagashima, Hitoshi

    2015-11-01

    To elucidate the mechanism underlying rubratoxin B toxicity, the effects of rubratoxin B on the secretion of CCR5 chemokines, CCL3, CCL4, and CCL5, in a human promyelocytic leukemia cell line, HL60, were investigated. In addition, to examine whether the molecular chaperone 90-kDa heat shock protein (Hsp90) contributes to rubratoxin B toxicity, the effects of Hsp90-specific inhibitors, radicicol and geldanamycin, were investigated. Exposure to rubratoxin B for 24h induced secretion of each CCR5 chemokine, although the effect on CCL5 secretion was modest, and it enhanced secretion of proinflammatory cytokines tumor necrosis factor-α, CXCL8, and CCL2. Concomitant treatment with radicicol abolished the rubratoxin-induced secretion of all cytokines investigated. Geldanamycin antagonized the rubratoxin B-induced effects on CCL3 and CCL5, but not CCL4; the effects of geldanamycin were less than that of radicicol. Taken together, the results suggest that rubratoxin B, with the contribution of Hsp90, induces secretion of CCR5 chemokines.

  10. DNA fragmentation induced by all-trans retinoic acid and its steroidal analogue EA-4 in C2 C12 mouse and HL-60 human leukemic cells in vitro.

    Science.gov (United States)

    Alakhras, Raghda S; Stephanou, Georgia; Demopoulos, Nikos A; Grintzalis, Konstantinos; Georgiou, Christos D; Nikolaropoulos, Sotirios S

    2014-08-01

    We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15-26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0-1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified. Copyright © 2013 John Wiley & Sons, Ltd.

  11. 铁剥夺和铁超负荷对白血病细胞株HL-60凋亡的影响机制%The influence of iron deprivation and rich iron on the apoptosis of human Leukemia-60 cells

    Institute of Scientific and Technical Information of China (English)

    王叨; 李四保; 刘玉峰

    2012-01-01

    目的 探讨铁剥夺和铰超负荷对白血病细胞株HL-60细胞凋亡的影响及其机制,为临床采用铁剥夺策略治疗或辅助治疗白血病提供理论依据.方法 在HL-60细胞培养基中分别加入不同浓度的去铁胺(DFO)或三氯化铁(FeCl3),造成细胞内铁剥夺或铁超负荷状态,采取噻唑蓝(MTT)法、DNA原位末端标记染色法(TUNEL)、免疫组化法检测铁剥夺和铁超负荷状态下HL-60细胞活力、凋亡率、细胞色素C(Cyt C)阳性细胞率.结果 DFO组细胞活力呈明显下降趋势,凋亡率呈显著上升趋势;FeCl3组细胞活力和凋亡率与对照组相比呈下降趋势;DFO组细胞胞浆内Cyt C阳性细胞率与对照组相比明显升高;而FeCl3组细胞浆内CytC阳性细胞率与对照组相比无明显差异.结论 铁剥夺可促进线粒体释放Cyt C,诱导HL-60细胞凋亡;铁超负荷对线粒体释放Cyt C无直接影响作用.%Objective To explore the influence and mechanism of iron deprivation and rich iron on the apoptotic process of human Leukemia-60 ( HL-60) cells, and provide the theoretical basis for the clinical therapy. Methods HL-60 cells were cultivated with different concentration of DFO and FeCl3 to establish intracellular iron-deprivated and rich-iron states. Cell viability, apoptotic rate and the positive rate of Cyt C were detected by MTT, TUNEL and immunohistochemistry. Results The cell viability in DFO-treated group decreased, while the apoptotic rate increased dramatically. In contrast, an opposite result was obtained in FeCL,-treated group. The positive rate of Cyt C in DFO-treated group was elevated, but no difference was found in FeCl3-treated group. Conclusions Iron deprivation induces apoptosis of HL-60 cells by releasing Cyt C from mitochondria, while rich iron has no direct impact on it. The mechanism of HL-60 cell proliferation might be related with the apoptosis-related genes in the upstream of the mitochondrial pathway.

  12. Characterization of Entamoeba histolytica-induced dephosphorylation in Jurkat cells

    Indian Academy of Sciences (India)

    J E Teixeira; B J Mann

    2002-11-01

    Entamoeba histolytica killing of host cells is contact dependent and mediated by a Gal/GalNAc lectin. Upon contact with amoeba a rapid and extensive dephosphorylation of tyrosine phosphorylated host cell proteins is observed. This effect is mediated by the Gal/GalNAc lectin. However, it requires intact cells, as purified lectin failed to induce dephosphorylation in Jurkat cells. The nonpathogenic, but morphologically identical amoeba, Entamoeba moshkovskii also did not induce dephosphorylation in target cells. Treatment of Jurkat cells with phosphotyrosine phosphatase inhibitors has shown that a host phosphatase is responsible for dephosphorylation. However, it was found that the CD45 phosphotase was not necessary for dephosphorylation of host cell proteins.

  13. Transcriptional targeting of sphingosine-1- phosphate receptor S1P2 by epigallocatechin- 3-gallate prevents sphingosine-1-phosphate- mediated signaling in macrophage-differentiated HL-60 promyelomonocytic leukemia cells

    Directory of Open Access Journals (Sweden)

    Chokor R

    2014-05-01

    Full Text Available Rima Chokor, Sylvie Lamy, Borhane AnnabiLaboratoire d'Oncologie Moléculaire, Centre de recherche BIOMED, Département de Chimie, Université du Québec à Montréal, Montreal, QC, CanadaBackground: Macrophage chemotaxis followed by blood–brain barrier transendothelial migration is believed to be associated with inflammation in the central nervous system. Antineuroinflammatory strategies have identified the dietary-derived epigallocatechin-3-gallate (EGCG as an efficient agent to prevent neuroinflammation-associated neurodegenerative diseases by targeting proinflammatory mediator signaling.Methods: Given that high levels of sphingosine kinase and its product, sphingosine-1-phosphate (S1P, are present in brain tumors, we used quantitative reverse-transcription polymerase chain reaction (qRT-PCR and immunoblotting to test whether EGCG may impact on S1P receptor gene expression and prevent S1P response in undifferentiated and in terminally differentiated macrophages.Results: Promyelomonocytic human leukemia (HL-60 cells were differentiated into macrophages, and S1P triggered phosphorylation in extracellular signal-regulated kinase (ERK, c-Jun N-terminal kinase (JNK, and P38 mitogen-activated protein kinase (MAPK intracellular signaling, as shown by Western blot analysis. Pretreatment of cells with EGCG prior to differentiation inhibited the response to S1P in all three pathways, while EGCG abrogated P38 MAPK phosphorylation when present only during differentiation. Terminally-differentiated macrophages were, however, insensitive to EGCG treatment. Using qRT-PCR, gene expression of the S1P receptors S1P1, S1P2, and S1P5 was predominantly induced in terminally-differentiated macrophages, while the S1P2 was decreased by EGCG treatment.Conclusion: Our data suggest that diet-derived EGCG achieves efficient effects as a preventive agent, targeting signaling pathways prior to cell terminal differentiation. Such properties could impact on cell chemotaxis

  14. File list: InP.Bld.50.AllAg.Jurkat [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Bld.50.AllAg.Jurkat hg19 Input control Blood Jurkat SRX326895,SRX032295,SRX8355...RX206836,SRX1162906,SRX732512,SRX709027,SRX080335,SRX190051,SRX725580 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Bld.50.AllAg.Jurkat.bed ...

  15. File list: ALL.Bld.50.AllAg.Jurkat [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.50.AllAg.Jurkat hg19 All antigens Blood Jurkat SRX725577,SRX725585,SRX72558...9,SRX725580 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.50.AllAg.Jurkat.bed ...

  16. The Putative Role of the Non-Gastric H+/K+-ATPase ATP12A (ATP1AL1 as Anti-Apoptotic Ion Transporter: Effect of the H+/K+ ATPase Inhibitor SCH28080 on Butyrate-Stimulated Myelomonocytic HL-60 Cells

    Directory of Open Access Journals (Sweden)

    Martin Jakab

    2014-10-01

    Full Text Available Background/Aims: The ATP12A gene codes for a non-gastric H+/K+ ATPase, which is expressed in a wide variety of tissues. The aim of this study was to test for the molecular and functional expression of the non-gastric H+/K+ ATPase ATP12A/ATP1AL1 in unstimulated and butyrate-stimulated (1 and 10 mM human myelomonocytic HL-60 cells, to unravel its potential role as putative apoptosis-counteracting ion transporter as well as to test for the effect of the H+/K+ ATPase inhibitor SCH28080 in apoptosis. Methods: Real-time reverse-transcription PCR (qRT-PCR was used for amplification and cloning of ATP12A transcripts and to assess transcriptional regulation. BCECF microfluorimetry was used to assess changes of intracellular pH (pHi after acute intracellular acid load (NH4Cl prepulsing. Mean cell volumes (MCV and MCV-recovery after osmotic cell shrinkage (Regulatory Volume Increase, RVI were assessed by Coulter counting. Flow-cytometry was used to measure MCV (Coulter principle, to assess apoptosis (phosphatidylserine exposure to the outer leaflet of the cell membrane, caspase activity, 7AAD staining and differentiation (CD86 expression. Results: We found by RT-PCR, intracellular pH measurements, MCV measurements and flow cytometry that ATP12A is expressed in human myelomonocytic HL-60 cells. Treatment of HL-60 cells with 1 mM butyrate leads to monocyte-directed differentiation whereas higher concentrations (10 mM induce apoptosis as assessed by flow-cytometric determination of CD86 expression, caspase activity, phosphatidylserine exposure on the outer leaflet of the cell membrane and MCV measurements. Transcriptional up-regulation of ATP12A and CD86 is evident in 1 mM butyrate-treated HL-60 cells. The H+/K+ ATPase inhibitor SCH28080 (100 µM diminishes K+-dependent pHi recovery after intracellular acid load and blocks RVI after osmotic cell shrinkage. After seeding, HL-60 cells increase their MCV within the first 24 h in culture, and subsequently

  17. Cytoskeletal forces during signaling activation in Jurkat T-cells

    Science.gov (United States)

    Hui, King Lam; Balagopalan, Lakshmi; Samelson, Lawrence E.; Upadhyaya, Arpita

    2015-01-01

    T-cells are critical for the adaptive immune response in the body. The binding of the T-cell receptor (TCR) with antigen on the surface of antigen-presenting cells leads to cell spreading and signaling activation. The underlying mechanism of signaling activation is not completely understood. Although cytoskeletal forces have been implicated in this process, the contribution of different cytoskeletal components and their spatial organization are unknown. Here we use traction force microscopy to measure the forces exerted by Jurkat T-cells during TCR activation. Perturbation experiments reveal that these forces are largely due to actin assembly and dynamics, with myosin contractility contributing to the development of force but not its maintenance. We find that Jurkat T-cells are mechanosensitive, with cytoskeletal forces and signaling dynamics both sensitive to the stiffness of the substrate. Our results delineate the cytoskeletal contributions to interfacial forces exerted by T-cells during activation. PMID:25518938

  18. XaNSoNS: GPU-accelerated simulator of diffraction patterns of nanoparticles

    Science.gov (United States)

    Neverov, V. S.

    XaNSoNS is an open source software with GPU support, which simulates X-ray and neutron 1D (or 2D) diffraction patterns and pair-distribution functions (PDF) for amorphous or crystalline nanoparticles (up to ∼107 atoms) of heterogeneous structural content. Among the multiple parameters of the structure the user may specify atomic displacements, site occupancies, molecular displacements and molecular rotations. The software uses general equations nonspecific to crystalline structures to calculate the scattering intensity. It supports four major standards of parallel computing: MPI, OpenMP, Nvidia CUDA and OpenCL, enabling it to run on various architectures, from CPU-based HPCs to consumer-level GPUs.

  19. The Multi-voicedness of Independence and Interdependence - The case of Cameroonian Nso

    DEFF Research Database (Denmark)

    Keller, Heidi; Demuth, Carolin; Yovsi, Relindis D.

    2008-01-01

    It is often claimed that independence and interdependence are two dimensions that are part of any culture and the psychology of any human being. While previous studies have considered these two concepts merely as a matter of degree, this article argues that, in fact, they can be of different...... quality and have a variety of meanings depending on the specific socio-cultural context. From a systemic approach, the study addresses the dialogical co-existence of these dimensions and views culture as an open system that allows for adaptation and constant reorganization according to the given context....... Interviews with 10 mothers from the ethnic group of the Cameroonian Nso on their ideas on childrearing revealed that different conceptions of autonomy and interpersonal relatedness not only co-exist in this ethnic group but may serve different purposes and change depending on the specific socio-cultural...

  20. Jurkat/A4 cells with multidrug resistance exhibit reduced sensitivity to quercetin.

    Science.gov (United States)

    Philchenkov, A; Zavelevich, M; Savinska, L; Blokhin, D

    2010-07-01

    While multidrug resistance of cancer cells is a well-known phenomenon, little is known on the cross resistance between cytotoxic chemotherapeutical agents and unrelated substances such as natural flavonoids. To compare the effects of cytotoxic drug, vepeside and natural flavonoid, quercetin in Jurkat cells and their multidrug-resistant subline Jurkat/A4, in particular to analyze the effector mechanisms of apoptosis and the profiles of several pro- and antiapoptotic proteins in these cells upon exposure to vepeside or quercetin. Apoptosis and poly (ADP-ribose) polymerase cleavage were assessed by flow cytometry. Expression of apoptosis-related proteins was analyzed by Western blotting. Jurkat/A4 cells are less sensitive to antiproliferative effects of quercetin as compared with the parental Jurkat cell line. While vepeside as well as quercetin initially induces apoptosis in both cell lines, the following survival of the exposed cells is essentially different. In resistant Jurkat/A4 cells, vepeside or quercetin treatment activates significantly less caspase-9 and -3 as compared with that in the parental cells. The expression of Bad and BNip1 proteins in Jurkat/A4 cells is lower than in the parental cell line. At the same time, XIAP and CAS levels in Jurkat/A4 cells increase. Upon apoptosis induction, XIAP and CAS levels in Jurkat cells decrease, this effect being negligible in resistant cells. Multidrug-resistant Jurkat/A4 cells exhibit reduced sensitivity to cytotoxic effects of quercetin. The expression profile of Jurkat/A4 cells is characterized by the increased levels of XIAP and CAS representing the endogenous inhibitors of apoptosis.

  1. File list: Unc.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR hg19 Unclassified Blood Jurkat (...Clone E6-1) http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR.bed ...

  2. File list: Unc.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: DNS.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: His.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: DNS.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  8. File list: ALL.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: ALL.Bld.20.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Oth.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: His.Bld.20.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: ALL.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: Oth.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: DNS.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR hg19 DNase-seq Blood Jurkat (Clo...ne E6-1) http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR.bed ...

  15. File list: Unc.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Oth.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

    Full Text Available DNS.Bld.20.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR hg19 DNase-seq Blood Jurkat (Clo...ne E6-1) http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/DNS.Bld.20.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR.bed ...

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  20. Antitumor Effect of BCG on Growth of Transplanted Human Myeloid Leukemia HL-60 Cells in Nude Mice%卡介苗抑制裸鼠白血病移植瘤生长及抗肿瘤的实验研究

    Institute of Scientific and Technical Information of China (English)

    王媛媛; 王玲珍; 孙立荣

    2011-01-01

    This study was purposed to explore the anti-leukemia effect of bacillus calmette-guerin vaccine (BCG) on the human myeloid leukemia cell xenograft models.An animal model was established by inoculating the human myeloid leukemia HL-60 cells into the BALB/c (8 - 10 weeks of age) nude mice.The mice were randomly divided into two groups: control group and experimental group.Nude mice in control group were injected with physiological saline, while those of experimental group were given BCG and inactivated BCG respectively.The tumor growth was assayed by using caliper.The survival time of nude mice was determined.Necrotic extent and morphological changes of tumor were observed and examined by HE staining and immunohistochemical method.The results indicated that on 5th -7th days after tumor inoculated, 2 -3 mm tumor mass could be observed.The tumor volume increased over the time.HE staining of tumor tissues showed that there were different degrees of tumor necrosis in BCG group and inactivated BCG group.Immunohistochemistry results demonstrated that CD20 positive cells were obviously observed in the necrotic area of BCG group, compared with the control group and inactivated BCG group.It is concluded that human myeloid leukemia HL-60 cells have been successfully transplanted in nude mice, and the systemic metastasis occurs along with the prolongation of time.BCG inoculation can delay the tumor growth and prolong the survival time of nude mice with leukemia, suggesting that BCG has an antitumor effect.%本研究通过建立人白血病突变株细胞异种移植模型探讨卡介苗(bacillus calmette- guerin vaccine,BCG)的抗白血病作用.对8-10周龄的BALB/c裸鼠皮下接种1×107/ml人急性髓系白0细胞,于4-6天可形成皮下浸润的白血病裸鼠模型,随后将其分为2组:对照组和实验组.对照组于肿瘤内接种生理盐水,实验组又分为T1组(BCG组)、T2组(灭活的BCG组).观察各组裸鼠带瘤生存情况,以及通过对肿瘤组织

  1. Synergistic Effect of Decitabine and Valproic Acid on Induction of Differentiation in Leukemic HL-60 Cells%地西他滨联合丙戊酸钠对白血病细胞株的促分化效应研究

    Institute of Scientific and Technical Information of China (English)

    刘波; 邬露丹

    2012-01-01

    OBJECTIVE To investigate the synergistic effect of decitabine(DCA) and valproic acid(VPA) in differentiation induction in leuketnic HL-60 cells. METHODS The drug groups were set as follows: control group; DCA group A(1.0 umol·L‐1); DCA group B(4.0 μmol·L‐1); VPA group(2.0 mmol·L‐1); combination group A(DCA 1.0 μmolL‐1+VPA 2.0 mmol·L‐1); combination group B(DCA 4.0 umol·L‐1+VPA 2.0 mmol·L‐1). The cells were treated for 48 h. The CD117, CD34 mean fluorescence intensity(MFl) and CDI4, CDllb expression were detected by flow cytometry. RESULTS The CD117, CD34 MFI of combination group A and B were significantly lower than their corresponding concentration single drug group(P<0.01). Except CD14 of combination group B, the CD14, CDllb expressions of combination group A and B were significantly lower than their corresponding concentration single drug group(.P<0.01). CONCLUSION There is an enhanced differentiation activity of combinations of DCA and VPA in HL-60 cells.%目的 探讨地西他滨(decitabine,DCA)和丙戊酸钠(valproic acid,VPA)联用对白血病细胞株HL-60的促分化影响.方法 设立分组如下:对照组,DCAA组(1.0 μmol·L-1),DCA B组(4.0 μmol·L-1),VPA组(2.0mmol·L-1),联合用药A组(DCA1.0 μmol·L-1+VPA 2.0 mmol·L-1),联合用药B组(DCA4.0 μmol·L-1+VPA 2.0 mmol·L-1),作用48h.流式细胞术检测CD34、CD117 MF1以及CD11b、CD14表达率、结果 联合用药A、B组的CD1 17和CD34MFI显著低于其各自的单药组(P<0.01);联合用药A组的CD11b和CD14表达率和联合用药B组CDllb表达率显著低于其各自的单药组(P<0.01).结论 VPA与DCA联用对HL-60细胞其促分化作用.

  2. Role of NSO compounds during primary cracking of a Type II kerogen and a Type III lignite

    Science.gov (United States)

    Behar, F.; Lorant, F.; Lewan, M.

    2008-01-01

    The aim of this work is to follow the generation of NSO compounds during the artificial maturation of an immature Type II kerogen and a Type III lignite in order to determine the different sources of the petroleum potential during primary cracking. Experiments were carried out in closed system pyrolysis in the temperature range from 225 to 350 ??C. Two types of NSOs were recovered: one is soluble in n-pentane and the second in dichloromethane. A kinetic scheme was optimised including both kerogen and NSO cracking. It was validated by complementary experiments carried out on isolated asphaltenes generated from the Type II kerogen and on the total n-pentane and DCM extracts generated from the Type III lignite. Results show that kerogen and lignite first decompose into DCM NSOs with minor generation of hydrocarbons. Then, the main source of petroleum potential originates from secondary cracking of both DCM and n-pentane NSOs through successive decomposition reactions. These results confirm the model proposed by Tissot [Tissot, B., 1969. Premie??res donne??es sur les me??canismes et la cine??tique de la formation du pe??trole dans les bassins se??dimentaires. Simulation d'un sche??ma re??actionnel sur ordinateur. Oil and Gas Science and Technology 24, 470-501] in which the main source of hydrocarbons is not the insoluble organic matter, but the NSO fraction. As secondary cracking of the NSOs largely overlaps that of the kerogen, it was demonstrated that bulk kinetics in open system is a result of both kerogen and NSO cracking. Thus, another kinetic scheme for primary cracking in open system was built as a combination of kerogen and NSO cracking. This new kinetic scheme accounts for both the rate and amounts of hydrocarbons generated in a closed pyrolysis system. Thus, the concept of successive steps for hydrocarbon generation is valid for the two types of pyrolysis system and, for the first time, a common kinetic scheme is available for extrapolating results to natural

  3. Quantification of Natural Attenuation of N-S-O Heteroaromatic Compounds in Groundwater at Field Scale

    Science.gov (United States)

    Ptak, T.; Piepenbrink, M.; Grathwohl, P.

    2005-12-01

    N-S-O heteroaromatic compounds (HET) can typically be found in groundwater at tar oil contaminated sites. Despite the fact that most of these substances are toxic and potentially carcinogenic, they are not yet routinely monitored. Goal of this contribution is to present results from the investigation of the in-situ natural attenuation potential of HET in groundwater at field-scale. The research has been performed at the `Testfeld-Sed' (TFS) experimental site, a former gasworks site in southern Germany. At the TFS site, the ongoing quantitative determination of the natural attenuation potential of HET is performed using an integral investigation approach at multiple control planes (integral pumping tests, based on the inversion of concentration time series measured during pumping), which are positioned at different distances downgradient of the source zone. Natural attenuation processes result in a reduction of the total contaminant mass flow with increasing transport distance. Degradation rates can be obtained from (multi-)process-based reactive transport modeling which considers all processes contributing to mass flow reduction. Two control planes (CPs) at a distance of about 150 m were used for the integral measurements at the TFS site. The concentration time series already indicate that contaminants like BTEX and PAH almost vanished on their way from the upstream to the downstream CP, while HET were still present. The determined mass flow rates further indicate that some of the HET (especially Methylbenzofurane and Dimethylbenzofurane) are not only very mobile, but also highly persistent. This confirms the dominating role of HET as major contaminants at the TFS site. However, the observed decrease in the total Dimethylbenzofurane mass flow rates already indicates good chances for enhanced natural attenuation (ENA) measures (e.g. injection of H2O2 as additional electron acceptor to stimulate biodegradation) which are planned as next steps. Acknowledgement: The

  4. Nitric oxide donor NOC-5 increases XIAP and Aven level in Jurkat cells.

    Science.gov (United States)

    Starikova, Elena G; Tashireva, L A; Novitsky, V V; Ryazantseva, N V

    2014-07-01

    Mitochondrial permeabilisation after NO donor application did not activate caspase-9. We have studied the X-linked apoptosis inhibitor (XIAP) and Aven protein content in NO-treated Jurkat cells. The level of both proteins increased in NO-treated cells. Thus the increase in XIAP and Aven content could be the cause of the lack of caspase-9 activity after mitochondrial permeabilisation in NO-treated Jurkat cells.

  5. [Effects of Sam68 gene silence on proliferation of acute T lymphoblastic leukemia cell line Jurkat].

    Science.gov (United States)

    Wang, Chi-Juan; Xu, Hua; Zhang, Hai-Rui; Wang, Jian; Lin, Ya-Ni; Pang, Tian-Xiang; Li, Qing-Hua

    2014-08-01

    This study was purpose to investigate the effect of Sam68 gene silence on proliferation of human acute T lymphoblastic leukemia cell line Jurkat. The sequence of shRNA targeting the site 531-552 of Sam68 mRNA was designed and chemically synthesized, then a single-vector lentiviral, Tet-inducible shRNA-Sam68 system (pLKO-Tet-On) was constructed; next the Jurkat cells were infected with lentivirus to create stable cell clones with regulatable Sam68 gene expression. The inhibitory efficiency of Sam68 gene was assayed by Real-time PCR and Western blot; the cell activity of Jurkat cells was detected with MTT assay; the change of colony forming potential of Jurkat cells was analyzed by colony forming test; the cell cycle distribution was tested by flow cytometry. The results indicated that the expression of Sam68 in experimental cells was statistically decreased as compared with that of the control cells; the cells activity and colony forming capacity of the Jurkat cells with Sam68 gene silence were significantly inhibited; with Sam68 gene silencing, the percentage of S phase cells was significantly increased, while the percentage of G2 phase cells was significantly decreased. It is concluded that the silencing Sam68 gene using shRNA interference can effectively inhibit the proliferation of human acute T lymphoblastic leukemia cell line Jurkat.

  6. Student artistry sparks eclipse excitement on Maui: NSO/DKIST EPO for the 2016 Partial Solar Eclipse

    Science.gov (United States)

    Schad, Thomas A.; Penn, Matthew J.; Armstrong, James

    2016-05-01

    Local creativity and artistry is a powerful resource that enhances education programs and helps us generate excitement for science within our communities. In celebration of the 2016 Solar Eclipse, the National Solar Observatory (NSO) and its Daniel K Inouye Solar Telescope (DKIST) project were pleased to engage with students across Maui County, Hawai`i, via the 2016 Maui Eclipse Art Contest. With the help of the Maui Economic Development Board and the University of Hawai'is Institute for Astronomy, we solicited art entries from all K-12 schools in Maui County approximately 6 months prior to the eclipse. Along with divisional prizes, a grand prize was selected by a panel of local judges, which was subsequently printed on 25,000 solar eclipse viewing glasses and distributed to all Maui students. We found that the impact of a locally-sourced glasses design cannot be understated. Overall, the success of this program relied upon reaching out to individual teachers, supplying educational flyers to all schools, and visiting classrooms. On the day of the eclipse, all of the art entries were prominently displayed during a community eclipse viewing event at Kalama Beach Park in Kihei, HI, that was co-hosted by NSO and the Maui Science Center. This eclipse art contest was integral to making local connections to help promote science education on Maui, and we suggest that it could be adapted to the solar community's EPO activities for the upcoming 2017 Great American Solar Eclipse.

  7. Is Perceptual Priming Affected by Culture? A Study With German Middle-Class and Cameroonian Nso Farmer Children.

    Science.gov (United States)

    Vöhringer, Isabel Aline; Poloczek, Sonja; Graf, Frauke; Lamm, Bettina; Teiser, Johanna; Fassbender, Ina; Freitag, Claudia; Suhrke, Janina; Teubert, Manuel; Keller, Heidi; Lohaus, Arnold; Schwarzer, Gudrun; Knopf, Monika

    2015-01-01

    The authors explored priming in children from different cultural environments with the aim to provide further evidence for the robustness of the priming effect. Perceptual priming was assessed by a picture fragment completion task in 3-year-old German middle-class and Cameroonian Nso farmer children. As expected, 3-year-olds from both highly diverging cultural contexts under study showed a priming effect, and, moreover, the effect was of comparable size in both cultural contexts. Hence, the children profited similarly from priming, which was supported by the nonsignificant interaction between cultural background and identification performance as well as the analysis of absolute difference scores. However, a culture-specific difference regarding the level of picture identification was found in that German middle-class children identified target as well as control pictures with less perceptual information than children in the Nso sample. Explanations for the cross-cultural demonstration of the priming effect as well as for the culturally diverging levels on which priming occurs are discussed.

  8. Effects of canola and corn oil mimetic on Jurkat cells

    Directory of Open Access Journals (Sweden)

    Akinsete Juliana A

    2011-06-01

    Full Text Available Abstract Background The Western diet is high in omega-6 fatty acids and low in omega-3 fatty acids. Canola oil contains a healthier omega 3 to omega 6 ratio than corn oil. Jurkat T leukemia cells were treated with free fatty acids mixtures in ratios mimicking that found in commercially available canola oil (7% α-linolenic, 30% linoleic, 54% oleic or corn oil (59% linoleic, 24% oleic to determine the cell survival or cell death and changes in expression levels of inflammatory cytokines and receptors following oil treatment. Methods Fatty acid uptake was assessed by gas chromatography. Cell survival and cell death were evaluated by cell cycle analyses, propidium-iodide staining, trypan blue exclusion and phosphatidylserine externalization. mRNA levels of inflammatory cytokines and receptors were assessed by RT-PCR. Results There was a significant difference in the lipid profiles of the cells after treatment. Differential action of the oils on inflammatory molecules, following treatment at non-cytotoxic levels, indicated that canola oil mimetic was anti-inflammatory whereas corn oil mimetic was pro-inflammatory. Significance These results indicate that use of canola oil in the diet instead of corn oil might be beneficial for diseases promoted by inflammation.

  9. Rural N(SO) and German middle-class mothers' interaction with their 3- and 6-month-old infants: A longitudinal cross-cultural analysis.

    Science.gov (United States)

    Lamm, Bettina; Gudi, Helene; Fassbender, Ina; Freitag, Claudia; Graf, Frauke; Goertz, Claudia; Spangler, Sibylle; Teubert, Manuel; Knopf, Monika; Lohaus, Arnold; Schwarzer, Gudrun; Keller, Heidi

    2015-08-01

    This study aims to analyze culture-specific development of maternal interactional behavior longitudinally. Rural Cameroonian Nso mothers (n = 72) and German middle-class mothers (n = 106) were observed in free-play interactions with their 3- and 6-month-old infants. Results reveal the expected shift from a social to a nonsocial focus only in the German middle-class mothers' play interactions but not the rural Nso mothers' play. Nso mothers continue their proximal interactional style with a focus on body contact and body stimulation, whereas German middle-class mothers prefer a distal style of interaction with increasing object-centeredness. These cultural differences are in line with broader cultural models and become more accentuated as the infants grow older. (c) 2015 APA, all rights reserved).

  10. ANTI-INFLAMMATORY AND CYTOTOXICITY EFFECTS OF SALVADORA PERSICA (MESWAK EXTRACTS ON JURKAT T-CELLS

    Directory of Open Access Journals (Sweden)

    Farimah Sardari

    2015-04-01

    Full Text Available Salvadora persica (S. persica, Meswak, is an evergreen shrub to 6-7 m. It has many biological activities such as antipyretic, anti-inflammatory and antifungal activities. This study evaluated in vitro cytotoxic and anti-inflammatory effects of S. persica extracts on human oral Jurkat (T leukemia cells. Extracts from Meswak stick and leaves were tested in different concentrations for their cytotoxic and anti-inflammatory activities on human oral Jurkat T- cells. So treated cells viability with increasing concentrations of S. persica stick extract (0.008-0.2 μg/ml and leaves extract (0.016-0.5 μg/ml for 24, 48 or 72 hours was assessed by using the mitochondrial dependent reduction of yellow MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide to purple formazan. Also Enzyme-linked immunosorbent assay (ELISA was performed on supernatants from treated Jurkat T-cells with phytohemagglutinin (PHA and both extracts to quantify IL-6, IL-8 pro-inflammatory cytokines. Statistically significant differences were indicated by p <0.05. Incubation of Jurkat cells with sterile distilled water, negative control, didn't show any mortality through the incubation period. Against PHA, positive control, both stick and leaves extracts of S. persica like resulted in a dose-dependent decrease of IL-6 and IL-8 secretion (p <0.01. Although both extracts significantly inhibited survival of Jurkat cells (p < 0.01 in a dose- and time-dependent manner, stick extract exerted more cytotoxic effects on Jurkat cells than leaves extract of S. persica (p <0.03. In conclusion, although with increasing concentrations of both extracts anti-inflammatory properties were boosted, S. persica extracts had dose-dependent cytotoxic effects on human oral Jurkat T-cells.

  11. Galectin-1 and Galectin-3 induce mitochondrial apoptotic pathway in Jurkat cells

    Science.gov (United States)

    Vasil'eva, O. A.; Isaeva, A. V.; Prokhorenko, T. S.; Zima, A. P.; Novitsky, V. V.

    2016-08-01

    Cellular malignant transformation is often accompanied by increased gene expression of low-molecular proteins of lectins family-galectins. But it is unknown how galectins promote tumor growth and malignization. Galectins-1 and galectin-3 are thought to be possible immunoregulators exerting their effects by regulating the balance of CD4+ lymphocytes. In addition it is known that tumor cells overexpressing galectins are capable of escaping immunological control, causing apoptosis of lymphocytes. The aim of the study is to investigate the role of galectin-1 and galectin-3 in the implementation of mitochondrial apoptotic pathway in Jurkat cells. Methods: Jurkat cells were used as a model for the study of T-lymphocytes. Jurkat cells were activated with antibodies to CD3 and CD28 and cultured with recombinant galectin-1 and -3. Apoptosis of Jurkat cells and depolarization of the mitochondrial membrane were assessed by flow cytometry. It was found that galectin-1 and galectin-3 have a dose-dependent pro-apoptotic effect on Jurkat cells in vitro and enlarge the number of cells with decreased mitochondrial membrane potential compared with intact cells.

  12. Good Child is a Calm Child: Mothers' Social Status, Maternal Conceptions of Proper Demeanor, and Stranger Anxiety in One-Year Old Cameroonian Nso Children

    Directory of Open Access Journals (Sweden)

    Hiltrud Otto

    2015-04-01

    Full Text Available Our article questions the assumption that stranger anxiety develops universally in children; thereby our study is rooted in a long tradition in psychological anthropology of testing the universality of theories formulated in Western society. We argue that the infant's behavior towards strangers is the product of socialization processes that represent adaptations to cultural contexts. Our study investigates the ethnotheory of childrearing and the development of stranger anxiety in a Cameroonian community of traditional Nso farmers. The participants of the study were 29 Cameroonian Nso mothers with one-year old children. Using a multi-method approach, we demonstrate that Nso mothers value inexpressive infants that adjust easily to others. Accordingly, a considerably large number of one-year old Nso infants showed no stranger anxiety when encountered by a stranger. Maternal social status and her social support system proved crucial to successful implementation of the socialization goal of a calm child. Our data support the view that child behavior is a product of culturally constructed experiences of daily life. The acknowledgement of the cultural construction of stranger anxiety carries implications for developmental theories, especially for attachment theory, which relies on the universality of stranger anxiety in their most acclaimed paradigm, the Strange Situation.

  13. Deferred imitation in 18-month-olds from two cultural contexts: the case of Cameroonian Nso farmer and German-middle class infants.

    Science.gov (United States)

    Borchert, Sonja; Lamm, Bettina; Graf, Frauke; Knopf, Monika

    2013-12-01

    Imitative learning has been described in naturalistic studies for different cultures, but lab-based research studying imitative learning across different cultural contexts is almost missing. Therefore, imitative learning was assessed with 18-month-old German middle-class and Cameroonian Nso farmer infants - representing two highly different eco-cultural contexts associated with different cultural models, the psychological autonomy and the hierarchical relatedness - by using the deferred imitation paradigm. Study 1 revealed that the infants from both cultural contexts performed a higher number of target actions in the deferred imitation than in the baseline phase. Moreover, it was found that German middle-class infants showed a higher mean imitation rate as they performed more target actions in the deferred imitation phase compared with Cameroonian Nso farmer infants. It was speculated that the opportunity to manipulate the test objects directly after the demonstration of the target actions could enhance the mean deferred imitation rate of the Cameroonian Nso farmer infants which was confirmed in Study 2. Possible explanations for the differences in the amount of imitated target actions of German middle-class and Cameroonian Nso farmer infants are discussed considering the object-related, dyadic setting of the imitation paradigm with respect to the different learning contexts underlying the different cultural models of learning. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Effects of gravitational perturbation on the expression of genes regulating metabolism in Jurkat cells.

    Science.gov (United States)

    Singh, Kanika; Cubano, Luis; Lewis, Marian

    2010-01-01

    Gravitational pertubation altered gene expression and increased glucose consumption in spaceflown Jurkat cells. The purpose of this study was to determine if the acceleration experienced during launch was responsible for these changes. In ground-based studies, cells were subjected to typical launch centrifugal acceleration (3g of force for eight minutes) and centrifugal force of 90g for five minutes (commonly used to sediment cells) in a laboratory centrifuge. Controls consisted of static cultures. Gene expression was analyzed by RT-PCR. pH and glucose concentrations were evaluated to monitor metabolic changes. Comparison with controls indicated no significant change in pH or glucose use. Gene expression of Jurkat cells subjected to 3g or 90g of force was altered for only two genes out of seven tested. This research suggests that the changes observed in Jurkat cells flown on STS-95 were not a result of launch acceleration but to other conditions experienced during space flight.

  15. m iR-21反义寡核苷酸增强地西他滨体外抗白血病效应%Enhanced anti-leukemic activity of decitabine to leukemia HL-60 cells by anti-miR-21 oligonucleotide

    Institute of Scientific and Technical Information of China (English)

    王晔恺; 于倩; 林奇龙; 姚燕珍; 梅佩玉; 李翊卫

    2015-01-01

    目的:研究 miR-21反义寡核苷酸(anti-miR-21 oligonucleotide, AMO)对地西他滨(decitabine, DCA)抗白血病效应的影响及可能机制。方法:将AMO和无义寡核苷酸( scramble oligonucleotide , SCR)通过脂质体转染导入HL-60细胞,实时荧光定量PCR( real-time PCR)验证转染效率,再分别与DCA 0.5、2.0和4.0μmol/L作用48 h。 Real-time PCR分别检测人周期节律蛋白3(hPer3) mRNA表达,Annexin V/PI法检测凋亡,流式细胞术检测CD117和CD11b平均荧光强度(MFI)。结果: AMO转染组miR-21表达(0.35±0.07)低于空白组(0.71±0.07)和SCR转染组(0.66±0.05),差异有统计学意义(P<0.05)。 AMO转染组的HL-60细胞DCA的IC50低于空白组和SCR转染组(P<0.01)。同一浓度下,AMO组的早期凋亡率、CD11b的MFI和hPer3 mRNA均高于同一浓度药物作用的空白组和SCR组,CD117 MFI均低于同一浓度药物作用的空白组和SCR组,差异均具有统计学意义(P<0.01)。结论:AMO能显著促进DCA体外抗白血病效应,其机制可能与其协助激活hPer3的表达有关。%AIM:To investigate the role of anti-miR-21 oligonucleotide ( AMO) in the anti-leukemic activity of decitabine (DCA) in vitro.METHODS:AMO and scramble oligonucleotide (SCR) were constructed and transfected into HL-60 cells.The miR-21 expression was analyzed by real-time PCR to identify the transfection efficiency .The cells were treated with DCA at gradient concentrations (0.5, 2.0 and 4.0 μmol/L) for 48 h.The mRNA expression of human period circadian protein 3 (hPer3) was detected by real-time PCR.The early apoptotic rates were determined by flow cy-tometry with Annexin V/PI staining.Mean fluorescence intensities ( MFI) of CD117 and CD11b were also measured by flow cytometry.RESULTS:The miR-21 relative expression level in AMO group was significantly lower than that in blank group and SCR group (P<0.01).IC50 of DCA in AMO group was

  16. File list: InP.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR hg19 Input control Blood Jurkat ...(Clone E6-1) SRX015828,SRX128786,SRX024358 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR.bed ...

  17. File list: InP.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR hg19 No description Blood Jurkat... (Clone E6-1) http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.05.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR.bed ...

  19. File list: NoD.Bld.20.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.20.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR hg19 No description Blood Jurkat... (Clone E6-1) http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.20.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR.bed ...

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    Lifescience Database Archive (English)

    Full Text Available InP.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR hg19 Input control Blood Jurkat ...(Clone E6-1) SRX024358,SRX128786,SRX015828,SRX128785 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/InP.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR.bed ...

  1. File list: NoD.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR hg19 No description Blood Jurkat... (Clone E6-1) http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/NoD.Bld.50.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR.bed ...

  2. File list: InP.Bld.20.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: NoD.Bld.10.AllAg.Jurkat_BRACKETLClone_E6-1BRACKETR [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. Influence of expressed TRAIL on biophysical properties of the human leukemic cell line Jurkat

    Institute of Scientific and Technical Information of China (English)

    Kai CHEN; Zong Yao WEN; Shu CHIEN; Dan LI; Yu Hui JIANG; Wei Juan YAO; Xin Juan WANG; Xiao Chao WEI; Jing GAO; Li De XIE; Zong Yi YAN

    2004-01-01

    The cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) was cloned into RevTet-On, a Tetregulated and high-level gene expression system. The gene expression system was constructed in a human leukemic cell line: Jurkat. By using RevTet-On TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the changes of cellular apoptosis before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the cellular apoptosis ratio was largely dependent on the TRAIL gene expression level. Moreover, it was found that the apoptosis-inducing TRAIL could cause significant changes in the biophysical properties of Jurkat cells. The cell surface charge density decreased, the membrane fluidity declined, the elastic coefficients K1 increased, and the proportion of o-helix in membrane protein secondary structure decreased. Thus, the apoptosis-inducing TRAIL gene caused significant changes on the biomechanic properties of Jurkat cells.

  5. Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells).

    Science.gov (United States)

    Zhang, Min; Zhu, Qi; Shi, Ming; Liu, Yang; Ma, Lei; Yang, Yining; Feng, Dongyun; Dai, Wen; Zhang, Lin; Kang, Tao; Chen, Ping; He, Ying; Liu, Tingting; Zhao, Qing; Wang, Wenjing; Zhi, Jin; Feng, Guodong; Zhao, Gang

    2015-08-01

    This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.

  6. Cytotoxic effects of Fisturalin-3 and 11-Deoxyfisturalin-3 on Jurkat and U937 cell lines.

    Science.gov (United States)

    Mijares, Michael Rodney; Ochoa, Mariana; Barroeta, Amairelys; Martinez, Gricelis Patricia; Suarez, Alirica Isabel; Compagnone, Reinaldo Santi; Chirinos, Perla; Avila, Ramona; De Sanctis, Juan Bautista

    2013-09-01

    Fisturalines are bromotyrosine compounds isolated from marine sponges. Previous studies have shown antineoplasic, antiviral and antibacterial effects in Vitro; however, the possible effects of these compounds in hematologic malignancies have not been assessed. In the present study, the antiproliferative and pro apoptotic effects of Fistularin-3 (F) and 11-Deoxyfistularin-3 (DF) were assessed using the MTT method and annexin V/propidium iodide by flow cytometry using the cell lines: Jurkat E6.1 and U937. In addition, the cell cycle was assessed by flow cytometry. Inhibition of the proliferative response was concentration and time dependent. The IC50 of F was 7.39 and 8.10 µM for Jurkat E6.1 and U937 respectively. At 24 and 48 h, in the U937 cell line, but not in the Jurkat cell line, both compounds induced up to 35% annexin V increase. Necrosis was not observed in any case. Compound F induced, in both cell lines, a decrease in the number of cells in the S phase and increase in the G0/G1 phase. In the Jurkat cell line only, there was an increase in the number of cells in the G2/M phase. Compound DF was not as effective as F. F is more active than DF in repressing the cell cycle and inducing apoptosis. Both compounds are potentially useful in the development of new drugs to treat hematologic malignancies.

  7. Comparison of Total Solar Irradiance with NASA/NSO Spectromagnetograph Data in Solar Cycles 22 and 23

    Science.gov (United States)

    Jones, Harrison P.; Branston, Detrick D.; Jones, Patricia B.; Popescu, Miruna D.

    2002-01-01

    An earlier study compared NASA/NSO Spectromagnetograph (SPM) data with spacecraft measurements of total solar irradiance (TSI) variations over a 1.5 year period in the declining phase of solar cycle 22. This paper extends the analysis to an eight-year period which also spans the rising and early maximum phases of cycle 23. The conclusions of the earlier work appear to be robust: three factors (sunspots, strong unipolar regions, and strong mixed polarity regions) describe most of the variation in the SPM record, but only the first two are associated with TSI. Additionally, the residuals of a linear multiple regression of TSI against SPM observations over the entire eight-year period show an unexplained, increasing, linear time variation with a rate of about 0.05 W m(exp -2) per year. Separate regressions for the periods before and after 1996 January 01 show no unexplained trends but differ substantially in regression parameters. This behavior may reflect a solar source of TSI variations beyond sunspots and faculae but more plausibly results from uncompensated non-solar effects in one or both of the TSI and SPM data sets.

  8. Sulphamic Acid (H2NSO3H)-Catalyzed Multicomponent Reaction of β-Naphthol: An Expeditious Synthesis of Amidoalkyl Naphthols

    Institute of Scientific and Technical Information of China (English)

    NAGAWADE Rahul R.; SHINDE Devanand B.

    2007-01-01

    Sulphamic acid (H2NSO3H) was found to be an efficient catalyst for the multicomponent condensation reaction of β-naphthol, aromatic aldehydes and urea or amides to afford the corresponding a-amidoalkyl-β-naphthols in good yields. The remarkable features of this new procedure are high conversions, short reaction time, clean reaction profiles and simple experimental and work-up procedures.

  9. Induction of Apoptosis by Recombinant Soluble Human TRAIL in Jurkat Cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate the therapeutic potential of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, and to analyze TRAIL-induced apoptosis in Jurkat cells. Methods Expression of TRAIL receptors (DR4 and DR5) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Cytotoxic effects were determined by colony formation assay and a cell counting kit. The effects of recombinant TRAIL on apoptosis of Jurkat cells were determined by DNA fragmentation (DNA ladder) and PI staining. Changes in mitochondrial membrane potential were detected with JC-1 fluorescence. Results TRAIL inhibited the proliferation and induced internucleosomal DNA fragmentation (characteristic of apoptosis) and loss of mitochondrial membrane potential. Conclusion Recombinant soluble TRAIL can be used as a therapy for cancer.

  10. RHAMNAZIN INHIBITS PROLIFERATION AND INDUCES APOPTOSIS OF HUMAN JURKAT LEUKEMIA CELLS IN VITRO.

    Science.gov (United States)

    Philchenkov, A A; Zavelevych, M P

    2015-01-01

    Antiproliferative and apoptogenic effects of rhamnazin, a dimethoxylated derivative of quercetin, were studied in human acute lymphoblastic leukemia Jurkat cells. The cytotoxicity and apoptogenic activity of rhamnazin in vitro are inferior to that of quercetin. The apoptogenic activity of rhamnazin is realized via mitochondrial pathway and associated with activation of caspase-9 and -3. The additive apoptogenic effect of rhamnazin and suboptimal doses of etoposide, a DNA topoisomerase II inhibitor, is demonstrated. Therefore, methylation of quercetin modifies its biological effects considerably.

  11. Reactive oxygen species-dependent necroptosis in Jurkat T cells induced by pathogenic free-living Naegleria fowleri.

    Science.gov (United States)

    Song, K-J; Jang, Y S; Lee, Y A; Kim, K A; Lee, S K; Shin, M H

    2011-07-01

    Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N. fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N. fowleri induces ROS-dependent necroptosis in Jurkat T cells.

  12. The associated regulators and signal pathway in rILl-16/CD4 mediated growth regulation in Jurkat cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    IL-16 is a ligand and chemotactic factor for CD4+ T cells. IL-16 inhibits the CD3 mediated lymphocyteactivation and proliferation. The effects of IL-16 on the target cells are dependent on the cell type, thepresence of co-activators etc. To understand the regulation function and mechanism of IL-16 on targetcells, we used a 130 a.a. recombinant IL-16 to study its effects on the growth of Jurkat T leukemia cellsin vitro. We found that the rIL-16 stimulated the proliferation of Jurkat cells at low dose (10-9M), butinhibited the growth of the cells at higher concentration (10-5M). Results showed that 10-5 M of rIL-16treatment induced an enhanced apoptosis in Jurkat cells. The treatment blocked the expression of FasL, butup-regulated the c-myc and Bid expression in the cells. Pre-treatment of PKC inhibitor or MEK1 inhibitormarkedly increased or decreased the rIL-16 induced growth-inhibiting effects on Jurkat cells, respectively.The results suggested that the rIL-16 might be a regulator for the growth or apoptosis of Jurkat cells ata dose-dependent manner. The growth-inhibiting effects of rIL-16 might be Fas/FasL independent, but,associated with the activation of PKC, up-regulated expression of c-Myc and Bid, and the participation ofthe ERK signal pathway in Jurkat cells.

  13. Pro-apoptotic effect of Persea americana var. Hass (avocado) on Jurkat lymphoblastic leukemia cells.

    Science.gov (United States)

    Bonilla-Porras, Angelica R; Salazar-Ospina, Andrea; Jimenez-Del-Rio, Marlene; Pereañez-Jimenez, Andres; Velez-Pardo, Carlos

    2013-11-05

    Abstract Context: Therapy for leukemia has a limited efficacy. There is a need to search for alternative anti-leukemia therapies. Persea americana Mill var. Hass (Lauraceae) is a tropical fruit (avocado) that might be used against cancer. Objective: To investigate whether P. americana induces death in Jurkat lymphoblastic leukemia cells. Materials and methods: Four ethanol extracts (0.1, 0.5, 1, 2 and 5 mg/mL) from avocado fruit (endocarp, whole seed, seed and leaves) were analyzed against Jurkat cells. Hydrogen peroxide generation by oxidation of 2',7'-dichlorodihydrofluorescein diacetate to the fluorescent compound 2',7'-dichlorfluorescein assay, acridine orange/ethidium bromide staining, flow cytometry analysis of annexin-V/7-amino-actinomycin, mitochondrial membrane potential and immunocytochemistry detection of transcription factor p53, caspase-3 and apoptosis-inducing factor (AIF) were evaluated. Results: Endocarp, seed, whole seed, and leaf (0.1 mg/mL) extracts induced significant apoptosis in Jurkat cells (p americana extracts function as a pro-apoptotic compound. Leukemic cells are eliminated through an oxidative stress mechanism. This study contributes to the understanding of the molecular mechanism of the avocado and its therapeutic action on leukemia.

  14. Constituents of French Marigold (Tagetes patula L. Flowers Protect Jurkat T-Cells against Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Irakli Chkhikvishvili

    2016-01-01

    Full Text Available The flowers of French marigold (Tagetes patula L. are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μM H2O2. Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10 in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers.

  15. n-Hexane toxicity in Jurkat T-cells is mediated by reactive oxygen species.

    Science.gov (United States)

    McDermott, Catherine; O'Donoghue, Maria Hutch; Heffron, James J A

    2008-03-01

    Here we assess the role of reactive oxygen species (ROS) formation in the manifestation of n-hexane toxicity in Jurkat T-cells and the chemo-protective potential of the antioxidants epigallocatechin-3-gallate (EGCG) and thymoquinone (TQ) against n-hexane toxicity in vitro. n-Hexane is an important industrial solvent and ambient air pollutant. Subchronic exposure to n-hexane results in a concentration-dependent increase in ROS formation with a corresponding decrease in Jurkat T-cell proliferation. Results from time-course studies indicate that ROS formation plays a causal role in n-hexane induced alterations in Jurkat T-cell proliferation and membrane integrity. Treatment of cells with EGCG, at a concentration reached in plasma, reduced the ROS formation caused by exposure to n-hexane and inhibited the decrease in cell proliferation. Similar effects were obtained with TQ. Both EGCG and TQ significantly reduced n-hexane-induced LDH leakage to control levels. The combined results show that oxidative stress plays a role in the development of n-hexane toxicity.

  16. Polarization imaging and classification of Jurkat T and Ramos B cells using a flow cytometer.

    Science.gov (United States)

    Feng, Yuanming; Zhang, Ning; Jacobs, Kenneth M; Jiang, Wenhuan; Yang, Li V; Li, Zhigang; Zhang, Jun; Lu, Jun Q; Hu, Xin-Hua

    2014-09-01

    Label-free and rapid classification of cells can have awide range of applications in biology. We report a robust method of polarization diffraction imaging flow cytometry (p-DIFC) for achieving this goal. Coherently scattered light signals are acquired from single cells excited by a polarized laser beam in the form of two cross-polarized diffraction images. Image texture and intensity parameters are extracted with a gray level co-occurrence matrix (GLCM) algorithm to obtain an optimized set of feature parameters as the morphological "fingerprints" for automated cell classification. We selected the Jurkat T cells and Ramos B cells to test the p-DIFC method's capacity for cell classification. After detailed statistical analysis, we found that the optimized feature vectors yield accuracies of classification between the Jurkat and Ramos ranging from 97.8% to 100% among different cell data sets. Confocal imaging and three-dimensional reconstruction were applied to gain insights on the ability of p-DIFC method for classifying the two cell lines of highly similar morphology. Based on these results we conclude that the p-DIFC method has the capacity to discriminate cells of high similarity in their morphology with "fingerprints" features extracted from the diffraction images, which may be attributed to subtle but statistically significant differences in the nucleus-to-cell volume ratio in the case of Jurkat and Ramos cells.

  17. Exosomes derived from renal cancer cells induce Jurkat T cell apoptosis in vitro%肾癌细胞来源的exosomes诱导Jurkat T细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    杨林; 吴小候; 罗春丽; 王丹; 陈力学

    2013-01-01

    Objective To investigate the underlying mechanism of exosomes derived from renal cancer cell lines 786-0 to mediate tumor immune escape in vitro. Methods CCK-8 assay was used to determine the effects of exosomes on proliferation in Jurkat T cells. Morphological changes were by wright-giemsa staining;flow cytometry with Annexin V-FITC/PI double staining was used to detect the apoptosis; secretion functions of Jurkat T cell were detected by ELISA assay; effects of exosomes on apoptosis of Jurkat T cell were detected by soluble Fas block experiment; effects on the protein expression of FasL, caspase, Bax and Bcl-2 were assessed by Western blot analysis. Results Exosomes could inhibit Jurkat T cell proliferation, 10 μg/mL exosomes act on Jurkat T cell for 24 and 72 h, growth inhibition rate was (19. 64 ±0. 92)% and (36. 24 ± 1. 12)% ; while 400 μg/mL exosomes act on it for 24 h and 72 h, growth inhibition rate was (55.96 ± 1.35)% and (76.51 ± 1. 37)% respectively. Exosomes could induce Jurkat T cell apoptosis, 10 μg/mL exosomes act on Jurkat T cell for 8 h, apoptosis rate was (7. 31 ±1.32)% , extending this monitoring to 24 h, apoptosis rate was (20. 19 ± 1.47)% ; while 400μg/mL exosomes act on it for 8 and 24 h, apoptosis rate was (27. 28 ± 1. 29)% and (41.72 ±0.88)% respectively. Exosomes also suppressed IL-2, IFN-γ, IL-6 and IL-10 secretion obviously. FasL was highly expressed in exosomes, soluble Fas block could reverse Jurkat T cell apoptosis. In this course, caspase-3 , caspase-8, caspase-9 were activated, and the ratio of Bax/Bcl-2 increased. Conclusion Exosomes could inhibit the growth of Jurkat T cell and induce apoptosis. It could mediate tumor immune escape.%目的 体外研究肾癌786-0细胞来源的exosomes介导肿瘤免疫逃逸的机制.方法 采用CCK-8法检测肾癌786-0细胞来源的exosomes对Jurkat T细胞生长的影响,瑞氏-姬姆萨染色检测Jurkat T细胞形态变化,Annexin V-FITC/PI双染色流式细胞术检测Jurkat T细胞凋亡率,ELISA法检测Jurkat

  18. Down-regulation of β-catenin Nuclear Localization by Aspirin Correlates with Growth Inhibition of Jurkat Cell Line

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    In this study, we examined the effects of aspirin on the growth rates, subcellar distribution of β-catenin protein, the expression of β-catenin/TCF signaling pathway target gene cyclinD1 mRNA,and cell cycle of Jurkat cell line (Human T-acute lymphoblastic leukemia). Our results showed that the treatment with aspirin inhibited the growth of Jurkat cell line. Jurkat cells treated with 3 mmol/L of aspirin could significantly decrease nuclear localization of β-catenin, and at 5 mmol/L of aspirin,the nuclear localization of β-catenin was undetectable. QRT-PCR showed that the target gene cyclinD1 mRNA expression was gradually decreased with the dosage of aspirin. Aspirin induced G0/G1cell cycle arrest in Jurkat cells. We are led to conclude that aspirin acts through β-catenin-independent mechanisms. The effects of aspirin include down-regulation of β-catenin nuclear localization and G0/G1 cell cycle arrest, which might serve as a means of growth inhibition in aspirin-treated human Jurkat cell line.

  19. Amoebic PI3K and PKC is required for Jurkat T cell death induced by Entamoeba histolytica.

    Science.gov (United States)

    Lee, Young Ah; Kim, Kyeong Ah; Min, Arim; Shin, Myeong Heon

    2014-08-01

    The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.

  20. Effect of Cyclosporine A on the expression of GSTO2 metabolizing enzyme in Jurkat cell line

    Directory of Open Access Journals (Sweden)

    Nioosha Nekooie-Marnany

    2012-01-01

    Full Text Available Cyclosporine A (CsA, a cyclic polypeptide metabolite extracted from the fungus, is used clinically to combat organ graft rejection in transplant subjects. Previous studies have shown that CsA exposure enhances the production of reactive oxygen species (ROS and lipid peroxidation, which are directly involved in CsA toxicity. To protect cells and organs against ROS, the human body has evolved a highly antioxidant protection system to neutralize free radicals. The aim of this study was to investigate the effect of CsA on mRNA expression of anti-oxidant GSTO2. To do this, Jurkat cells were incubated for 24 h with different doses of CsA, ranging from 1-80 µg/ml, and the IC50 of CsA was calculated to be 40 µg/ml. Subsequently, Jurkat cells were treated with 3 µg/ml CsA for 24 h and the gene expression of GSTO2 was quantified by quantitative Real-time PCR. Results showed that the mean (SD expression of the GSTO2 gene in CsA treated cells was 1.10 (0.07 (when assuming an expression level in untreated cells of 1.0. However, statistical analyses showed that the alterations were not significant (t=2.29, df=2, P=0.149. These findings suggest that at this concentration of CsA, other antioxidant enzymes are up-regulated in Jurkat cell lines to detoxify free radicals induced by CsA.

  1. Different effects of two cyclic chalcone analogues on redox status of Jurkat T cells.

    Science.gov (United States)

    Rozmer, Zsuzsanna; Berki, Tímea; Maász, Gábor; Perjési, Pál

    2014-12-01

    Chalcones are intermediary compounds of the biosynthetic pathway of the naturally flavonoids. Previous studies have demonstrated that chalcones and their conformationally rigid cyclic analogues have tumour cell cytotoxic and chemopreventive effects. It has been shown that equitoxic doses of the two cyclic chalcone analogues (E)-2-(4'-methoxybenzylidene)-(2) and (E)-2-(4'-methylbenzylidene)-1-benzosuberone (3) have different effect on cell cycle progress of the investigated Jurkat cells. It was also found that the compounds affect the cellular thiol status of the treated cells and show intrinsic (non-enzyme-catalyzed) reactivity towards GSH under cell-free conditions. In order to gain new insights into the cytotoxic mechanism of the compounds, effects on the redox status and glutathione level of Jurkat cells were investigated. Detection of intracellular ROS level in Jurkat cells exposed to 2 and 3 was performed using the dichlorofluorescein-assay. Compound 2 did not influence ROS activity either on 1 or 4h exposure; in contrast, chalcone 3 showed to reduce ROS level at both timepoints. The two compounds had different effects on cellular glutathione status as well. Compound 2 significantly increased the oxidized glutathione (GSSG) level showing an interference with the cellular antioxidant defence. On the contrary, chalcone 3 enhanced the reduced glutathione level, indicating enhanced cellular antioxidant activity. To investigate the chalcone-GSH conjugation reactions under cellular conditions, a combination of a RP-HPLC method with electrospray ionization mass spectrometry (ESI-MS) was performed. Chalcone-GSH adducts could not be observed either in the cell supernatant or the cell sediment after deproteinization. The investigations provide further details of dual - cytotoxic and chemopreventive - effects of the cyclic chalcone analogues.

  2. 金雀异黄素诱导人白血病Jurkat E6-1细胞凋亡作用%Induction of apoptosis by genistein in human leukemia cell line Jurkat E6-1

    Institute of Scientific and Technical Information of China (English)

    刘素芳; 何坚; 杨静静; 李万里

    2011-01-01

    Objective To investigate the effect and its mechanism of genistein (Gen) on human leukemia cell line Jurkat E6-1. Methods Jurkat E6-1 cells were treated with different concentrations of Gen. Inhibitive effect of Gen on cell growth was determined with methy thiazoly tetazolium(MTT) test. DNA-ladder was used to measure the effect of Gen on apoptosis of Jurkat E6-1 cells. Alternative of bcl-2 and bax genes were detected with reverse transcription polymerase chain reaction(RT-PCR). Results At a concentration of higher than 0. 5 μmol / L,Gen could inhibit the growth of Jurkat E6-1 cells. After 24 hours, the inhibition ratio was 5.9% compared with that of the control group, with a significant difference (P < 0.01 ) in a time-does-dependent manner( P < 0. 01 ). After 72 hours, the inhibition ratio was 24.9%, with a significant difference compared to that of at 24 hours(P <0. 01 ). The expression rate of bcl-2 decreased after the treatment with the increasing concentration of Gen. However, the expression rate of bax increased after the treatment with the decreased concentrations of Gen. Conclusion Gen can significantly inhibit the growth of human leukemia line Jurkat E6-1. Its mechanism is up-regulation of the bax expression and down-regulation of the bcl-2 expression.%目的 探讨金雀异黄素(genistein,Gen)诱导人白血病Jurkat E6-1细胞凋亡机制.方法 以不同浓度Gem作用于Jurkat E6-1细胞,采用四甲基偶氮噻唑蓝(MTT)法检测Gen对Jurkat E6-1细胞增殖抑制作用;采用DNA-ladder检测Gen对细胞凋亡影响;采用实时定量PCR(RT-PCR)检测凋亡相关基因bax、bcl-2表达水平.结果与对照组比较,P>0.5 μmol/L浓度的Gen明显抑制Jurkat E6-1细胞增殖,培养24 h,10 μmoL/L Gen组抑制率为5.9%,与对照组比较,差异有统计学意义(P<0.01),随Gen浓度增加和培养时间延长,抑制作用逐渐增强,72 h后,10μmol/L Gen组抑制率为24.9%;Gen使Jurkat E6-1细胞Bax表达上调,Bcl-2表达下调,

  3. Rhamnazin inhibits proliferation and induces apoptosis of human jurkat leukemia cells in vitro

    Directory of Open Access Journals (Sweden)

    А. А. Philchenkov

    2015-12-01

    Full Text Available Antiproliferative and apoptogenic effects of rhamnazin, a dimethoxylated derivative of quercetin, were studied in human acute lymphoblastic leukemia Jurkat cells. The cytotoxicity and apoptogenic activity of rhamnazin in vitro are inferior to that of quercetin. The apoptogenic activity of rhamnazin is realized via mitochondrial pathway and associated with activation of caspase-9 and -3. The additive apoptogenic effect of rhamnazin and suboptimal doses of etoposide, a DNA topoisomerase II inhibitor, is demonstrated. Therefore, methylation of quercetin modifies its biological effects considerably.

  4. Detection of the apoptosis of Jurkat cell using an electrorotation chip

    Institute of Scientific and Technical Information of China (English)

    Long Quan; Xing Wanli

    2006-01-01

    The apoptosis of cells is one of the fields that attract increasing attention in biology today.Usually,the cells are treated with chemicals when detecting apoptosis.It is highly desired to detect apoptosis in a real-time basis.Apoptosis of Jurkat cells was studied using a real-time electrorotation chip.This chip allows the detection of the cell membrane capacitance changes during the course of apoptosis and therefore facilitates the analysis of apoptosis in a real-time basis without involving any chemical treatment.

  5. Fenugreek extract as an inducer of cellular death via autophagy in human T lymphoma Jurkat cells

    Directory of Open Access Journals (Sweden)

    Al-Daghri Nasser M

    2012-10-01

    Full Text Available Abstract Background Drugs used both in classical chemotherapy and the more recent targeted therapy do not have cancer cell specificity and, hence, cause severe systemic side effects. Tumors also develop resistance to such drugs due to heterogeneity of cell types and clonal selection. Several traditional dietary ingredients from plants, on the other hand, have been shown to act on multiple targets/pathways, and may overcome drug resistance. The dietary agents are safe and readily available. However, application of plant components for cancer treatment/prevention requires better understanding of anticancer functions and elucidation of their mechanisms of action. The current study focuses on the anticancer properties of fenugreek, a herb with proven anti-diabetic, antitumor and immune-stimulating functions. Method Jurkat cells were incubated with 30 to 1500 μg/mL concentrations of 50% ethanolic extract of dry fenugreek seeds and were followed for changes in viability (trypan blue assay, morphology (microscopic examination and autophagic marker LC3 transcript level (RT-PCR. Results Incubation of Jurkat cells with fenugreek extract at concentrations ranging from 30 to 1500 μg/mL for up to 3 days resulted in cell death in a dose- and time-dependent manner. Jurkat cell death was preceded by the appearance of multiple large vacuoles, which coincided with transcriptional up-regulation of LC3. GC-MS analysis of fenugreek extract indicated the presence of several compounds with anticancer properties, including gingerol (4.82%, cedrene (2.91%, zingerone (16.5%, vanillin (1.52% and eugenol (1.25%. Conclusions Distinct morphological changes involving appearance of large vacuoles, membrane disintegration and increased expression of LC3 transcripts indicated that fenugreek extract induced autophagy and autophagy-associated death of Jurkat cells. In addition to the already known apoptotic activation, induction of autophagy may be an additional mechanism

  6. Molecular regulation of MICA expression after HDAC-inhibitor treatment of Jurkat T cells

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Pedersen, Marianne T.

    pathways that lead to MICA expression after HDAC-inhibitor treatment of Jurkat T cells. Chelating Calcium with Bapta-AM or EGTA potently inhibited HDAC-inhibitor mediated MICA/B expression. It was further observed that ER Calcium stores were depleted after HDAC-inhibitor treatment. NF-kB activity can......, we made a promoter construct consisting of ~3kb of the proximal MICA promoter in front of GFP. Deletion analysis showed that a GC-box containing a putative Sp1 site from position -113 to -93 relative to the mRNA start site, was important for HDAC-inhibitor induced promoter activity. Sp1...

  7. A comprehensive characterization of the impact of mycophenolic acid on the metabolism of Jurkat T cells.

    Science.gov (United States)

    Fernández-Ramos, Ana A; Marchetti-Laurent, Catherine; Poindessous, Virginie; Antonio, Samantha; Petitgas, Céline; Ceballos-Picot, Irène; Laurent-Puig, Pierre; Bortoli, Sylvie; Loriot, Marie-Anne; Pallet, Nicolas

    2017-09-05

    Metabolic reprogramming is critical for T cell fate and polarization and is regulated by metabolic checkpoints, including Myc, HIF-1α, AMPK and mTORC1. Our objective was to determine the impact of mycophenolic acid (MPA) in comparison with rapamycin (Rapa), an inhibitor of mTORC1, on the metabolism of Jurkat T cells. We identified a drug-specific transcriptome signature consisting of the key enzymes and transporters involved in glycolysis, glutaminolysis or nucleotide synthesis. MPA produced an early and transient drop in the intracellular ATP content related to the inhibition of de novo synthesis of purines, leading to the activation of the energy sensor AMPK. MPA decreases glycolytic flux, consistent with a reduction in glucose uptake, but also in the oxidation of glutamine. Additionally, both drugs reduce aerobic glycolysis. The expression of HIF-1α and Myc, promoting the activation of glycolysis and glutaminolysis, was inhibited by MPA and Rapa. In conclusion, we report that MPA profoundly impacts the cellular metabolism of Jurkat T cells by generating an energetic distress, decreasing the glycolytic and glutaminolytic fluxes and by targeting HIF-1α and Myc. These findings open interesting perspectives for novel combinatorial therapeutic strategies targeting metabolic checkpoints to block the proliferation of T cells.

  8. Effect of polyamine analogues on hypusine content in JURKAT T-cells.

    Science.gov (United States)

    Bergeron, R J; Weimar, W R; Müller, R; Zimmerman, C O; McCosar, B H; Yao, H; Smith, R E

    1998-09-24

    The availability of synthetic hypusine and deoxyhypusine has made it possible to develop analytical methods which allow for the measurement of these compounds in various tissues. The methods involve dansylation of extracts from the pellet remaining after perchloric acid precipitation of cell or tissue homogenates, followed by high-performance liquid chromatography. To demonstrate the utility of this approach, the impact of four polyamine analogues, N1,N11-diethylnorspermine (DENSPM), N1,N14-diethylhomospermine (DEHSPM), 1,6,12-triazadodecane [(4,5) triamine], and 1,7, 13-triazatridecane [(5,5) triamine], on hypusine levels in a human T-cell line (JURKAT) is evaluated. All four analogues are active in controlling cell growth and compete well with spermidine for the polyamine transport apparatus. After 144 h of exposure to JURKAT cells, DENSPM reduces putrescine to below detectable limits and spermidine to 10% of the level in control cells. The other three analogues diminish both putrescine and spermidine to below detectable limits. The effectiveness with which the compounds lower spermine levels is DENSPM > DEHSPM > (4,5) triamine > (5,5) triamine. The analogues decrease the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase in a similar fashion. Of the four polyamines, DENSPM and DEHSPM are potent at lowering intracellular hypusine levels after 144 h: 59 +/- 9% and 73 +/- 12% of control levels, respectively. The other two analogues have marginal effects.

  9. Caspase-resistant vimentin suppresses apoptosis after photodynamic treatment with a silicon phthalocyanine in Jurkat cells.

    Science.gov (United States)

    Belichenko, I; Morishima, N; Separovic, D

    2001-06-01

    Oxidative stress, such as photodynamic therapy, is an apoptosis inducer. Apoptosis, as well as photosensitization, have been associated with disruption of the cytoskeletal network. The purpose of the present study was to assess the role of vimentin, a major cytoskeletal protein, in apoptosis after photodynamic treatment (PDT) with the silicon phthalocyanine Pc 4 in human Jurkat T cells. Here we show for the first time that photosensitization with Pc 4 initiates vimentin cleavage and that this event precedes poly(ADP-ribose) polymerase (PARP) degradation. Similar findings were obtained in the presence of C2-ceramide, an inducer of oxidative stress and apoptosis. In the presence of benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, a pan-caspase inhibitor, Pc 4-PDT-induced vimentin and PARP cleavage were abolished. In Jurkat cells transfected with a caspase-resistant vimentin apoptosis was partly suppressed and delayed post-Pc 4-PDT. We suggest that the full-length vimentin confers resistance to nuclear apoptosis after PDT with Pc 4.

  10. Probing mechanical properties of Jurkat cells under the effect of ART using oscillating optical tweezers.

    Directory of Open Access Journals (Sweden)

    Samaneh Khakshour

    Full Text Available Acute lymphoid leukemia is a common type of blood cancer and chemotherapy is the initial treatment of choice. Quantifying the effect of a chemotherapeutic drug at the cellular level plays an important role in the process of the treatment. In this study, an oscillating optical tweezer was employed to characterize the frequency-dependent mechanical properties of Jurkat cells exposed to the chemotherapeutic agent, artesunate (ART. A motion equation for a bead bound to a cell was applied to describe the mechanical characteristics of the cell cytoskeleton. By comparing between the modeling results and experimental results from the optical tweezer, the stiffness and viscosity of the Jurkat cells before and after the ART treatment were obtained. The results demonstrate a weak power-law dependency of cell stiffness with frequency. Furthermore, the stiffness and viscosity were increased after the treatment. Therefore, the cytoskeleton cell stiffness as the well as power-law coefficient can provide a useful insight into the chemo-mechanical relationship of drug treated cancer cells and may serve as another tool for evaluating therapeutic performance quantitatively.

  11. Development and application of a simultaneous SPE-method for polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, heterocyclic PAHs (NSO-HET) and phenols in aqueous samples from German Rivers and the North Sea.

    Science.gov (United States)

    Siemers, Anne-Kathrin; Mänz, Jan Sebastian; Palm, Wolf-Ulrich; Ruck, Wolfgang K L

    2015-03-01

    Polycyclic aromatic hydrocarbons (PAHs), heterocyclic PAHs (NSO-HETs), alkylated PAHs and phenols are known as the prevailing contaminants in groundwater at tar contaminated sites. Besides these local sources, the concentrations and the distribution in particular of NSO-HETs in environmental samples, such as rivers, have received notably less attention. To investigate their occurrence in river basins two sensitive analytical methods for the simultaneous extraction of 86 substances including NSO-HETs, classical EPA-PAHs, alkylated PAHs and phenols were developed: liquid-liquid extraction for the whole water phase and solid phase extraction for the dissolved water phase only. Solely GC-MS or additionally LC-MSMS for fractionated basic nitrogen heterocycles (N-HETs) were used for quantification. Limits of quantification were in the low ngL(-1) range. Concentrations were determined in 29 aqueous samples from 8 relatively large rivers located in Lower Saxony (Germany) and the North Sea. NSO-HETs had comparable or even higher sum concentrations than EPA-PAHs. N-HETs, especially acridine and quinolines with concentrations of up to 20ngL(-1) per substance, were predominant.

  12. N-(1-Pyrenyl Maleimide Induces Bak Oligomerization and Mitochondrial Dysfunction in Jurkat Cells

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    Pei-Rong Huang

    2015-01-01

    Full Text Available N-(1-pyrenyl maleimide (NPM is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm, and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

  13. Butterfat fatty acids differentially regulate growth and differentiation in Jurkat T-cells.

    Science.gov (United States)

    Bergamo, Paolo; Luongo, Diomira; Maurano, Francesco; Rossi, Mauro

    2005-10-01

    Synthetic Conjugated Linoleic Acid mixture (CLA; c9,t11; t10,c12-18:2) has been previously shown to inhibit growth, and enhance apoptosis and IL-2 mRNA synthesis in human lymphoblastic Jurkat T-cells. In this study, two different butterfat types were evaluated and compared for their effects on Jurkat cell viability, oxidative stress, pro-apoptotic activity, and cytokine synthesis: the conventionally produced butterfat (CBF), and organic butterfat (OBF) containing significantly higher amounts of c9,t11 (Rumenic Acid, RA), trans-vaccenic acid (VA; t11-18:1), alpha-linolenic acid (ALA), and lower levels of linoleic acid (LA). Results from cell treatment with both butterfat mixtures showed comparable oxidative stress (superoxide production, intracellular GSH depletion,and lipid peroxides yield), NADPH oxidase activation, cytotoxicity (LDH release), and IL-2 transcript level, whereas the effects of enhanced growth-inhibitory and pro-apoptotic activities were associated with OBF treatment. To then investigate each butterfat-induced effect caused by RA, VA, LA, and ALA, cells were exposed to synthetic FA concentrations similar to those from the different butterfats. Higher oxidative stress (superoxide production, intracellular GSH depletion) was induced by alpha-linolenic (ALA) and linoleic (LA) incubation (P<0.01) and superoxide production was suppressed by specific PKCalpha inhibitor (Gö 6976) and linked to increased toxicity and IL-2 synthesis inhibition. By contrast, cell treatment with RA increased apoptosis and IL-2 synthesis. These results suggest that a supply of ALA and LA is responsible for BF-induced oxidative stress via PKCalpha-NADPH oxidase pathway, and that enhanced antiproliferative effects in OBF treated cells is essentially determined by RA-induced pro-apoptotic activity. Copyright (c) 2005 Wiley-Liss, Inc.

  14. Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and Jurkat cells

    Science.gov (United States)

    Mahlke, Megan A.; Navara, Christopher; Ibey, Bennett L.

    2014-03-01

    Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to

  15. Cyclophosphamide Perturbs Cytosine Methylation in Jurkat-T Cells through LSD1-mediated Stabilization of DNMT1 Protein

    OpenAIRE

    ZHANG Jing; Yuan, Bifeng; Zhang, Fan; Xiong, Lei; Wu, Jiang; Pradhan, Sriharsa; Wang, Yinsheng

    2011-01-01

    Aberrant cytosine methylation is known to be associated with cancer development. Here we assessed how common cancer chemotherapeutic agents perturb cytosine methylation in Jurkat-T acute lymphoblastic leukemia cells. We tested six anti-tumor agents and found that cyclophosphamide induced the most pronounced increase in global DNA cytosine methylation after a 24-hr treatment. Long-term treatment with cyclophosphamide led to a time-dependent increase in cytosine methylation level with up to 4 d...

  16. Effects of in vitro Brevetoxin Exposure on Apoptosis and Cellular Metabolism in a Leukemic T Cell Line (Jurkat

    Directory of Open Access Journals (Sweden)

    John W. Sleasman

    2008-06-01

    Full Text Available Harmful algal blooms (HABs of the toxic dinoflagellate, Karenia brevis, produce red tide toxins, or brevetoxins. Significant health effects associated with red tide toxin exposure have been reported in sea life and in humans, with brevetoxins documented within immune cells from many species. The objective of this research was to investigate potential immunotoxic effects of brevetoxins using a leukemic T cell line (Jurkat as an in vitro model system. Viability, cell proliferation, and apoptosis assays were conducted using brevetoxin congeners PbTx-2, PbTx-3, and PbTx-6. The effects of in vitro brevetoxin exposure on cell viability and cellular metabolism or proliferation were determined using trypan blue and MTT (1-(4,5-dimethylthiazol-2-yl-3,5- diphenylformazan, respectively. Using MTT, cellular metabolic activity was decreased in Jurkat cells exposed to 5 - 10 μg/ml PbTx-2 or PbTx-6. After 3 h, no significant effects on cell viability were observed with any toxin congener in concentrations up to 10 μg/ml. Viability decreased dramatically after 24 h in cells treated with PbTx-2 or -6. Apoptosis, as measured by caspase-3 activity, was significantly increased in cells exposed to PbTx-2 or PbTx-6. In summary, brevetoxin congeners varied in effects on Jurkat cells, with PbTx-2 and PbTx-6 eliciting greater cellular effects compared to PbTx-3.

  17. MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

    Science.gov (United States)

    Mukherjee, P; Tinder, T L; Basu, G D; Gendler, S J

    2005-01-01

    MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

  18. Reduced scytonemin isolated from Nostoc commune induces autophagic cell death in human T-lymphoid cell line Jurkat cells.

    Science.gov (United States)

    Itoh, Tomohiro; Tsuzuki, Ryosuke; Tanaka, Toshiomi; Ninomiya, Masayuki; Yamaguchi, Yuji; Takenaka, Hiroyuki; Ando, Masashi; Tsukamasa, Yasuyuki; Koketsu, Mamoru

    2013-10-01

    Nostoc commune is a terrestrial benthic blue-green alga that often forms an extended mucilaginous layer on the soil, accumulates on stones and mud in aquatic environments. Reduced-scytonemin (R-scy), isolated from N. commune Vaucher, has been shown to suppress the human T-lymphoid Jurkat cell growth. To reveal the mechanisms underlying the R-scy-mediated inhibition of Jurkat cell growth, we examined cell morphology, DNA fragmentation, and microtubule-associated protein light chain 3 (LC3) modification in these cells. We observed multiple vacuoles as well as the conversion of LC3-I to LC3-II in R-scy-treated cells. These results suggest that the R-scy induced Jurkat cell growth inhibition is attributable to the induction of type II programmed cell death (PCD II; autophagic cell death or autophagy). We further examined the mechanisms underlying R-scy-induced PCDII. The cells treated with R-scy produced large amounts of reactive oxygen species (ROS), leading to the induction of mitochondrial dysfunction. However, the elimination of R-scy-induced ROS by treatment with N-acetyl-L-cysteine (NAC) markedly opposed R-scy-induced PCDII. Based on these results, we conclude that ROS formation plays a critical role in R-scy-induced PCDII. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Specific Inhibition of CD147 Gene Expression in Jurkat T Lymphocytes Decreased Colony Forming Capability%抑制CD147表达对Jurkat T淋巴细胞集落形成的影响

    Institute of Scientific and Technical Information of China (English)

    魏冬; 张俊峰; 王汉伟; 郭竹英; 徐芒华; 高丰厚

    2011-01-01

    Objective To investigate the effect of inhibiting CD147 gene expression by small interfering RNA(siRNA) in Jurkat T lymphocytes on the colony forming capability. Methods Specific siRNA targeting CD147 gene was transfected into Jurkat cells by LipofectimineTM 2000. The changes of CD147 mRNA level were determined by semi-quantitative RT-PCR,and total proteins level and cell surface protein were respectively detected by Western blot and flow cytometry at 48 h after transfection. By fluorescence inverted microscope,the changes of colony forming in Jurkat cells between the specificly inhibit ed CD147 group and the normal group were compared. Results Compared with control group,the CD147 mRNA and protein level in siRNA interference group were reduced by( 38.57 ± 1.55 ) % and (47.4 ± 1.47 ) % respectively ,and the mean fluorescence intensity of CD147 in Jurkat cells was descended by(50.5 ±4.7) %. Along with the inhibition of CD147, the colony forming of siRNA interference group obviously reduced(P <0.05 ). Conclusion siRNA can specifically inhibit the expression of CD147 in Jurkat cells,which can decrease the colony forming of the Jurkat cells, indicating that CD147 may potentially facilitate inflammatory cell adhesion and aggregation.%目的 研究小干扰RNA抑制CD147基因对Jurkat T淋巴细胞集落形成的影响.方法 将CD147特异的siRNA,经LipofectamineTM2000转染Jurkat细胞48 h后,用半定量RT-PCR检测CD147 mRNA水平的变化,Western blot和流式细胞术(FCM)分别检测总蛋白和细胞表面蛋白水平的变化.通过荧光倒置显微镜比较特异性抑制CDl47表达后Jurkat细胞集落形成的变化.结果 与对照组相比siRNA干扰组细胞CD147 mRNA和蛋白水平的表达均降低,抑制率分别为(38.57±1.55)%和(47.4±1.47)%,细胞表面CD147的平均荧光强度(MFI)下降(50.5±4.7)%(P<0.05).随着CD147被抑制,siRNA干扰组细胞集落形成明显少于对照组(P<0.05).结论

  20. Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells.

    Science.gov (United States)

    Hage-Sleiman, Rouba; Hamze, Asmaa B; El-Hed, Aimée F; Attieh, Randa; Kozhaya, Lina; Kabbani, Sarah; Dbaibo, Ghassan

    2016-08-01

    Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase's translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation-where PKCθ plays a critical role.

  1. Inhibition of Kv1.3 Channels in Human Jurkat T Cells by Xanthohumol and Isoxanthohumol.

    Science.gov (United States)

    Gąsiorowska, Justyna; Teisseyre, Andrzej; Uryga, Anna; Michalak, Krystyna

    2015-08-01

    Using whole-cell patch-clamp technique, we investigated influence of selected compounds from groups of prenylated chalcones and flavonoids: xanthohumol and isoxanthohumol on the activity of Kv1.3 channels in human leukemic Jurkat T cells. Obtained results provide evidence that both examined compounds were inhibitors of Kv1.3 channels in these cells. The inhibitory effects occurred in a concentration-dependent manner. The estimated value of the half-blocking concentration (EC50) was about 3 μM for xanthohumol and about 7.8 μM for isoxanthohumol. The inhibition of Kv1.3 channels by examined compounds was not complete. Upon an application of the compounds at the maximal concentrations equal to 30 μM, the activity of Kv1.3 channels was inhibited to about 0.13 of the control value. The inhibitory effect was reversible. The application of xanthohumol and isoxanthohumol did not change the currents' activation and inactivation rate. These results may confirm our earlier hypothesis that the presence of a prenyl group in a molecule is a factor that facilitates the inhibition of Kv1.3 channels by compounds from the groups of flavonoids and chalcones. The inhibition of Kv1.3 channels might be involved in antiproliferative and proapoptotic effects of the compounds observed in cancer cell lines expressing these channels.

  2. Cell retention by encapsulation for the cultivation of Jurkat cells in fixed and fluidized bed reactors.

    Science.gov (United States)

    Kaiser, P; Werner, M; Jérôme, V; Hübner, H; Buchholz, R; Freitag, R

    2014-12-01

    Jurkat cells are accepted model cells for primary human T lymphocytes, for example, in medical research. Their growth to tissue-like cell densities (up to 100 × 10(6)  cells/mLcapsule ) in semi-permeable (molecular weight cut off cultivations, that is, under conditions where both encapsulated and non-encapsulated cells can be cultivated under otherwise identical conditions, showed that maximum specific growth rates were higher for the encapsulated than for the non-encapsulated cells. In the subsequent batch and repeated batch bioreactor experiments (only encapsulated cells), growth rates were similar, with the exception of the fixed bed batch reactor, where growth kinetics were significantly slower. Concomitantly, a significant fraction of the cells towards the bottom of the bed were no longer metabolically active, though apparently not dead. In the repeated batch fluidized bed reactor cellular division could be maintained for more than two weeks, albeit with a specific growth rate below the maximum one, leading to final cell densities of approximately 180 × 10(6)  cell/gcapsule . At the same time, the cell cycle distribution of the cells was shifted to the S and G2/M phases.

  3. Differential induction of apoptosis by type A and B trichothecenes in Jurkat T-lymphocytes.

    Science.gov (United States)

    Nasri, Thanakarn; Bosch, Remko R; Voorde, Sandra ten; Fink-Gremmels, Johanna

    2006-09-01

    Several studies have shown that the mycotoxins T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON) and nivalenol (NIV) affect lymphocyte functioning. However, the molecular mechanisms underlying the immunomodulatory effects of these trichothecenes are not defined yet. In this study, the potency of the type A trichothecenes T-2 toxin and DAS, and the type B trichothecenes DON (and its metabolite de-epoxy-deoxynivalenol; DOM-1) and NIV to reduce mitochondrial activity and to induce apoptosis of Jurkat T cells (human T lymphocytes) were examined. T-2 toxin and DAS are much more cytotoxic at low concentrations than DON and NIV as shown by the AlamarBlue cytotoxicity assay. In addition, the mechanism whereby DON and NIV induced cytotoxicity is mainly via apoptosis as we observed phosphatidylserine externalization, mitochondrial release of cytochrome c, procaspase-3 degradation and Bcl-2 degradation. In contrast, type A trichothecenes reduce the mitochondrial activity at approximately 1000-fold lower concentrations than the type B trichothecenes, resulting in necrosis. These data suggest that the mechanisms resulting in cytotoxic effects are different for type A and type B trichothecenes.

  4. Gene expression profile of Jurkat cells exposed to high power terahertz radiation

    Science.gov (United States)

    Grundt, Jessica E.; Roth, Caleb C.; Rivest, Benjamin D.; Doroski, Michael L.; Payne, Jason; Ibey, Bennett L.; Wilmink, Gerald J.

    2011-03-01

    Terahertz (THz) radiation sources are now being used in a host of military, defense, and medical applications. Widespread employment of these applications has prompted concerns regarding the health effects associated with THz radiation. In this study, we examined the gene expression profile of mammalian cells exposed to THz radiation. We hypothesized that if THz radiation couples directly to cellular constituents, then exposed cells may express a specific gene expression profile indicative of ensuing damage. To test this hypothesis, Jurkat cells were irradiated with a molecular gas THz laser (2.52 THz, 636 mWcm-2, durations: 5, 10, 20, 30, 40, or 50 minutes). Viability was assessed 24 h post-exposure using MTT assays, and gene expression profiles were evaluated 4 h post-exposure using mRNA microarrays. Comparable analyses were also performed for hyperthermic positive controls (44°C for 40 minutes). We found that cellular temperatures increased by ~6 °C during THz exposures. We also found that cell death increased with exposure duration, and the median lethal dose (LD50) was calculated to be ~44 minutes. The microarray data showed that THz radiation induced the transcriptional activation of genes associated with cellular proliferation, differentiation, transcriptional activation, chaperone protein stabilization, and apoptosis. For most genes, we found that the magnitude of differential expression was comparable for both the THz and thermal exposure groups; however, several genes were specifically activated by the THz exposure. These results suggest that THz radiation may elicit effects that are not exclusively due to the temperature rise created during THz exposures (i.e. thermal effects). In future work, we plan to verify the results of our microarray experiments using qPCR techniques.

  5. Apoptosis induction in Jurkat cells and sCD95 levels in women's sera are related with the risk of developing cervical cancer

    Directory of Open Access Journals (Sweden)

    Bravo-Cuellar Alejandro

    2008-04-01

    Full Text Available Abstract Background Currently, there is clear evidence that apoptosis plays an important role in the development and progression of tumors. One of the best characterized apoptosis triggering systems is the CD95/Fas/APO-1 pathway; previous reports have demonstrated high levels of soluble CD95 (sCD95 in serum of patients with some types of cancer. Cervical cancer is the second most common cancer among women worldwide. As a first step in an attempt to design a minimally invasive test to predict the risk of developing cervical cancer in patients with precancerous lesions, we used a simple assay based on the capacity of human serum to induce apoptosis in Jurkat cells. We evaluated the relationship between sCD95 levels and the ability to induce apoptosis in Jurkat cells in cervical cancer patients and controls. Methods Jurkat cells were exposed to serum from 63 women (20 healthy volunteers, 21 with cervical intraepithelial neoplasia grade I [CIN 1] and 22 with cervical-uterine carcinoma. The apoptotic rate was measured by flow cytometry using Annexin-V-Fluos and Propidium Iodide as markers. Serum levels of sCD95 and soluble CD95 ligand (sCD95L were measured by ELISA kits. Results We found that serum from almost all healthy women induced apoptosis in Jurkat cells, while only fifty percent of the sera from women with CIN 1 induced cell death in Jurkat cells. Interestingly, only one serum sample from a patient with cervical-uterine cancer was able to induce apoptosis, the rest of the sera protected Jurkat cells from this killing. We were able to demonstrate that elimination of Jurkat cells was mediated by the CD95/Fas/Apo-1 apoptotic pathway. Furthermore, the serum levels of sCD95 measured by ELISA were significantly higher in women with cervical cancer. Conclusion Our results demonstrate that there is a strong correlation between low levels of sCD95 in serum of normal women and higher apoptosis induction in Jurkat cells. We suggest that an analysis of

  6. Bcl-2 over-expression and activation of protein kinase C suppress the Trail-induced apoptosis in Jurkat T cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Trail,a tumor necrosis factor-related apoptosis-inducing ligand,is a novel potent endogenous activator of the cell death pathway through the activation of cell surface death receptors Trail-R1 and Trail-R2.Its role,like FasL in activation-induced cell death(AICD),has been demonstrated in immune system.However the mechanism of Trail induced apoptosis remains unclear.In this report,the recombinant Trail protein was expressed and purified.The apoptosis-inducing activity and the regulation mechanism of recombinant Trail on Jurkat T cells were explored in vitro.Trypan blue exclusion assay demonstrated that the recombinant Trail protein actively killed Jurkat T cells in a dose-dependent manner.Trail-induced apoptosis in Jurkat T cells were remarkably reduced by Bcl-2 over expression in Bcl-2 gene transfected cells.Treatment with PMA(phorbol 12-myristate 13-acetate),a PKC activator,suppressed Trail-induced apoptosis in Jurkat T cells.The inhibition of apoptosis by PMA was abolished by pretreatment with Bis,a PKC inhibitor.Taken together,it was suggested that Bcl-2 over-expression and PMA activated PKC actively down-regulated the Trail-mediated apoptosis in Jurkat T cell.

  7. Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by downregulation of PI3K/AKT and upregulation of p38 MAPK pathways.

    Science.gov (United States)

    Yang, Tingfang; Yao, Shuluan; Zhang, Xianfeng; Guo, Yan

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) as a prevalent hematologic malignancy is one of the most common malignant tumors worldwide in children. Andrographolide (Andro), the major active component from Andrographis paniculata, has been shown to possess antitumor activities in several types of cancer cells. However, whether Andro would inhibit T-ALL cell growth remains unclear. In this study, we investigated the cytotoxic effect of Andro on human T-ALL Jurkat cells and explored the mechanisms of cell death. Cell apoptosis was assayed by flow cytometry, and the signaling transduction for Andro was analyzed by Western blotting. The results indicated 10 μg/mL Andro could significantly induce Jurkat cells' apoptosis, depending on the inhibition of PI3K/AKT pathway. Moreover, Andro-induced apoptosis is enhanced by AKT-selective inhibitor LY294002. ERK- or JNK-selective inhibitors PD98059 and SP600125 had no effect on Andro-induced apoptosis. In addition, p38 inhibitor SB203580 could reverse Andro-induced apoptosis in Jurkat cells. We also found that the protein expression of p-p53 and p-p38 were increased after Andro treatments. The result of an in vivo study also demonstrated Andro's dose-dependent inhibition in subcutaneous Jurkat xenografts. In conclusion, our findings explained a novel mechanism of drug action by Andro in Jurkat cells and suggested that Andro might be developed into a new candidate therapy for T-ALL patients in the coming days.

  8. Decreases in CD31 and CD47 levels on the cell surface during etoposide-induced Jurkat cell apoptosis.

    Science.gov (United States)

    Azuma, Yutaro; Nakagawa, Hideaki; Dote, Kanae; Higai, Koji; Matsumoto, Kojiro

    2011-01-01

    Engulfment of apoptotic cells is regulated by 'eat me' and 'don't eat me' signals on the cell surface. Alterations to the 'eat me' signals have been well described; however, very little is known about the 'don't eat me' signals on the cell surface during apoptosis. In the present study, apoptosis of Jurkat cells was induced by treatment with topoisomerase II inhibitor etoposide, and then the CD31 and CD47 levels on the apoptotic cell surface and in microparticles were estimated by flow cytometry and immunoblotting methods in the presence of caspase, metalloproteinase, and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitors. The CD31 and CD47 levels on the cell surface of apoptotic Jurkat cells had decreased after treatment with etoposide. These decreases in CD31 and CD47 levels on the apoptotic cell surface were almost completely suppressed by the caspase 3 inhibitor, Ac-DEVD-CHO, and partially suppressed by caspase 8 (Ac-IETD-CHO) and caspase 9 (Ac-LEHE-CHO) inhibitors but not by the metalloproteinase inhibitors GM6001 and TAPI-0. Microparticle counts in culture supernatants were higher during etoposide-induced apoptosis. The ROCK1 inhibitor, Y27632, suppressed blebbing formation and microparticle release. Moreover, flow cytometry and immunoblotting revealed CD31 and CD47 in the microparticles. These results indicate that CD31 and CD47 were released by the apoptotic Jurkat cells into the culture supernatant in microparticles, but not in soluble forms, resulting in decreased levels on the apoptotic cell surface.

  9. ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

    Science.gov (United States)

    Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

    2017-01-01

    The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

  10. Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP)

    Science.gov (United States)

    2016-05-02

    77 (43%) genes changing due to nsEP exposure. For the Jurkat cells, the top canonical pathway was the “ Hepatic Fibrosis / Hepatic Stellate Cell...150kv nsEP vs. sham Hs.446125 male germ cell-associated kinase MAK 4.483 0.00081 Hs.155111 hepatitis A virus cellular receptor 2 HAVCR2 4.453 0.00479...Symbol Fold change 150kVnsEP vs. sham p-Value 150kv nsEP vs. sham Hs.25647 v-fos FBJ murine osteosarcoma viral oncogene homolog FOS 7.269 0.00031 Hs

  11. Arecoline inhibits interleukin-2 secretion in Jurkat cells by decreasing the expression of alpha7-nicotinic acetylcholine receptors and prostaglandin E2.

    Science.gov (United States)

    Hwang, G S; Hu, S; Lin, Y H; Chen, S T; Tang, T K; Wang, P S; Wang, S W

    2013-10-01

    The purpose of the present study was to explore the effect of arecoline on phytohemagglutinin (PHA)-stimulated interleukin-2 (IL-2) secretion, the expression of alpha7-nicotinic acetylcholine receptors (α7-nAChRs), prostaglandin E2(PGE2) protein, and IL-2 mRNA in human lymphocyte cells (Jurkat cell line). The IL-2 and PGE2 were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of phosphorylated extracellular signal-regulated kinase (ERK) and α7-nAChRs were determined by Western blotting. The level of IL-2 mRNA was determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Arecoline, in a dose-dependent manner, significantly decreased IL-2 and PGE2 secretion by Jurkat cells incubated with 0 or 5 μg/ml 5 μg/ml PHA. PGE2 also significantly inhibited IL-2 secretion by Jurkat cells in a dose-dependent manner. In addition, reduced expression of PHA-induced ERK phosphorylation was observed in Jurkat cells treated with arecoline. PHA-enhanced IL-2 mRNA expression was also inhibited by arecoline. These results imply that arecoline inhibits the release of PGE2 and PHA-induced IL-2 secretion by Jurkat cells and that these effects seem to occur, at least in part, either through the attenuation of ERK in conjunction with a decrease of PHA-induced IL-2 mRNA expression. These results imply that arecoline inhibits the protein expression of α7-nAChRs , the release of PGE2 and PHA-induced IL-2 secretion by Jurkat cells.

  12. Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by downregulation of PI3K/AKT and upregulation of p38 MAPK pathways

    Directory of Open Access Journals (Sweden)

    Yang T

    2016-04-01

    Full Text Available Tingfang Yang,1 Shuluan Yao,2 Xianfeng Zhang,3 Yan Guo2 1Department of Pediatrics, Jining No 1 People’s Hospital, Shandong Province, People’s Republic of China; 2Department of Respiratory Medicine, Jining Medical University Affiliated Hospital, Shandong Province, People’s Republic of China; 3Department of Psychiatry, Jining Psychiatric Hospital, Shandong Province, People’s Republic of China Abstract: T-cell acute lymphoblastic leukemia (T-ALL as a prevalent hematologic malignancy is one of the most common malignant tumors worldwide in children. Andrographolide (Andro, the major active component from Andrographis paniculata, has been shown to possess antitumor activities in several types of cancer cells. However, whether Andro would inhibit T-ALL cell growth remains unclear. In this study, we investigated the cytotoxic effect of Andro on human T-ALL Jurkat cells and explored the mechanisms of cell death. Cell apoptosis was assayed by flow cytometry, and the signaling transduction for Andro was analyzed by Western blotting. The results indicated 10 µg/mL Andro could significantly induce Jurkat cells’ apoptosis, depending on the inhibition of PI3K/AKT pathway. Moreover, Andro-induced apoptosis is enhanced by AKT-selective inhibitor LY294002. ERK- or JNK-selective inhibitors PD98059 and SP600125 had no effect on Andro-induced apoptosis. In addition, p38 inhibitor SB203580 could reverse Andro-induced apoptosis in Jurkat cells. We also found that the protein expression of p-p53 and p-p38 were increased after Andro treatments. The result of an in vivo study also demonstrated Andro’s dose-dependent inhibition in subcutaneous Jurkat xenografts. In conclusion, our findings explained a novel mechanism of drug action by Andro in Jurkat cells and suggested that Andro might be developed into a new candidate therapy for T-ALL patients in the coming days. Keywords: andrographolide, PI3K, AKT, Burkitt lymphoma, Jurkat cell

  13. Coumestrol, Bisphenol-A, DDT, and TCDD Modulation of Interleukin-2 Expression in Activated CD+4 Jurkat T Cells

    Directory of Open Access Journals (Sweden)

    Robert W. McMurray

    2004-02-01

    Full Text Available Endogenous estrogens are known to modulate several components of immune response, including interleukin-2 (IL-2 production. IL-2 is a cytokine that plays an important role in adaptive immune responses. These responses may be modulated by xenoestrogens such as coumestrol, bisphenol A (BPA, DDT, and TCDD. In this research, we examined the effects and potential mechanisms of action of these estrogenic compounds on IL-2 production in activated CD4+ Jurkat T cells. IL-2 production was analyzed by ELISA and Western Blot. At the transcriptional level, protein expression was examined by RT-PCR. Coumestrol, DDT and TCDD (but not BPA significantly suppressed IL-2 production in activated CD4+ Jurkat T cells, at the transcriptional and translational levels. The transcriptional suppression of IL-2 was associated with decreased protein levels of NF-κβ, an important IL-2 positive transcription factor, without affecting the expression of Iκ−Βα protein expression, an important inhibitor of NF-κβ nuclear translocation. Although the direct mechanisms of xenoestrogens modulation of the immune system remain to be elucidated, coumestrol-, DDT- and TCDD-induced suppression of IL-2 may have ramifications for our understanding of the impact of xenoestrogens on health and disease.

  14. Caspase-6 Induces 7A6 Antigen Localization to Mitochondria During FAS-induced Apoptosis of Jurkat Cells.

    Science.gov (United States)

    Suita, Hiroaki; Shinomiya, Takahisa; Nagahara, Yukitoshi

    2017-04-01

    Mitochondria are central to apoptosis. However, apoptosis progression involving mitochondria is not fully understood. A factor involved in mitochondria-mediated apoptosis is 7A6 antigen. 7A6 localizes to mitochondria from the cytosol during apoptosis, which seems to involve 'effector' caspases. In this study, we investigated the precise role of effector caspases in 7A6 localization to mitochondria during apoptosis. Human T-cell lymphoma Jurkat cells were treated with an antibody against FAS. 7A6 localization was analyzed by confocal laser scanning microscopy and flow cytometry. Caspases activation was determined by western blot analysis. 7A6 localization to mitochondria during anti-FAS-induced apoptosis was significantly reduced by the caspase-6 inhibitor, N-acetyl-Val-Glu-Ile-Asp-aldehyde, but not by the caspase-3 inhibitor, N-acetyl-Asp-Asn-Leu-Asp-aldehyde, nor caspase-7/3 inhibitor, N-acetyl-Asp-Gln-Thr-Asp-aldehyde. Moreover, caspase-6 down-regulation suppressed 7A6 localization to mitochondria. Caspase-6 regulates 7A6 localization to mitochondria during anti-FAS-induced apoptosis of Jurkat cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. Inhibition of TNF-alpha induced cell death in human umbilical vein endothelial cells and Jurkat cells by protocatechuic acid.

    Science.gov (United States)

    Zhou-Stache, J; Buettner, R; Artmann, G; Mittermayer, C; Bosserhoff, A K

    2002-11-01

    The Chinese herb radix Salviae miltiorrhizae (RSM) is used in traditional Chinese medicine as a treatment for cardiovascular and cerebrovascular diseases. Several components of the plant extract from salvia mitorrhiza bunge have been determined previously, one of which is protocatechuic acid (PAC). It has been found, in the study, that PAC inhibited TNF-alpha-induced cell death of human umbilical vein endothelial cells (HUVECs) and Jurkat cells in a concentration of 100 microM when applied 2 h prior to TNF-alpha exposure. Molecular studies revealed that PAC activated NF-kappaB with a maximum effect after 30 min of treatment. Inhibition of NF-kappaB action by MG132 and NF-kappaB inhibitory peptide suppressed the cell-protective effect of PAC. Further, degradation of IkBalpha occurred in response to PAC treatment. The results provide evidence that activation of NF-kappaB plays an important role in mediating the cell-protecting effect of PAC on HUVECs and Jurkat cells. Further studies are required to test whether PAC, a component of radix salviae miltiorrhizae, could be useful in preventing in vivo cell death resulting from cardiovascular or cerebrovascular diseases.

  16. Antiproliferative effect of rapamycin on human T-cell leukemia cell line Jurkat by cell cycle arrest and telomerase inhibition

    Institute of Scientific and Technical Information of China (English)

    Yan-min ZHAO; Qian ZHOU; Yun XU; Xiao-yu LAI; He HUANG

    2008-01-01

    Aim:To examine the ability of rapamycin to suppress growth and regulate telomerase activity in the human T-cell leukemia cell line Jurkat. Methods:Cell proliferation was assessed after exposure to rapamycin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were determined by flow cytometry. The proteins important for cell cycle progres-sion and Akt/mammalian target of rapamycin signaling cascade were assessed by Western blotting. Telomerase activity was quantified by telomeric repeat amplication protocol assay. The human telomerase reverse transcriptase (hTERT) mRNA levels were determined by semi-quantitative RT-PCR. Results:Rapamycin inhibited the proliferation of Jurkat, induced G1 phase arrest, unregulated the pro-tein level of p21 as well as p27, and downregulated cyclinD3, phospho-p70s6k, and phospho-s6, but had no effect on apoptosis. Treatment with rapamycin reduced telomerase activity, and reduced hTERT mRNA and protein expression. Conclusion:Rapamycin displayed a potent antileukemic effect in the human T-cell leukemia cell line by inhibition of cell proliferation through G1 cell cycle arrest and also through the suppression of telomerase activity, suggesting that rapamycin may have potential clinical implications in the treatment of some leukemias.

  17. In vitro study of AFB1 and AFM1 effects on human lymphoblastoid Jurkat T-cell model.

    Science.gov (United States)

    Luongo, D; Russo, R; Balestrieri, A; Marzocco, S; Bergamo, P; Severino, L

    2014-10-01

    Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by Aspergillus spp. that can occur as a natural contaminant in foods and feeds of vegetable origin. Post-ingestion, AFB(1) can be metabolized in the liver of mammals into hydroxylated aflatoxin M(1) (AFM(1)) that is excreted with milk. Although several studies have been carried out to evaluate effects of AFB(1) on the immune system, studies regarding AFM(1) are moreover lacking. The aim of the current study was to investigate effects of AFB(1) and AFM(1) on immune function using a lymphoblastoid Jurkat T-cell line as an experimental model. Both AFB(1) and AFM(1) produced significant decreases in Jurkat cell proliferation, whereas only minor effects were noted on interleukin (IL)-2 and interferon (IFN)-γ cytokines mRNA expression in stimulated cells that had been pre-incubated with AFB(1) and AFM(1). Particularly, AFB(1), but not AFM(1), at the highest concentration (50 µM) induced a marked increase in IL-8 mRNA expression. The results of the current study suggested the existence of a concentration threshold for AFB(1) and AFM(1) needed to exert biological activity on cell viability and innate immunity.

  18. Benzalkonium chloride (BAK induces apoptosis or necrosis, but has no major influence on the cell cycle of Jurkat cells

    Directory of Open Access Journals (Sweden)

    Piotr Pozarowski

    2011-07-01

    Full Text Available Benzalkonium chloride (BAK is a cationic detergent with a very slow turnover. Because of its strong antibacterial activities, BAK is widely used especially in dentistry and ophthalmology. It is the most commonly used preservative in topical ophthalmic medications. Due to chronicity and widespread use of such treatments, BAK’s side effects are of great importance. BAK toxicity for adherent cells, probably related to its pro-oxidative activities, is time- and dose-dependent. Although lymphocytes often infiltrate superficial eye tissues, the BAK influence on them is yet to be established. The aim of this study was to check BAK cytotoxicity on T lymphocytic Jurkat line cells and to verify the suggestion that BAK can induce G2M cell blocks. A dose- and time-dependent cytotoxic effect of BAK on lymphoid cells in relatively low concentrations was shown in this study. In lower concentrations, it shows a moderate apoptotic and minimal antiproliferative effect on Jurkat cells, while in higher concentrations it shows a rapid necrotic effect. No G2M cell blocks were observed. Our findings could suggest lymphoid dysfunction during intensive, prolonged topical BAK treatment, even at dosages relatively non-toxic to epithelial eye cells. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 2, pp. 225–230

  19. OPTIMIZATIONS FOR 5-AMINOLEVULINIC ACID BASED PHOTODYNAMIC THERAPY IN PURGING LEUKEMIA CELL HL60

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Photodynamictherapy(PDT),whichinvolves administrationofatumorlocalizing photosensiti zingagentthatmayrequiremetabolizingsynthesis(i.e.,aprodrug),followedbyactivationofthea gentbylightofaspecificwavelength.Thistherapy resultsinasequenceofphotochemicalandphotobio logicprocessthatcausesirreversiblephotodamageto tumortissue.SeveralphotosensitizersinPDThavesofar beenappliedtoeradicateresidualtumorcellsinau tograftsandtoreducecontaminationoftumorcells inhemopathy[1-3].However,alltheexogenous photosensitizershavea...

  20. Basic Apoptotic Mechanisms of Lead Toxicity in Human Leukemia (Hl-60) Cells

    OpenAIRE

    Tchounwou, Paul B.; Carolyn B. Howard; Milner, Jessica N.; Yedjou, Clement G.

    2010-01-01

    Lead exposure represents a medical and public health emergency, especially in children consuming high amounts of lead-contaminated flake paints. It may also cause hematological effects to people of all ages. Recent studies in our laboratory have indicated that apoptosis may be associated with the lead-induced oxidative stress and DNA damage. However, the mechanisms underlying its effect on lymphocytes are still largely unknown. Therefore, the aim of the present study was to investigate the ap...

  1. Quercus Suber L. Cork Extracts Induce Apoptosis in Human Myeloid Leukaemia HL-60 Cells.

    Science.gov (United States)

    Bejarano, Ignacio; Godoy-Cancho, Belén; Franco, Lourdes; Martínez-Cañas, Manuel A; Tormo, María A

    2015-08-01

    Quercus suber L. cork contains a diversity of phenolic compounds, mostly low molecular weight phenols. A rising number of reports support with convergent findings that polyphenols evoke pro-apoptotic events in cancerous cells. However, the literature related to the anti-cancer bioactivity of Q. suber L. cork extractives (QSE) is still limited. Herein, we aim to describe the antitumor potential displayed by cork extractives obtained by different extraction methods in the human promyelocytic leukaemia cells. In order to quantify the effects of QSE on cancer cells viability, phosphatidylserine exposure, caspase-3 activity, mitochondrial membrane potential and cell cycle were evaluated. The results indicated that the QSE present a time-dependent and dose-dependent cytotoxicity in the human promyelocytic leukaemia cells. Such a noxious effect leads these leukaemia cells to their death through apoptotic processes by altering the mitochondrial outer membrane potential, activating caspase-3 and externalizing phosphatidylserine. However, cells cycle progression was not affected by the treatments. This study contributes to open a new way to use this natural resource by exploiting its anti-cancer properties. Moreover, it opens new possibilities of application of cork by-products, being more efficient in the sector of cork-based agriculture. Copyright © 2015 John Wiley & Sons, Ltd.

  2. microRNA-181b targets MLK2 in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    microRNAs(miRNAs) play critical roles in many different cellular processes,including metabolism,apoptosis,differentiation and development.We showed miR-181b to be highly expressed in acute myeloid leukemia(AML).Furthermore,miR-181b contributed to proliferation of AML cells by targeting MLK2.Our results demonstrated that miR-181b plays an important role in the biology of AML and may be useful in developing therapies targeting miRNAs.

  3. Effect of silencing HOXA5 gene expression using RNA interference on cell cycle and apoptosis in Jurkat cells.

    Science.gov (United States)

    Huang, Hui-Ping; Liu, Wen-Jun; Guo, Qu-Lian; Bai, Yong-Qi

    2016-03-01

    Acute lymphocytic leukemia (ALL) is a common malignant tumor with a high morbidity rate among children, accounting for approximately 80% of leukemia cases. Although there have been improvements in the treatment of patients frequent relapse lead to a poor prognosis. The aim of the present study was to determine whether HOXA5 may be used as a target for gene therapy in leukemia in order to provide a new treatment. Mononuclear cells were extracted from the bone marrow according to the clinical research aims. After testing for ALL in the acute stage, the relative mRNA and protein expression of HOXA5 was detected in the ALL remission groups (n=25 cases per group) and the control group [n=20 cases, immune thrombocytopenia (ITP)]. Gene silencing by RNA interference (RNAi) was used to investigate the effect of silencing HOXA5 after small interfering RNA (siRNA) transfection to Jurkat cells. The HOXA5-specific siRNA was transfected to Jurkat cells using lipofectamine. The experiment was divided into the experimental group (liposomal transfection of HOXA5 targeting siRNA), the negative control group (liposomal transfection of cells with negative control siRNA) and the control group (plus an equal amount of cells and culture media only). Western blotting and quantitative fluorescent polymerase chain reaction (QF‑PCR) were used to detect the relative HOXA5 mRNA expression and protein distribution in each cell group. Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined using flow cytometry. The expression of HOXA5 at the mRNA and protein levels in the acute phase of ALL was significantly higher than that in ALL in the remission and control groups. In cells transfected with HOXA5-specific siRNA, the expression of HOXA5 at the mRNA and protein levels decreased significantly (Pcells in the cell cycle was also altered. Specifically, more cells were present in the G0/G1 phase compared to the S phase (Pcells transfected with HOXA5

  4. A synthesized nostocionone derivative potentiates programmed cell death in human T-cell leukemia Jurkat cells through mitochondria via the release of endonuclease G.

    Science.gov (United States)

    Itoh, Tomohiro; Muramatsu, Yuji; Masu, Masayo; Tsuge, Ayaka; Taniguchi, Masaki; Ninomiya, Masayuki; Ando, Masashi; Tsukamasa, Yasuyuki; Koketsu, Mamoru

    2014-01-01

    Nostocionone (Nost), a compound isolated from Nostoc commune, and its synthesized derivatives (NostDs) were evaluated for in vitro cytotoxicity against human T-cell leukemia Jurkat cells. NostD3 [(1E,4E)-1-(3,4-dihydroxyphenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4-dien-3-one] inhibited cell growth more potently than Nost. To elucidate the mechanisms of NostD3-induced cell death, we examined changes in cell morphology, the loss of mitochondrial membrane potential (MMT), and DNA fragmentation. From these results, the cytotoxic effects of NostD3 were found to be mainly due to Type I programmed cell death (PCDI; i.e., apoptosis). Using caspase inhibitors, we further found that NostD-3-induced PCDI occurred through a caspase-independent pathway. Moreover, NostD3 decreased MMT and modulated multiple signaling molecules (MAPKs, Akt, Bcl-2, Bax, and c-Myc) in Jurkat cells, thereby inducing the release of endonuclease G (Endo-G) from mitochondria. The level of intracellular reactive oxygen species (ROS) in cells treated with NostD3 was elevated up to 1 h after the treatment. However, suppression of ROS by N-acetyl-l-cysteine restored Jurkat cell growth. Taken together, our data suggested that ROS production acted as a trigger in NostD3-induced PCDI in Jurkat cells through release of Endo-G from the mitochondria.

  5. T-cell activation. V. Anti-major histocompatibility complex class I antibody-induced activation and clonal abortion in Jurkat T-leukaemic cells

    DEFF Research Database (Denmark)

    Claesson, M H; Dissing, S; Tscherning, T;

    1993-01-01

    We have studied activation-induced changes in intracellular calcium [Ca2+]i, interleukin-2 (IL-2) secretion, and clonal abortion of the human leukaemic T-cell line Jurkat and three T-cell receptor (TcR)/CD3 receptor negative clones deficient for the TcR alpha, TcR beta and CD3 gamma chains respec...

  6. New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment.

    Science.gov (United States)

    Cardoso, F M; Tomkova, M; Petrovajova, D; Bubanova, M; Ragac, O; Hornakova, T

    2017-01-31

    The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs failed to rescue Jurkat cells viability in presence of +Aza, as +DLE treatment did in our MTT assay. In conclusion, our new cell-based MTT assay has excellent DLE biological activity consistency, robustness and is cost effective, presenting important advantages over previous DLE activity in vitro and in vivo assays.

  7. UVB-Radiation-Induced Apoptosis in Jurkat Cells: A Coordinated Fourier Transform Infrared Spectroscopy-Flow Cytometry Study

    CERN Document Server

    Pozzi, Deleana; Gaudenzi, Silvia; Di Giambattista, Lucia; Silvestri, Ida; Morrone, Stefania; Castellano, Agostina Congiu

    2010-01-01

    We studied the induction of apoptosis in Jurkat cells by UVB radiation (wavelength 290-320 nm) at a dose of 310 mJ/cm^2. We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide new and improved information about cell modifications. To do this, we looked for correspondences and correlations between spectroscopy and flow cytometry data and found three highly probable spectroscopic markers of apoptosis. The behavior of the wave number shift of both the Amide I beta-sheet component and the area of the 1083 cm^-1 band reproduced, with a high correlation, the behavior of the early apoptotic cell population, while the behavior of the Amide I area showed a high correlation with the early plus late apoptotic cell population.

  8. High ACSL5 Transcript Levels Associate with Systemic Lupus Erythematosus and Apoptosis in Jurkat T Lymphocytes and Peripheral Blood Cells

    Science.gov (United States)

    2011-01-01

    Background Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which increased apoptosis and decreased apoptotic cells removal has been described as most relevant in the pathogenesis. Long-chain acyl-coenzyme A synthetases (ACSLs) have been involved in the immunological dysfunction of mouse models of lupus-like autoimmunity and apoptosis in different in vitro cell systems. The aim of this work was to assess among the ACSL isoforms the involvement of ACSL2, ACSL4 and ACSL5 in SLE pathogenesis. Findings With this end, we determined the ACSL2, ACSL4 and ACSL5 transcript levels in peripheral blood mononuclear cells (PBMCs) of 45 SLE patients and 49 healthy controls by quantitative real time-PCR (q-PCR). We found that patients with SLE had higher ACSL5 transcript levels than healthy controls [median (range), healthy controls = 16.5 (12.3–18.0) vs. SLE = 26.5 (17.8–41.7), P = 3.9×10 E-5] but no differences were found for ACSL2 and ACSL4. In in vitro experiments, ACSL5 mRNA expression was greatly increased when inducing apoptosis in Jurkat T cells and PBMCs by Phorbol-Myristate-Acetate plus Ionomycin (PMA+Io). On the other hand, short interference RNA (siRNA)-mediated silencing of ACSL5 decreased induced apoptosis in Jurkat T cells up to the control levels as well as decreased mRNA expression of FAS, FASLG and TNF. Conclusions These findings indicate that ACSL5 may play a role in the apoptosis that takes place in SLE. Our results point to ACSL5 as a potential novel functional marker of pathogenesis and a possible therapeutic target in SLE. PMID:22163040

  9. High ACSL5 transcript levels associate with systemic lupus erythematosus and apoptosis in Jurkat T lymphocytes and peripheral blood cells.

    Directory of Open Access Journals (Sweden)

    Antonio Catalá-Rabasa

    Full Text Available BACKGROUND: Systemic lupus erythematosus (SLE is a prototypical autoimmune disease in which increased apoptosis and decreased apoptotic cells removal has been described as most relevant in the pathogenesis. Long-chain acyl-coenzyme A synthetases (ACSLs have been involved in the immunological dysfunction of mouse models of lupus-like autoimmunity and apoptosis in different in vitro cell systems. The aim of this work was to assess among the ACSL isoforms the involvement of ACSL2, ACSL4 and ACSL5 in SLE pathogenesis. FINDINGS: With this end, we determined the ACSL2, ACSL4 and ACSL5 transcript levels in peripheral blood mononuclear cells (PBMCs of 45 SLE patients and 49 healthy controls by quantitative real time-PCR (q-PCR. We found that patients with SLE had higher ACSL5 transcript levels than healthy controls [median (range, healthy controls = 16.5 (12.3-18.0 vs. SLE = 26.5 (17.8-41.7, P = 3.9×10 E-5] but no differences were found for ACSL2 and ACSL4. In in vitro experiments, ACSL5 mRNA expression was greatly increased when inducing apoptosis in Jurkat T cells and PBMCs by Phorbol-Myristate-Acetate plus Ionomycin (PMA+Io. On the other hand, short interference RNA (siRNA-mediated silencing of ACSL5 decreased induced apoptosis in Jurkat T cells up to the control levels as well as decreased mRNA expression of FAS, FASLG and TNF. CONCLUSIONS: These findings indicate that ACSL5 may play a role in the apoptosis that takes place in SLE. Our results point to ACSL5 as a potential novel functional marker of pathogenesis and a possible therapeutic target in SLE.

  10. Down-regulation of 11β-hydroxysteroid dehydrogenase type 2 by bortezomib sensitizes Jurkat leukemia T cells against glucocorticoid-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Yi Tao

    Full Text Available 11β-Hydroxysteroid dehydrogenases type 2 (11β-HSD2, a key regulator for pre-receptor metabolism of glucocorticoids (GCs by converting active GC, cortisol, to inactive cortisone, has been shown to be present in a variety of tumors. But its expression and roles have rarely been discussed in hematological malignancies. Proteasome inhibitor bortezomib has been shown to not only possess antitumor effects but also potentiate the activity of other chemotherapeutics. In this study, we demonstrated that 11β-HSD2 was highly expressed in two GC-resistant T-cell leukemic cell lines Jurkat and Molt4. In contrast, no 11β-HSD2 expression was found in two GC-sensitive non-hodgkin lymphoma cell lines Daudi and Raji as well as normal peripheral blood T cells. Inhibition of 11β-HSD2 by 11β-HSD inhibitor 18β-glycyrrhetinic acid or 11β-HSD2 shRNA significantly increased cortisol-induced apoptosis in Jurkat cells. Additionally, pretreatment of Jurkat cells with low-dose bortezomib resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis, more cells arrested at G1 stage and up-regulation of GC-induced leucine zipper which is an important mediator of GC action. Furthermore, we clarified that bortezomib could dose-dependently inhibit 11β-HSD2 messenger RNA and protein levels as well as activity (cortisol-cortisone conversion through p38 mitogen-activated protein kinase signaling pathway. Therefore, we suggest 11β-HSD2 is, at least partially if not all, responsible for impaired GC suppression in Jurkat cells and also indicate a novel mechanism by which proteasome inhibitor bortezomib may influence GC action.

  11. Anti-proliferative properties of commercial Pelargonium sidoides tincture, with cell-cycle G0/G1 arrest and apoptosis in Jurkat leukaemia cells.

    Science.gov (United States)

    Pereira, Andreia; Bester, Megan; Soundy, Puffy; Apostolides, Zeno

    2016-09-01

    Context Pelargonium sidoides DC (Geraniaceae) is an important medicinal plant indigenous to South Africa and Lesotho. Previous studies have shown that root extracts are rich in polyphenolic compounds with antibacterial, antiviral and immunomodulatory activities. Little is known regarding the anticancer properties of Pelargonium sidoides extracts. Objective This study evaluates the anti-proliferative effects of a Pelargonium sidoides radix mother tincture (PST). Materials and methods The PST was characterized by LC-MS/MS. Anti-proliferative activity was evaluated in the pre-screen panel of the National Cancer Institute (NCI-H460, MCF-7 and SF-268) and the Jurkat leukaemia cell line at concentrations of 0-150 μg/mL. The effect on cell growth was determined with sulphorhodamine B and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays after 72 h. The effect on cell cycle and apoptosis induction in Jurkat cells was determined by flow cytometry with propidium iodide and Annexin V: fluorescein isothiocyanate staining. Results Dihydroxycoumarin sulphates, gallic acid as well as gallocatechin dimers and trimers were characterized in PST by mass spectrometry. Moderate anti-proliferative effects with GI50 values between 40 and 80 μg/mL were observed in the NCI-pre-screen panel. Strong activity observed with Jurkat cells with a GI50 value of 6.2 μg/mL, significantly better than positive control 5-fluorouracil (GI50 value of 9.7 μg/mL). The PST arrested Jurkat cells at the G0/G1 phase of the cell cycle and increased the apoptotic cells from 9% to 21%, while the dead cells increased from 4% to 17%. Conclusion We present evidence that P. sidoides has cancer cell type-specific anti-proliferative effects and may be a source of novel anticancer molecules.

  12. Systems biology approach to transplant tolerance: proof of concept experiments using RNA interference (RNAi) to knock down hub genes in Jurkat and HeLa cells in vitro.

    Science.gov (United States)

    Lwin, Wint Wah; Park, Ken; Wauson, Matthew; Gao, Qin; Finn, Patricia W; Perkins, David; Khanna, Ajai

    2012-07-01

    Systems biology is gaining importance in studying complex systems such as the functional interconnections of human genes [1]. To investigate the molecular interactions involved in T cell immune responses, we used databases of physical gene-gene interactions to constructed molecular interaction networks (interconnections) with R language algorithms. This helped to identify highly interconnected "hub" genes AT(1)P5C1, IL6ST, PRKCZ, MYC, FOS, JUN, and MAPK1. We hypothesized that suppression of these hub genes in the gene network would result in significant phenotypic effects on T cells and examined this in vitro. The molecular interaction networks were then analyzed and visualized with Cytoscape. Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BrdU labeling, which were measured 36, 72, and 96 h after activation. Following T cell stimulation, we found a significant decrease in ATP production (P cells. However, HeLa cells showed a significant (P cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN, and FOS were knocked down. In both Jurkat and HeLa cells, targeted gene knockdown using siRNA showed decreased cell proliferation and ATP production in both Jurkat and HeLa cells. However, Jurkat T cells and HELA cells use different hub genes to regulate activation responses. This experiment provides proof of principle of applying siRNA knockdown of T cell hub genes to evaluate their proliferative capacity and ATP production. This novel concept outlines a systems biology approach to identify hub genes for targeted therapeutics. Published by Elsevier Inc.

  13. Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein

    Science.gov (United States)

    Sanclemente, Manuel; Iturralde, María; Naval, Javier; Alava, María Angeles; Martínez-Lostao, Luis; Thierse, Hermann-Josef; Anel, Alberto

    2016-01-01

    We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion. PMID:27086912

  14. Down-regulation of Bmi-1 by RNA interference in Jurkat cells%RNAi抑制Bmi-1基因表达对Jurkat细胞的影响

    Institute of Scientific and Technical Information of China (English)

    Shangen Zheng; Qibin Jing; Yaqiong Zheng; Yinjuan Ding; Qianchuan Huang; Guoqiang Zhao

    2012-01-01

    Objective: The aim of our study was to investigate the effects of down-regulation Bmi-1 by RNA interference (RNAi) in T Lymphocytic leukemia Jurkat cells. Methods: Two complementary oligonucleotide strands were synthesized based on the siRNA sequence targeting Bmi-1 gene. After annealing, siRNA strands were recombined into the pRNATU6.2 vector, and then DNA sequencing was carried out following transformation and amplification. The recombinant was transfected into Jurkat cells with liposomes. Positive colonies were obtained through G418 selection. The mRNA and protein expressions of Bmi-1 were detected by RT-PCR and Western-blot, respectively. Effects of Bmi-1 silence on cell proliferation, cell cycle and cell aging of Jurkat cells were detected by MTT assay, flow cytometry, colony formation assay and SA-β-Gal staining, respectively. Results: The siRNA recombinant targeting Bmi-1 gene was successfully constructed. All three siRNA recombinants could significantly inhibit the expression of Bmi-1. The siRNA targeting 825nt-843nt (GACCAGACCACTACT GAAT) has the strongest inhibitory effect on Bmi-1 expression, with almost complete inhibition on Bmi-1 mRNA and protein expressions. Compared with the non-transfection group and the empty vector group, growth velocity and colony formation ability were significantly decreased, while the proportion of cells in G1 phase and the percentage of senile cells were significantly increased in highly transfected group (P < 0.05). Conclusion: Down-regulation Bmi-1 by RNA interference (RNAi) could significantly inhibit the growth of Jurkat cells in vitro.

  15. An agonistic monoclonal antibody against DR5 induces ROS production, sustained JNK activation and Endo G release in Jurkat leukemia cells

    Institute of Scientific and Technical Information of China (English)

    Caifeng Chen; Yanxin Liu; Dexian Zheng

    2009-01-01

    We have previously reported that AD5-10, a novel agonistic monoclonal antibody against DRS, possessed a strong cytotoxic activity in various tumor cells, via induction of caspase-dependent and-independent signaling pathways. The present study further demonstrates that reactive oxygen species (ROS) were generated in abundance in Jurkat leukemia cells upon ADS-10 stimulation and that ROS accumulation subsequently evoked sustained activation of c-Jun N-terminal kinase (JNK), loss of mitochondrial membrane potential, and release of endonuclease G (Endo G) from mitochondria into the cytosol. The reducing agent, N-acetylcysteine (NAC), effectively inhibited the sustained activation of JNK, release of Endo G, and cell death in Jurkat cells treated by ADS-10. Moreover, a dominant-nega-tive form of JNK (but not of p38) enhanced NF-KB activation, suppressed caspase-8 recruitment in death-inducing signaling complexes (DISCs), and reduced adverse effects on mitochondria, thereby inhibiting AD5-10-induced cell death in Jurkat leukemia cells. These data provide novel information on the DRS-mediated cell death-signaling path-way and may shed new light on effective strategies for leukemia and solid tumor therapies.

  16. Combined treatment with fenretinide and indomethacin induces AIF-mediated, non-classical cell death in human acute T-cell leukemia Jurkat cells

    Energy Technology Data Exchange (ETDEWEB)

    Hojka-Osinska, Anna, E-mail: hojka@immuno.iitd.pan.wroc.pl [Laboratory of Tumor Molecular Immunobiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, 53-114 Wroclaw (Poland); Ziolo, Ewa, E-mail: ziolo@immuno.iitd.pan.wroc.pl [Laboratory of Tumor Molecular Immunobiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, 53-114 Wroclaw (Poland); Rapak, Andrzej, E-mail: rapak@immuno.iitd.pan.wroc.pl [Laboratory of Tumor Molecular Immunobiology, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, 53-114 Wroclaw (Poland)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer The combination of fenretinide and indomethacin induces a high level of cell death. Black-Right-Pointing-Pointer Apoptotic pathway is caspase-independent. Black-Right-Pointing-Pointer Jurkat cells undergo AIF-mediated cell death. -- Abstract: Currently used cytotoxic drugs in cancer therapy have a similar mechanism of action and low specificity. Applied simultaneously, they show an additive effect with strong side effects. Clinical trials with the use of different agents in cancer therapy show that the use of these compounds alone is not very effective in fighting cancer. An alternative solution could be to apply a combination of these agents, because their combination has a synergistic effect on some cancer cells. Therefore, in our investigations we examined the effects of a synthetic retinoid-fenretinide when combined with a non-steroidal anti-inflammatory drug-indomethacin on the process of apoptosis in the acute human T-cell leukemia cell line Jurkat. We demonstrate that treatment with the combination of the tested compounds induces the death of cells, that is peculiar and combines features of apoptosis as well as non-apoptotic cell death. In detail we observed, cell membrane permeabilization, phosphatydylserine exposure, no oligonucleosomal DNA fragmentation, no caspase-3 activation, but apoptosis inducing factor (AIF) nuclear translocation. Taken together these results indicate, that Jurkat cells after treatment with a combination of fenretinide and indomethacin undergo AIF-mediated programmed cell death.

  17. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    Energy Technology Data Exchange (ETDEWEB)

    Iri-Sofla, Farnoush Jafari [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ahmadvand, Davoud [Center of Pharmaceutical Nanotechnology and Nanotoxicology, Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen O (Denmark); Rasaee, Mohammad J. [Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of)

    2011-11-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  18. DNA electrophoretic migration patterns change after exposure of Jurkat cells to a single intense nanosecond electric pulse.

    Directory of Open Access Journals (Sweden)

    Stefania Romeo

    Full Text Available Intense nanosecond pulsed electric fields (nsPEFs interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a development of biomedical applications of nsPEFs, including cancer therapy, and b better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake. Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns.

  19. Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP.

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    Caleb C Roth

    Full Text Available Nanosecond electrical pulse (nsEP exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

  20. Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells

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    Yao Pan

    2015-08-01

    Full Text Available Trichloroethylene (TCE is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA and dichloroacetic acid (DCA, on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

  1. Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells.

    Science.gov (United States)

    Pan, Yao; Wei, Xuetao; Hao, Weidong

    2015-08-28

    Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

  2. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

    Energy Technology Data Exchange (ETDEWEB)

    Katika, Madhumohan R. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Hendriksen, Peter J.M. [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Shao, Jia [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Loveren, Henk van [Department of Health Risk Analysis and Toxicology, Maastricht University (Netherlands); National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Netherlands Toxicogenomics Centre (Netherlands); Peijnenburg, Ad, E-mail: ad.peijnenburg@wur.nl [RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen (Netherlands); Netherlands Toxicogenomics Centre (Netherlands)

    2012-10-01

    Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

  3. Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells

    Science.gov (United States)

    Jazi, Marie Saghaeian; Mohammadi, Saeed; Yazdani, Yaghoub; Sedighi, Sima; Memarian, Ali; Aghaei, Mehrdad

    2016-01-01

    Objective(s): T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA) as a recently emerged anti-neoplastic histone deacetylase (HDAC) inhibitor and pioglitazone (PGZ) as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ) agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. Materials and Methods: Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. Results: Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after 24 hr; however, PGZ 400 μM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A) phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27) expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. Conclusion: These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies. PMID:27635203

  4. Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells

    Directory of Open Access Journals (Sweden)

    Marie Saghaeian Jazi

    2016-07-01

    Full Text Available Objective(s: T-cell acute lymphoblastic leukemia (T-ALL is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA as a recently emerged anti-neoplastic histone deacetylase (HDAC inhibitor and pioglitazone (PGZ as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. Materials and Methods: Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. Results: Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after   24 hr; however, PGZ 400 μM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27 expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. Conclusion: These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies.

  5. Integrated mRNA and micro RNA profiling reveals epigenetic mechanism of differential sensitivity of Jurkat T cells to AgNPs and Ag ions.

    Science.gov (United States)

    Eom, Hyun-Jeong; Chatterjee, Nivedita; Lee, Jeongsoo; Choi, Jinhee

    2014-08-17

    In our previous in vitro study of the toxicity on silver nanoparticles (AgNPs), we observed a dramatically higher sensitivity of Jurkat T cells to AgNPs than to Ag ions, and DNA damage and apoptosis were found to be involved in that toxicity. In this study, to understand underlying mechanism of different sensitivity of Jurket T cells to AgNPs and Ag ions, mRNA microarray and micro RNA microarray were concomitantly conducted on AgNPs and Ag ions exposed Jurkat T cells. Surprisingly only a small number of genes were differentially expressed by exposure to each of the silver (15 altered mRNA by AgNPs exposure, whereas 4 altered mRNA by Ag ions exposure, as determined 1.5-fold change as the cut-off value). miRNA microarray revealed that the expression of 63 miRNAs was altered by AgNPs exposure, whereas that of 32 miRNAs was altered by Ag ions exposure. An integrated analysis of mRNA and miRNA expression revealed that the expression of hsa-miR-219-5p, was negatively correlated with the expression of metallothionein 1F (MT1F) and tribbles homolog 3 (TRIB3), in cells exposed to AgNPs; whereas, the expression of hsa-miR-654-3p was negatively correlated with the expression of mRNA, endonuclease G-like 1 (EDGL1) in cells exposed to Ag ions. Network analysis were further conducted on mRNA-miRNA pairs, which revealed that miR-219-5p-MT1F and -TRIB3 pairs by AgNPs are being involved in various cellular processes, such as, oxidative stress, cell cycle and apoptosis, whereas, miR-654-3p and ENDOGL1 pair by Ag ions generated a much simpler network. The putative target genes of AgNPs-induced miR-504, miR-33 and miR-302 identified by Tarbase 6.0 are also found to be involved in DNA damage and apoptosis. These results collectively suggest that distinct epigenetic regulation may be an underlying mechanism of different sensitivity of Jurkat T cells to AgNPs and Ag ion. Further identification of putative target genes of DE miRNA by AgNPs and Ag ions may provide additional clues for the

  6. Molecular regulation of MHC class I chain-related protein A expression after HDAC-inhibitor treatment of Jurkat T cells

    DEFF Research Database (Denmark)

    Andresen, Lars; Jensen, Helle; Pedersen, Marianne T

    2007-01-01

    /B expression. It was further observed that endoplasmic reticulum calcium stores were depleted after HDAC treatment. NF-kappaB activity can be induced by HDAC treatment. However, nuclear translocation of NF-kappaB p65 was not observed after HDAC treatment of Jurkat T cells and even though we could effectively...... inhibit p65 expression by siRNA, it did not modify MICA/B expression. To identify important elements in MICA regulation, we made a promoter construct consisting of approximately 3 kb of the proximal MICA promoter in front of GFP. Deletion analysis showed that a germinal center-box containing a putative Sp...

  7. Docosahexaenoic acid and other fatty acids induce a decrease in pHi in Jurkat T-cells

    Science.gov (United States)

    Aires, Virginie; Hichami, Aziz; Moutairou, Kabirou; Khan, Naim Akhtar

    2003-01-01

    Docosahexaenoic acid (DHA) induced rapid (t1/2=33 s) and dose-dependent decreases in pHi in BCECF-loaded human (Jurkat) T-cells. Addition of 5-(N,N-dimethyl)-amiloride, an inhibitor of Na+/H+ exchanger, prolonged DHA-induced acidification as a function of time, indicating that the exchanger is implicated in pHi recovery. Other fatty acids like oleic acid, arachidonic acid, eicosapentaenoic acid, but not palmitic acid, also induced a fall in pHi in these cells. To assess the role of calcium in the DHA-induced acidification, we conducted experiments in Ca2+-free (0% Ca2+) and Ca2+-containing (100% Ca2+) buffer. We observed that there was no difference in the degree of DHA-induced transient acidification in both the experimental conditions, though pHi recovery was faster in 0% Ca2+ medium than that in 100% Ca2+ medium. In the presence of BAPTA, a calcium chelator, a rapid recovery of DHA-induced acidosis was observed. Furthermore, addition of CaCl2 into 0% Ca2+ medium curtailed DHA-evoked rapid pHi recovery. In 0% Ca2+ medium, containing BAPTA, DHA did not evoke increases in [Ca2+]i, though this fatty acid still induced a rapid acidification in these cells. These observations suggest that calcium is implicated in the long-lasting DHA-induced acidosis. DHA-induced rapid acidification may be due to its deprotonation in the plasma membrane (flip-flop model), as suggested by the following observations: (1) DHA with a –COOH group induced intracellular acidification, but this fatty acid with a –COOCH3 group failed to do so, and (2) DHA, but not propionic acid, -induced acidification was completely reversed by addition of fatty acid-free bovine serum albumin in these cells. These results suggest that DHA induces acidosis via deprotonation and Ca2+ mobilization in human T-cells. PMID:14645139

  8. Pandemic Influenza A (H1N1) Virus Infection Increases Apoptosis and HIV-1 Replication in HIV-1 Infected Jurkat Cells.

    Science.gov (United States)

    Wang, Xue; Tan, Jiying; Biswas, Santanu; Zhao, Jiangqin; Devadas, Krishnakumar; Ye, Zhiping; Hewlett, Indira

    2016-02-02

    Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.

  9. Lipopolysaccharides and the Enhanced Expression of TACE Mrna in HL-60 Cells and Adhesive Cells from Human Spleen

    Institute of Scientific and Technical Information of China (English)

    DING; Tingbo(

    2001-01-01

    [1]Flad H D Loppnow H.Agonists and antagonists for LPS-induced cytokines.Immunology 1993 187:303[2]Robache-Gallea S Morand V Bruneau J H et al.In vitro processing of human tumor necrosis factor-α.J Bio Chem 1995 270(40):23 688[3]Kim K U Kwon O J Jue D M.Pro-tumor necrosis factor cleaving enzyme in macrophage membrane/particulate.Immunology 1993 80(2) :134[4]Moss M L.Cloning of a disintegrin metalloproteinase that processes precursor tumor necrosis factor-α.Nature 1997 385 (6):733[5]Flick D A Gilford G E.The detection of biological activity of tumor necrosis factor-α.J Immunol Methods 1984 68:167[6]Sambrook J Fritsch E F Maniatis F.In Vitro Amplification of DNA by the Polymerase Chain Reaction.In:Molecular Cloning A Laboratory Manual.2nd edition.New York:Cold Spring Harbor Laboratory Press 1989.10.1-10.70[7]吴朝栋 李明珍 张艳萍等.中药热毒清对内毒素DIC家兔血浆肿瘤坏死因子α白细胞介素水平影响的研究.中国中西医结合杂志 1995 15:365

  10. SUBSTITUTED 1,3-PHENYL(PYRIDYL PROPENONES AND DERIVATIVES WITH THIOSEMICARBAZIDIC GROUPS. STRUCTRURE – (HL-60 ANTILEUKEMIA ACTIVITY RELATIONSHIP

    Directory of Open Access Journals (Sweden)

    Ana Popusoi

    2014-12-01

    Full Text Available 3-(4-(Dimethylaminophenyl-1-(4-isothiocyanatophenylprop-2-en-1-one was obtained from the corresponding N,N-dimethylthyoureas by elimination of dimethylamine at heating with gaseous hydrogen chloride in chloroform and 1-(4-isothiocyanatophenyl-3-(pyridin-2-ilprop-2-en-1-one by treating 1,1-dimethyl-3-(4-(3-(pyridin-2-il-acryloyl-phenylthyourea with acetic anhydride. The difference in the reactivity of the groups >C=O and NCS in the synthesis with hydrazine hydrate and its derivatives allows the synthesis of some 1,3-disubstituted propenones with thiosemicarbazide groups (4- and 1,4-disubstituted in good yields. From 4-substituted thiosemicarbazides and 2-formilpyridine thiosemicarbazones were obtained. In the case of some derivatives, the propenone group in the reaction with hydrazine hydrate allows the formation of pyrazole derivatives. All obtained compounds were investigated for antileukemia activity. It was found that this activity is more pronounced for thiosemicarbazide derivatives with two pyridine nuclei at concentrations 10-5-10-7 mol/L.

  11. CD147 and CD98 complex-mediated homotypic aggregation attenuates the CypA-induced chemotactic effect on Jurkat T cells.

    Science.gov (United States)

    Guo, Na; Zhang, Kui; Lv, Minghua; Miao, Jinlin; Chen, Zhinan; Zhu, Ping

    2015-02-01

    Homotypic cell aggregation plays important roles in physiological and pathological processes, including embryogenesis, immune responses, angiogenesis, tumor cell invasion and metastasis. CD147 has been implicated in most of these phenomena, and it was identified as a T cell activation-associated antigen due to its obvious up-regulation in activated T cells. However, the explicit function and mechanism of CD147 in T cells have not been fully elucidated. In this study, large and compact aggregates were observed in Jurkat T cells after treatment with the specific CD147 monoclonal antibody HAb18 or after the expression of CD147 was silenced by RNA interference, which indicated an inhibitory effect of CD147 in T cell homotypic aggregation. Knocking down CD147 expression resulted in a significant decrease in CD98, along with prominent cell aggregation, similar to that treated by CD98 and CD147 monoclonal antibodies. Furthermore, decreased cell chemotactic activity was observed following CD147- and CD98-mediated cell aggregation, and increased aggregation was correlated with a decrease in the chemotactic ability of the Jurkat T cells, suggesting that CD147- and CD98-mediated homotypic cell aggregation plays a negative role in T cell chemotaxis. Our data also showed that p-ERK, p-ZAP70, p-CD3ζ and p-LCK were significantly decreased in the CD147- and CD98-knocked down Jurkat T cells, which suggested that decreased CD147- and/or CD98-induced homotypic T cell aggregation and aggregation-inhibited chemotaxis might be associated with these signaling pathways. A role for CD147 in cell aggregation and chemotaxis was further indicated in primary CD4(+) T cells. Similarly, low expression of CD147 in primary T cells induced prominent cell aggregation and this aggregation attenuated primary T cell chemotactic ability in response to CypA. Our results have demonstrated the correlation between homotypic cell aggregation and the chemotactic response of T cells to CypA, and these data

  12. CD45 immunoaffinity depletion of vesicles from Jurkat T cells demonstrates that exosomes contain CD45: no evidence for a distinct exosome/HIV-1 budding pathway

    Directory of Open Access Journals (Sweden)

    Ott David E

    2008-07-01

    Full Text Available Abstract The presence of relatively high levels of cellular protein contamination in density-purified virion preparations is a confounding factor in biochemical analyses of HIV and SIV produced from hematopoietic cells. A major source of this contamination is from vesicles, either microvesicles or exosomes, that have similar physical properties as virions. Thus, these particles can not be removed by size or density fractionation. Although virions and vesicles have similar cellular protein compositions, CD45 is excluded from HIV-1 yet is present in vesicles produced from hematopoietic cells. By exploiting this finding, we have developed a CD45 immunoaffinity depletion procedure that removes vesicles from HIV-1 preparations. While this approach has been successfully applied to virion preparations from several different cell types, some groups have concluded that "exosomes" from certain T cell lines, specifically Jurkat, do not contain CD45. If this interpretation is correct, then these vesicles could not be removed by CD45 immunoaffinity depletion. Here we show that dense vesicles produced by Jurkat and SupT1/CCR5 cells contain CD45 and are efficiently removed from preparations by CD45-immunoaffinity depletion. Also, contaminating cellular proteins were removed from virion preparations produced by these lines. Previously, the absence of CD45 from both "exosomes" and virions has been used to support the so called Trojan exosome hypothesis, namely that HIV-1 is simply an exosome containing viral material. The presence of CD45 on vesicles, including exosomes, and its absence on virions argues against a specialized budding pathway that is shared by both exosomes and HIV-1.

  13. Kaempferol Activates G₂-Checkpoint of the Cell Cycle Resulting in G₂-Arrest and Mitochondria-Dependent Apoptosis in Human Acute Leukemia Jurkat T Cells.

    Science.gov (United States)

    Kim, Ki Yun; Jang, Won Young; Lee, Ji Young; Jun, Do Youn; Ko, Jee Youn; Yun, Young Ho; Kim, Young Ho

    2016-02-01

    The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G2-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G2- arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G1 cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G2-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G2-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G2-checkpoint activation, which caused not only G2-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondriadependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δpsim loss, and caspase cascade activation.

  14. Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1.

    Science.gov (United States)

    Alhosin, Mahmoud; Abusnina, Abdurazzag; Achour, Mayada; Sharif, Tanveer; Muller, Christian; Peluso, Jean; Chataigneau, Thierry; Lugnier, Claire; Schini-Kerth, Valérie B; Bronner, Christian; Fuhrmann, Guy

    2010-05-01

    The salvage anti-tumoral pathway which implicates the p53-related p73 gene is not yet fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the p73-dependent pro-apoptotic pathway, by focusing on the anti-apoptotic and epigenetic integrator UHRF1 which is essential for cell cycle progression. For this purpose, we analyzed the effects of a known anti-neoplastic drug, thymoquinone (TQ), on the p53-deficient acute lymphoblastic leukemia (ALL) Jurkat cell line. Our results showed that TQ inhibits the proliferation of Jurkat cells and induces G1 cell cycle arrest in a dose-dependent manner. Moreover, TQ treatment triggers programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (DeltaPsim). TQ-induced apoptosis, confirmed by the presence of hypodiploid G0/G1 cells, is associated with a rapid and sharp re-expression of p73 and dose-dependent changes of the levels of caspase-3 cleaved subunits. These modifications are accompanied by a dramatic down-regulation of UHRF1 and two of its main partners, namely DNMT1 and HDAC1, which are all involved in the epigenetic code regulation. Knockdown of p73 expression restores UHRF1 expression, reactivates cell cycle progression and inhibits TQ-induced apoptosis. Altogether our results showed that TQ mediates its growth inhibitory effects on ALL p53-mutated cells via the activation of a p73-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets UHRF1.

  15. The oncogenic 70Z Cbl mutation blocks the phosphotyrosine binding domain-dependent negative regulation of ZAP-70 by c-Cbl in Jurkat T cells.

    Science.gov (United States)

    van Leeuwen, J E; Paik, P K; Samelson, L E

    1999-10-01

    T-cell receptor (TCR) engagement results in the activation of Src family (Lck and Fyn) and ZAP-70 protein tyrosine kinases, leading to tyrosine phosphorylation of multiple cellular substrates including the complex adapter protein c-Cbl. Moreover, Cbl is tyrosine phosphorylated upon engagement of growth factor receptors, cytokine receptors, and immunoreceptors and functions as a negative regulator of tyrosine kinase signalling pathways. Cbl associates via its phosphotyrosine binding (PTB) domain to the ZAP-70 pY292 negative regulatory phosphotyrosine. We recently demonstrated that the oncogenic Cbl mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in unstimulated Jurkat T cells. Here, we demonstrate that kinase-dead but not wild-type forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT activation. Moreover, 70Z Cbl does not upregulate NFAT in the ZAP-70-deficient P116 Jurkat T-cell line. The requirement for Fyn, Lck, and ZAP-70 is not due to tyrosine phosphorylation of 70Z Cbl, as mutation of all tyrosines in, or deletion of, the C-terminal region of 70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling pathways. Finally, the upregulation of NFAT observed upon ZAP-70 overexpression is blocked by Cbl in a PTB domain-dependent manner. We conclude that oncogenic 70Z Cbl acts as a dominant negative to block the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constitutive ZAP-70 signalling and activation of transcription factors.

  16. 甲基乙二醛对Jurkat细胞氧化应激及分泌细胞因子的影响%Effects of methylglyoxal on oxidative stress and cytokine profiles in cultured Jurkat cells

    Institute of Scientific and Technical Information of China (English)

    游捷; 余洪根; 彭雪峰; 刘晓红; 刘礼斌

    2011-01-01

    Objective:To investigate the effects of mthylglyoxal(MGO) on oxidative stress and cytokine profiles in cultured Jurkat cells for understanding more about the mechanism of diabetes accelerated athevosclerosis.Method:Jurkat cell which pre-stimulated by PHA were incubated with MGO for 24h.The cells survial rate was detected by MTT.ROS was quantitated by flow cytometry.Total and phosphory-lated p38 and JNK were assessed using Western blot.After incubation with MGO,the expression of TNFF-α and IFN-γ were tested by ELISA.Results:No changes in cells viability was observed when Jurkat cells were treated with 0-60 μmol/1Jurkat cells with MGO significantly induced ROS production.Co-incubation with NAC or AG decreased MGO-induced ROS production.Incubation of Jurkat cells with MGO results in significant increase in p38 and JNK phosphorylation.In addition MGO significantly increased the production of TNF-α and IFN-γ while p38,JNK inhibitor and NAC reversed the secretion of pro-inflammatory cytokines.Conclusion:These data demonstrate that MGO in-duced the release of TNF-α and IFN-γ in Jurkat cells viaoxidative stress,p38,JNK signaling pathway.%目的:探讨甲基乙二醛(MGO)对Jurkat细胞氧化应激及分泌细胞因子的影响.方法:不同浓度MGO作用PHA预刺激的Jurkat细胞,MTT检测细胞生长,流式细胞检测细胞内活性氧(ROS),Western blot检测p38 MAPK、JNK磷酸化水平,ELISA检测TNF-α、IFN-γ.结果:15~60 μmol/L MGO对Jurkat细胞生长无影响,但能使ROS呈浓度依赖性升高;N-乙酰-L-半胱氨酸(NAC)和氨基胍(AG)能明显抑制此作用.MGO作用30分钟,pp38/p38、pJNK/JNK明显升高.MGO诱导Jurkat细胞分泌TNF-α、IFN-γ;P38、JNK抑制剂及NAC能降低TNF-α、IFN-γ的分泌.结论:MGO能诱导Jurkat分泌TNF-α、IFN-γ;其机制可能通过氧化应激、P38、JNK信号通路.

  17. Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells.

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    Sylvie Rato

    Full Text Available HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.

  18. 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.

  19. Detailed Analysis of Apoptosis and Delayed Luminescence of Human Leukemia Jurkat T Cells after Proton Irradiation and Treatments with Oxidant Agents and Flavonoids

    Directory of Open Access Journals (Sweden)

    Irina Baran

    2012-01-01

    Full Text Available Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H2O2. 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H2O2 and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(PH level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G2/M arrest. Quercetin reduced apoptosis and prolonged the G2/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site.

  20. Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

    Science.gov (United States)

    Cubano, L. A.; Lewis, M. L.

    2001-01-01

    Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

  1. Increase of RhoB in {gamma}-radiation-induced apoptosis is regulated by c-Jun N-terminal kinase in Jurkat T cells

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    Kim, Chun-Ho [Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706 (Korea, Republic of); Won, Misun; Choi, Chung-Hae; Ahn, Jiwon; Kim, Bo-Kyung [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of); Song, Kyung-Bin [Department of Food Science and Technology, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Kang, Chang-Mo, E-mail: kangcm@kcch.re.kr [Laboratory of Cytogenetics and Tissue Regeneration, KIRAMS, Seoul 139-706 (Korea, Republic of); Chung, Kyung-Sook, E-mail: kschung@kribb.re.kr [Genome Research Center, KRIBB, Daejeon 305-806 (Korea, Republic of)

    2010-01-08

    The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in {gamma}-radiation-induced apoptosis. However, it is unclear how JNK activation and {gamma}-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by {gamma}-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. {gamma}-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the {gamma}-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to {gamma}-radiation.

  2. Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation*

    Science.gov (United States)

    Norambuena, Andrés; Metz, Claudia; Vicuña, Lucas; Silva, Antonia; Pardo, Evelyn; Oyanadel, Claudia; Massardo, Loreto; González, Alfonso; Soza, Andrea

    2009-01-01

    Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity. PMID:19276072

  3. Transcriptional regulation of the human IL5 gene by ionizing radiation in Jurkat T cells: evidence for repression by an NF-AT-like element.

    Science.gov (United States)

    Lu-Hesselmann, J; Messer, G; van Beuningen, D; Kind, P; Peter, R U

    1997-12-01

    Eosinophilia is often observed in patients with parasitic infections and atopic diseases like allergic asthma and atopic dermatitis. Additionally, it is a typical feature of the inflammatory reaction after therapeutic and accidental exposure to ionizing radiation. This uniquely specific phenomenon regulated by the cytokine interleukin 5 (IL-5) suggests specific control for IL5 gene expression. In this study, we generated promoter-CAT constructs containing different human IL-5 promoter regions spanning from positions -507 to +43. Transfection experiments in Jurkat T cells revealed that the promoter sequence from -57 to +43 was required for constitutive and inducible IL-5 promoter activity. Low baseline CAT activity could be enhanced by treatment with phenylmercuric acetate (PMA) or the combination of PMA and calcium ionophore. The promoter region between positions -97 and +43 showed responsiveness to low-dose X rays. Electrophoretic mobility shift assays demonstrated that the region from -117 to -97 was responsive to irradiation. Transcription factors specifically bound to this sequence showed a dose-dependent response to single doses of X rays between 1 and 8 Gy. Competition analysis indicated that the protein-DNA complexes at this region were related to the nuclear factor of activated T cells (NF-AT). Further confirmation was obtained by the addition of specific antibodies into protein-DNA reactions. For the first time, we have demonstrated that specific DNA binding of NF-ATp at the promoter region from -117 to -97 is involved in transcriptional regulation of the human IL5 gene in response to ionizing radiation.

  4. The molecular mechanism of curcumol on inducing cell growth arrest and apoptosis in Jurkat cells, a model of CD4⁺ T cells.

    Science.gov (United States)

    Wang, Heng; Wang, Yong; Jiang, Xiaoji; Wang, Zhizhong; Zhong, Bing; Fang, Yongfei

    2014-08-01

    CD4(+) T cells in rheumatoid arthritis (RA) express growth signaling pathway in association with deregulated growth and resistance to apoptosis. The janus kinase (Jak) 3 and signal transducer and activator of transcription (STAT) pathway play a critical role in interleukin-2 (IL-2)-induced CD4(+) T cell proliferation. The present study aimed to explore the anti-cell proliferation mechanism of curcumol, a pure monomer extracted from Chinese medical plant Rhizoma curcumae. Cell proliferation was determined using WST-1 assay after curcumol treatment. The cell cycle distribution and Bcl-2 protein expression were assessed by flow cytometry. The cellular morphology of apoptosis was evaluated by Hoechst 33258 staining. The expressions of phosphorylated-Jak3 (p-Jak3), p-STAT3, and p-STAT5a following IL-2 stimulation were determined by western blot analysis. The Electrophoretic Mobility Shift Assay was used to detect the DNA binding activities of transcription factors STAT3 and STAT5. The study results showed that curcumol could inhibit the IL-2-induced Jurkat cell proliferation in a concentration- and time-dependent manner in vitro. Curcumol could cause cell cycle arrest at the S phase, induce cell apoptosis, and inhibit the expression of Bcl-2 in a dose-dependent manner. Curcumol at 50μg/mL and Jak3 inhibitor ZM39923 could inhibit the phosphorylation of Jak3 and STAT5a. In conclusion, the underlying mechanism of curcumol on suppressing CD4(+) T cell proliferation and inducing apoptosis might partly be mediated by inhibition of Jak3-STAT5-related molecular activities and Bcl-2 expression, respectively; further studies are required in vivo to test the use of curcumol as a promising therapeutic option for RA.

  5. 夏枯草提取物对人T淋巴瘤Jurkat细胞增殖的影响及机制探讨%The effects and mechanism of action of Prunella vulgaris L extract on Jurkat human T lymphoma cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Changying Chen; Gang Wu; Mingzhi Zhang

    2009-01-01

    Objective: The aim of this study was to observe the effect of the Prunella vulgaris L extract on the Jurkat human T lymphoma cell line. Methods: Jurkat cells were cultivated with different concentrations of the extract from Prunella vulgaris L. The MTT assay and flow cytometry were employed to determine the cells' proliferation inhibition ratio and the apoptosis rates, respectively. Agarose gel electrophoresis was used to observe cellular DNA fragmentation, and westem blotting was used to observe changes in Bcl-2 and Bax protein expression. Results: The Prunella vulgaris L extract remarkably inhibited the proliferation of Jurkat cells. This inhibition exhibited dose dependence, with an IC50 of 20.23 + 0.31 IJg/mL. Agarose gel electrophoresis showed that the apoptosis strap became wider and brighter, and flow cytometry showed that the apoptosis rate increased in a concentration-dependent manner. Western blotting showed that Bcl-2 protein was down-regulated and Bax protein was up-regulated during apoptosis. Conclusion: The extract from Prunella vulgaris L induced apoptosis of Jurkat cells by down-regulating Bcl-2 protein and up-regulating Bax protein. These actions inhibited the growth of Jurkat ceils.

  6. Prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim reduces the association of Bcl-2 with Bak or Bim, provoking Bak activation and mitochondrial apoptosis in nocodazole-treated Jurkat T cells.

    Science.gov (United States)

    Han, Cho Rong; Jun, Do Youn; Lee, Ji Young; Kim, Young Ho

    2014-01-01

    Treatment of Jurkat T cells with the microtubule-depolymerizing agent nocodazole (NOC) caused prometaphase arrest and apoptosis. NOC-induced mitochondrial apoptotic events including Bak activation, Δψm loss, cytochrome c release, and caspase cascade activation were blocked by Bcl-2 overexpression. However, mitotic arrest, Cdc25C activation, upregulation of cyclin B1 levels, Cdk1 activation, Bcl-2 phosphorylation at Thr-56 and Ser-70, and Bim phosphorylation were retained. The treatment of Jurkat T cells concomitantly with NOC and the G1/S-blocking agent hydroxyurea resulted in G1/S arrest and complete abrogation of all apoptotic events. The association of Bcl-2 with Bim or Bak declined after the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, whereas the association of Bcl-2 with Bax remained relatively constant. Although Bax was redistributed from the cytosol to the mitochondria, resulting in an increase in the mitochondrial level of Bax following NOC treatment, the subcellular localization of Bcl-2, Bim, Bak and apoptosis-inducing factor was confined to the mitochondrial fraction irrespective of NOC treatment. Experiments using selective caspase inhibitors showed that mitochondria-dependent activation of caspase-9 and -3 was crucial for NOC-induced apoptosis. NOC-induced phosphorylation of Bcl-2 and Bim, Δψm loss, and mitochondria-dependent apoptotic events were significantly suppressed by a Cdk1 inhibitor roscovitine, but not by the JNK inhibitor SP600125 or the p38 MAPK inhibitor SB203580. These results show that the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, which was mediated by Cdk1, could reduce the association of Bcl-2 with Bak or Bim to allow Bak activation and mitochondrial apoptotic events in Jurkat T cells exposed to NOC.

  7. Effects of parathyroid hormone-related protein and macrophage inflammatory protein-1α in Jurkat T-cells on tumor formation in vivo and expression of apoptosis regulatory genes in vitro.

    Science.gov (United States)

    Shu, Sherry T; Dirksen, Wessel P; Lanigan, Lisa G; Martin, Chelsea K; Thudi, Nanda K; Werbeck, Jillian L; Fernandez, Soledad A; Hildreth, Blake E; Rosol, Thomas J

    2012-04-01

    Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) have been implicated in the pathogenesis of adult T-cell leukemia/lymphoma, but their effects on T-cells have not been well studied. Here we analyzed the functions of PTHrP and MIP-1α on T-cell growth and death both in vitro and in vivo by overexpressing either factor in human Jurkat T-cells. PTHrP or MIP-1α did not affect Jurkat cell growth in vitro, but PTHrP increased their sensitivity to apoptosis. Importantly, PTHrP and MIP-1α decreased both tumor incidence and growth in vivo. To investigate possible mechanisms, polymerase chain reaction (PCR) arrays and real-time reverse transcription (RT)-PCR assays were performed. Both PTHrP and MIP-1α increased the expression of several factors including signal transducer and activator of transcription 4, tumor necrosis factor α, receptor activator of nuclear factor κB ligand and death-associated protein kinase 1, and decreased the expression of inhibitor of DNA binding 1, interferon γ and CD40 ligand in Jurkat cells. In addition, MIP-1α also increased the expression of transcription factor AP-2α and PTHrP increased expression of the vitamin D3 receptor. These data demonstrate that PTHrP and MIP-1α exert a profound antitumor effect presumably by increasing the sensitivity to apoptotic signals through modulation of transcription and apoptosis factors in T-cells.

  8. Study on Up-Regulated Expressions of Anti-HIV Genes by vMIP-Ⅰin Jurkat Cell%vMIP-Ⅰ激活Jurkat细胞抗-HIV基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    尹小菲; 陈彬; 谭晓华; 罗燕; 杨磊

    2012-01-01

    本研究通过构建真核表达载体pEGFP-N3-vMIP-Ⅰ,电穿孔法将其转染至Jurkat细胞,荧光定量PCR检测vMIP-Ⅰ基因对Jurkat细胞内CCL5、APOBEC3G、APOBEC3F、等抗-HIV基因表达水平的影响,从而探讨vMIP-Ⅰ抗HIV感染的机制.结果显示:成功构建了pEGFP-N3-vMIP-Ⅰ载体,电穿孔转染效率达到40%左右,与转染空载体组相比,vMIP-Ⅰ转染组的Jurkat细胞内CCL5、A3G、A3F和MX1分别上调7.37倍、1.58倍、2.42倍和2.06倍.研究结果表明:vMIP-Ⅰ基因可激活Jurkat细胞内一些抗HIV相关基因的表达,这可能是vMIP-Ⅰ基因抗HIV感染的机制之一.%In this study, through the construction of eukaryotic expressive vector, the recombinant plasmids Pegfp-N3-Vmip- 玉 was transfected into Jurkat cells by electroporation. Then, by QRT-PCR technique, we detected the expression levels of anti-HIV genes: CCL5, APOBEC3F, MX1 in Jurkat cells which influenced by Vmip- 玉 gene and explored the mechanisms of Vmip- 玉 against HIV infection. The results he recombinant plasmids of Pegfp-N3-Vmip- 玉 was successfully constructed and electroporation transfection efficiency reached about 40%. In comparison with non-transfected gourp, the transfected Vmip- 玉 group can increase CCL5, A3G, A3F, and MX1 of Jurkat cells by 7.37, 1.58, 2.42 and 2.064 times seperately. The results suggest Vmip- 玉 can activate expressions of some relative anti-HIV genes in Jurkat cells, which probably is one of the mechanisms for anti-HIV infection.

  9. Study on Up-Regulated Expressions of Anti-HIV Genes by vMIP-Ⅰin Jurkat Cell%vMIP-Ⅰ激活Jurkat细胞抗-HIV基因表达的研究

    Institute of Scientific and Technical Information of China (English)

    尹小菲; 陈彬; 谭晓华; 罗燕; 杨磊

    2012-01-01

    In this study,through the construction of eukaryotic expressive vector,the recombinant plasmids pEGFP-N3-vMIP-Ⅰ was transfected into Jurkat cells by electroporation.Then,by QRT-PCR technique,we detected the expression levels of anti-HIV genes:CCL5,APOBEC3F,MX1 in Jurkat cells which influenced by vMIP-Ⅰ gene and explored the mechanisms of vMIP-Ⅰ against HIV infection.The results he recombinant plasmids of pEGFP-N3-vMIP-Ⅰ was successfully constructed and electroporation transfection efficiency reached about 40%.In comparison with non-transfected gourp,the transfected vMIP-Ⅰ group can increase CCL5,A3G,A3F,and MX1 of Jurkat cells by 7.37,1.58,2.42 and 2.064 times seperately.The results suggest vMIP-Ⅰ can activate expressions of some relative anti-HIV genes in Jurkat cells,which probably is one of the mechanisms for anti-HIV infection.%本研究通过构建真核表达载体pEGFP-N3-vMIP-Ⅰ,电穿孔法将其转染至Jurkat细胞,荧光定量PCR检测vMIP-Ⅰ基因对Jurkat细胞内CCL5、APOBEC3G、APOBEC3F、等抗-HIV基因表达水平的影响,从而探讨vMIP-Ⅰ抗HIV感染的机制。结果显示:成功构建了pEGFP-N3-vMIP-Ⅰ载体,电穿孔转染效率达到40%左右,与转染空载体组相比,vMIP-Ⅰ转染组的Jurkat细胞内CCL5、A3G、A3F和MX1分别上调7.37倍、1.58倍、2.42倍和2.06倍。研究结果表明:vMIP-Ⅰ基因可激活Jurkat细胞内一些抗HIV相关基因的表达,这可能是vMIP-Ⅰ基因抗HIV感染的机制之一。

  10. Benzo[a]pyrene affects Jurkat T cells in the activated state via the antioxidant response element dependent Nrf2 pathway leading to decreased IL-2 secretion and redirecting glutamine metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Murugaiyan, Jayaseelan; Rockstroh, Maxie; Wagner, Juliane [Department of Proteomics, Helmholtz-Centre for Environmental Research — UFZ, Permoserstr. 15, 04318 Leipzig (Germany); Baumann, Sven [Department of Metabolomics, Helmholtz-Centre for Environmental Research — UFZ, Permoserstr. 15, 04318 Leipzig (Germany); Schorsch, Katrin [Department of Proteomics, Helmholtz-Centre for Environmental Research — UFZ, Permoserstr. 15, 04318 Leipzig (Germany); Trump, Saskia; Lehmann, Irina [Department of Environmental Immunology, Helmholtz-Centre for Environmental Research — UFZ, Permoserstr. 15, 04318 Leipzig (Germany); Bergen, Martin von [Department of Proteomics, Helmholtz-Centre for Environmental Research — UFZ, Permoserstr. 15, 04318 Leipzig (Germany); Department of Environmental Immunology, Helmholtz-Centre for Environmental Research — UFZ, Permoserstr. 15, 04318 Leipzig (Germany); Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Aalborg (Denmark); Tomm, Janina M., E-mail: Janina.tomm@ufz.de [Department of Proteomics, Helmholtz-Centre for Environmental Research — UFZ, Permoserstr. 15, 04318 Leipzig (Germany)

    2013-06-15

    There is a clear evidence that environmental pollutants, such as benzo[a]pyrene (B[a]P), can have detrimental effects on the immune system, whereas the underlying mechanisms still remain elusive. Jurkat T cells share many properties with native T lymphocytes and therefore are an appropriate model to analyze the effects of environmental pollutants on T cells and their activation. Since environmental compounds frequently occur at low, not acute toxic concentrations, we analyzed the effects of two subtoxic concentrations, 50 nM and 5 μM, on non- and activated cells. B[a]P interferes directly with the stimulation process as proven by an altered IL-2 secretion. Furthermore, B[a]P exposure results in significant proteomic changes as shown by DIGE analysis. Pathway analysis revealed an involvement of the AhR independent Nrf2 pathway in the altered pr