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Sample records for hl-60 cells exposed

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

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  5. Gossypol-Induced Differentiation in Human Leukemia HL-60 Cells

    PubMed Central

    Wang, Wen-Qing; Li, Rong; Bai, Qing-Xian; Liu, Yu-Hong; Zhang, Wei-Ping; Wang, Juan-Hong; Wang, Zhe; Li, Yuan-Fei; Chen, Xie-Qun; Huang, Gao-Sheng

    2006-01-01

    The main treatment of leukemia is traditional radiochemotherapy, which is associated with serious side effects. In the past twenty years, differentiation was found as an important effective measure to treat leukemia with fewer side effects. Gossypol, a natural compound which has been used as an effective contraceptive drug, has been proposed to be a potent drug to treat leukemia, but the differentiation effect has not been studied. In the present study, we investigated the pro-differentiated effects, in vitro, of gossypol on the classic human myeloid leukemia HL-60 cell line. The effects of gossypol were investigated by using morphological changes, nitroblue tetrazolium (NBT) reduction, surface markers, cell-cycle analysis and Western blot analysis, etc. When HL-60 cells were incubated with low concentrations of gossypol (2-5μM) for 48hr, a prominent G0/G1 arrest was observed. At 96 hr of treatment, 90% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, and increase in cell surface expression of some molecules were detected. This study is the first to identify gossypol’s pro-differentiated effects on the leukemia cell line, and it induced differentiation through the PBK (PDZ-binding kinase)/TOPK (T-LAKcell-originated protein kinase) (PBK/TOPK) pathway. It is concluded that gossypol could induce differentiation in the leukemia HL-60 cells, and it may be a potential therapeutic agent, chemoprevention or chemotherapeutic adjuvant especially in combination drug therapy for leukemia. PMID:23675007

  6. Changes of reactive oxygen and nitrogen species and mitochondrial functioning in human K562 and HL60 cells exposed to ionizing radiation.

    PubMed

    Saenko, Yuriy; Cieslar-Pobuda, Artur; Skonieczna, Magdalena; Rzeszowska-Wolny, Joanna

    2013-10-01

    Free radicals generated by mitochondria are candidates for mediating long-lasting effects of radiation on cells, including genetic instability. To better understand the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in these long-term effects we assayed ROS and RNS levels, the mitochondrial membrane potential and mass, and the frequency of DNA strand breaks, apoptosis and necrosis in human leukemic cells (K562 and HL60) after 12 Gy of X irradiation. An increase in intracellular ROS level was observed immediately post-irradiation, and about 24 h later a second increase of ROS was accompanied by increase in nitrogen oxide, mitochondrial potential and mitochondrial mass in both cell types. The second peak of ROS level was partially inhibited by rotenone, an inhibitor of mitochondrial complex I, in K562 but not in HL60 cells suggesting that the sources of ROS differed in the two cell types. The frequency of DNA breaks showed kinetics similar to ROS levels, with a sharp peak immediately after irradiation and a second increase 24 and 48 h later, which was significantly higher in K562 cells. Forty-eight hours after irradiation an increase in the frequency of apoptotic cells was observed in both cell lines, which became larger and statistically significant in K562 cells after inhibition of mitochondrial complex I. Our results show that ionizing radiation activates cellular processes which produce long-lasting ROS and RNS radicals, which may have different sources in different cell types and could participate in cellular signaling networks important for radiosensitivity and mode of cell death.

  7. The role of copper in HL-60 cell differentiation

    SciTech Connect

    Bae, B.; Percival, S.S. )

    1991-03-15

    Copper deficiency in humans has been shown to result in neutropenia. This research asks what is copper's function in the development of neutrophils HL-60 cells, a promyelocyte cell line, was induced to differentiate towards the granulocytic lineage with 1 {mu}M retinoic acid for 5 days. Both noninduced and induced cells were incubated in either complete medium or in medium supplemented with 8 {mu}M copper. Intracellular copper levels, Cu/Zn superoxide dismutase activity and the respiratory burst (RB) activity of the cells were measured. The respiratory burst of neutrophils is a measure of cellular function and degree of differentiation. Induced cells, as expected, showed greater RB activity than the non-induced cells. Copper supplementation, however, had no effect on this activity. Differentiated HL-60 cells had two times more intracellular copper but ten times less Cu/Zn-SOD activity. Copper supplementation enhanced Cu/Zn-SOD activity in both noninduced and induced cells. This suggests that the availability of intracellular copper is important in expressing Cu/ZN-SOD activity and that differentiated cells, although they have more intracellular copper under basal conditions, cannot utilize that copper for Cu/Zn-SOD enzyme activity. When supplemental copper was provided during differentiation, Cu/Zn-SOD activity was maintained.

  8. Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells.

    PubMed

    Aquino, A; Warren, B S; Omichinski, J; Hartman, K D; Glazer, R I

    1990-01-30

    The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells. PMID:2302237

  9. Glycyrrhizin induces apoptosis in human stomach cancer KATO III and human promyelotic leukemia HL-60 cells.

    PubMed

    Hibasami, Hiroshige; Iwase, Hiroshi; Yoshioka, Kazumi; Takahashi, Hidehisa

    2005-08-01

    We have investigated the effects of glycyrrhizin (GL) on cell proliferations of human stomach cancer KATO III and promyelotic leukemia HL-60 cells, and on DNA of those cell lines. GL displayed growth inhibitory effect against KATO III and HL-60 cells. Morphological change showing apoptotic bodies was observed in the KATO III and HL-60 cells treated with GL. The fragmentation of DNA by GL to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in both cell lines. Caspase inhibitors such as Z-VAD-FMK and Z-Asp-CH2-DCB suppressed the DNA fragmentation induced by GL. The data of the present study show that the suppression of KATO III and HL-60 cell-growth by GL results from the induction of apoptosis by GL, and that caspase is involved in the induction of apoptosis by GL in these cells. PMID:16012754

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

    PubMed

    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.

  11. Identification of a retinoic acid responsive aldoketoreductase expressed in HL60 leukaemic cells.

    PubMed

    Mills, K I; Gilkes, A F; Sweeney, M; Choudhry, M A; Woodgate, L J; Bunce, C M; Brown, G; Burnett, A K

    1998-11-27

    Neutrophil and monocyte differentiation can be induced in HL60 leukaemia cells by all-trans-retinoic acid (ATRA) and 1alpha,25-dihydroxyvitamin D3 (D3), respectively, whose differentiating effects can be enhanced by exposure to 'anti-inflammatory agents' and steroids. We have provided evidence that this potentiation is via inhibition of the activity of an enzyme of the aldoketoreductase (AKR) family, but had failed to identify expression of known AKRs in HL60 cells. In this study, we have identified a previously unclassified aldoketoreductase family member (termed HAKR e) that is expressed in HL60 cells. HAKR e is dramatically and transiently up-regulated in HL60 cells within 24 h of exposure to ATRA, further supporting the proposition that a member(s) of this family of enzymes play(s) a role in controlling cell growth and/or differentiation.

  12. Activation of tracheal smooth muscle responsiveness by fMLP-treated HL-60 cells and neutrophils.

    PubMed

    Munoz, N M; Hamann, K J; Vita, A; Cozzi, P J; Baranowski, S; Solway, J; Leff, A R

    1993-03-01

    We assessed the effects of cultured human promyelocytic leukemia (HL-60) cells and polymorphonuclear leukocytes (neutrophils) isolated from peripheral human blood on tracheal smooth muscle responsiveness in 40 male Hartley guinea pigs. Undifferentiated HL-60 cells (16-25 passages) were activated in vitro by incubation with 1 microM f-Met-Leu-Phe (fMLP), and force of contraction was measured isometrically using an in situ preparation of tracheal smooth muscle. Increasing concentrations of acetylcholine (ACh; 10(-10) to 10(-6) mol/cm2 tracheal surface) were applied topically to the epithelial surface pretreated with 4 x 10(6) fMLP-activated HL-60 cells, 4 x 10(6) fMLP-activated neutrophils, 4 x 10(6) sham-activated HL-60 cells, fMLP+vehicle, or vehicle control. Topical application of fMLP-activated HL-60 cells caused a maximum active tension (AT) of 1.13 +/- 0.2 g/cm after 5 min; fMLP-activated neutrophils, sham-activated HL-60 cells, or fMLP+vehicle had no effect. The fMLP-activated HL-60 cells also caused substantial augmentation of tracheal contraction to ACh (P < 0.05 vs. sham-activated cells for all concentrations > 10(-9) mol/cm2). Although fMLP treatment caused 247 +/- 28% increase from baseline level in O2-. production, neither direct contraction nor augmentation of muscarinic stimulation was demonstrated after topical application of 4 x 10(6) neutrophils. In 12 other preparations, fMLP-activated HL-60 cells were pretreated with either 10 microM indomethacin (Indo) or 100 microM A63162, a 5-lipoxygenase inhibitor. Pretreatment with Indo caused complete blockade of direct tracheal contraction and 88 +/- 13% blockade of muscarinic augmentation; there was no effect after A63162.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8460711

  13. Xestospongin C induces monocytic differentiation of HL60 cells through activation of the ERK pathway.

    PubMed

    Moon, Dong-Oh; Asami, Yukihiro; Kim, Mun-Ock; Jang, Jae-Hyuk; Kim, Bo Yeon; Ahn, Jong Seog; Kim, Gi-Young; Yun, Sung Gyu

    2013-05-01

    Xestospongin C (XC), which is a group of macrocyclic bis-1-oxaquinolizidines, is a potent inhibitor of sarcoendoplasmic reticulum calcium transport ATPase and IP3 receptor. Nevertheless, very less information is available regarding whether XC induces AML differentiation. We investigated the potential role of XC in the differentiation of human leukemia HL60 cells and mechanisms underlying XC actin. XC treatment inhibited proliferation by inducing G1-phase cell cycle arrest in the HL60 cells. In addition, XC induced differentiation of HL60 cells into the CD14(+) monocytic lineage, which was indicated by morphological changes, nitroblue tetrazolium reduction assay, and expressions of CD11b and CD14 surface antigens. Our results also showed that XC promotes phagocytic activity and granularity in HL60 cells, suggesting that the cells are functionally activated. Furthermore, XC enhanced tumor necrosis factor (TNF)-α-mediated cytotoxic effect by increasing the numbers of TNF receptors. Moreover, we showed that XC activates extracellular signal-regulated kinase (ERK) pathway in the differentiation stages. Inhibition of ERK activation using PD98059 significantly decreased NBT+HL60 cells induced by XC treatment. Taken together, the results show that XC promotes monocytic differentiation of HL60 cells via ERK pathway activation, suggesting that XC could be a candidate for use as a differentiation-inducing agent for AML treatment.

  14. α-tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells

    PubMed Central

    HUANG, HUARONG; CHEN, SHAOHUA; VAN DOREN, JEREMIAH; LI, DONGLI; FARICHON, CHELSEA; HE, YAN; ZHANG, QIUYAN; ZHANG, KUN; CONNEY, ALLAN H; GOODIN, SUSAN; DU, ZHIYUN; ZHENG, XI

    2015-01-01

    α-tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α-tomatine on human myeloid leukemia HL-60 cells were investigated. Treatment of HL-60 cells with α-tomatine resulted in growth inhibition and apoptosis in a concentration-dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL-60 cells. Growth inhibition and apoptosis induced by α-tomatine in HL-60 cells was partially abrogated by addition of cholesterol indicating that interactions between α-tomatine and cell membrane-associated cholesterol may be important in mediating the effect of α-tomatine. Activation of nuclear factor-κB by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate failed to prevent apoptosis in HL-60 cells treated with α-tomatine. In animal experiments, it was found that treatment of mice with α-tomatine inhibited the growth of HL-60 xenografts in vivo. Results from the present study indicated that α-tomatine may have useful anti-leukemia activities. PMID:25625536

  15. Apoptosis induction by aluminum phthalocyanine tetrasulfonate-based sonodynamic therapy in HL-60 cells

    NASA Astrophysics Data System (ADS)

    Iwase, Yumiko; Yumita, Nagahiko; Nishi, Koji; Kuwahara, Hiroyuki; Fukai, Toshio; Ikeda, Toshihiko; Chen, Fu-shih; Momose, Yasunori; Umemura, Shin-ichiro

    2015-07-01

    The present study aims to investigate sonodynamically-induced apoptosis using the phthalocyanine, chloroaluminum phthalocyanine tetrasulfonate (AlPcTS). HL-60 cells were exposed to ultrasound for up to 3 min in the absence and presence of AlPcTS. Apoptosis was analyzed by cell morphology, DNA fragmentation, and caspase-3 activity. Electron spin resonance was used to measure reactive oxygen species. The number of apoptotic cells showing membrane blebbing and cell shrinkage after combined treatment (ultrasound and AlPcTS) was significantly higher than following other treatments, including ultrasound alone and AlPcTS alone. Furthermore, DNA ladder formation, caspase-3 activation and enhanced nitroxide generation were observed in cells treated with ultrasound and AlPcTS. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. The significant reduction by histidine indicated that ultrasonically generated reactive oxygen species, such as singlet oxygen, is an important mediator of sonodynamically-induced apoptosis.

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

    PubMed

    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

  17. Manipulation of neutrophil-like HL-60 cells for the study of directed cell migration.

    PubMed

    Millius, Arthur; Weiner, Orion D

    2010-01-01

    Many cells undergo directed cell migration in response to external cues in a process known as chemotaxis. This ability is essential for many single-celled organisms to hunt and mate, the development of multicellular organisms, and the functioning of the immune system. Because of their relative ease of manipulation and their robust chemotactic abilities, the neutrophil-like cell line (HL-60) has been a powerful system to analyze directed cell migration. In this chapter, we describe the maintenance and transient transfection of HL-60 cells and explain how to analyze their behavior with two standard chemotactic assays (micropipette and EZ-TAXIS). Finally, we demonstrate how to fix and stain the actin cytoskeleton of polarized cells for fluorescent microscopy imaging.

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

    PubMed

    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.

  19. Differentiated HL-60 promyelocytic leukaemia cells produce a factor inducing differentiation.

    PubMed

    Djulbegović, B; Christmas, S E; Moore, M

    1987-01-01

    The bipotential human promyelocytic leukaemia cell line HL-60 can be induced to differentiate into monocytic or granulocytic cells by treatment with 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or dimethylsulphoxide (DMSO) respectively. Conditioned media (CM) from 1,25(OH)2D3- or DMSO-treated cells were able to induce monocytic differentiation in fresh HL-60 cells as measured by induction of non-specific esterase and macrophage surface markers. CM from 1,25(OH)2D3-treated cells also led to a dose dependent loss of proliferative capacity in soft agar colony assays. These effects were not due to a toxic effect of the CM or to residual inducer present in the CM. gamma-interferon and GM-CSF were apparently not responsible for these effects. CM from the human histiocytic lymphoma cell line U937 led to only a low level of induction of macrophage differentiation in fresh HL-60 cells. The defect in HL-60 leukaemic cells may therefore be at the level of induction of an autonomously-produced differentiation factor.

  20. Nuclear CD38 in retinoic acid-induced HL-60 cells

    SciTech Connect

    Yalcintepe, Leman . E-mail: lemany@istanbul.edu.tr; Albeniz, Isil; Adin-Cinar, Suzan; Tiryaki, Demir; Bermek, Engin; Graeff, Richard M.; Lee, Hon Cheung

    2005-02-01

    The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD{sup +} and hydrolysis of either NAD{sup +} or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD{sup +} glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a {approx}43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the {approx}43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.

  1. Viability study of HL60 cells in contact with commonly used microchip materials.

    PubMed

    Wolbers, Floor; ter Braak, Paul; Le Gac, Severine; Luttge, Regina; Andersson, Helene; Vermes, Istvan; van den Berg, Albert

    2006-12-01

    This paper presents a study in which different commonly used microchip materials (silicon oxide, borosilicate glass, and PDMS) were analyzed for their effect on human promyelocytic leukemic (HL60) cells. Copper-coated silicon was analyzed for its toxicity and therefore served as a positive control. With quantitative PCR, the expression of the proliferation marker Cyclin D1 and the apoptosis marker tissue transglutaminase were measured. Flow cytometry was used to analyze the distribution through the different phases of the cell cycle (propidium iodide, PI) and the apoptotic cascade (Annexin V in combination with PI). All microchip materials, with the exception of Cu, appeared to be suitable for HL60 cells, showing a ratio apoptosis/proliferation (R(ap)) comparable to materials used in conventional cell culture (polystyrene). These results were confirmed with cell cycle analysis and apoptosis studies. Precoating the microchip material surfaces with serum favor the proliferation, as demonstrated by a lower R(ap) as compared to uncoated surfaces. The Cu-coated surface appeared to be toxic for HL60 cells, showing over 90% decreased viability within 24 h. From these results, it can be concluded that the chosen protocol is suitable for selection of the cell culture material, and that the most commonly used microchip materials are compatible with HL60 culturing. PMID:17124709

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

    PubMed

    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 < 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

  3. [Enhancement of Fas-mediated apoptosis in leukemic cell line HL-60 by Bay 11 - 7082].

    PubMed

    Wang, Li; Liu, Ling-Bo; Li, Lei; Zou, Ping

    2007-10-01

    The aim of study was to explore the effects of NF-kappaB inhibitor Bay 11 - 7082 on Fas/FasL system and Fas-mediated apoptosis in HL-60 cells. The mRNA and protein expression levels of Fas, FasL and XIAP after treatment with Bay 11 - 7082 were detected by RT-PCR and FCM respectively. The level of sFasL was detected by ELISA before and after treatment with Bay 11 - 7082; apoptosis was detected by FCM before and after treatment with Bay 11 - 7082. The results showed that after treating HL-60 cells with Bay 11 - 7082, the mRNA and protein levels of FasL and XIAP were lower than that of controls, the difference was significant by statistic analysis (p < 0.05). Neither the mRNA and protein levels of Fas, nor the level of sFasL changed significantly (p > 0.05). Apoptotic rate of HL-60 cells treated with Bay 11 - 7082 was significantly higher as compared with controls (p < 0.05). It is concluded that Bay 11 - 7082 can enhance Fas-mediated apoptosis in HL-60 cells by down-regulation of FasL and XIAP levels.

  4. [Effect of nimodipine on mechanisms of HL-60 cell apoptosis induced by cytarabine].

    PubMed

    Sun, Li-Rong; Gao, Bin-Chang; Pang, Xiu-Ying; Lu, Yuan; Li, Xue-Rong; Song, Ai-Qin

    2007-02-01

    The aim was to study the mechanisms of HL-60 cell apoptosis induced by nimodipine (NMDP) and cytarabine (Ara-C). The DNA fragment was detected by agarose gel electrophoresis. The expressions of bcl-2 and bax gene proteins related with apoptosis were investigated by immunohistochemistry. The results showed that HL-60 cell apoptosis rate had been increasing in the experimental groups compared with the control group since culturing 8 hours. The expression of Bcl-2 protein was lower and the expression of Bax protein was higher in the experimental groups than that in the control group, while ratio of bcl-2/bax was lower in the experimental groups than that in the control group. It is concluded that NMDP and Ara-C induce apoptosis of HL-60 cells, and the mechanism of apoptosis induced by them may down-regulate the expression of bcl-2 gene and up-regulate the expression of bax gene. The mechanism of HL-60 cell apoptosis induced by Ara-C and NMDP is probably associated with the down-regulation of Bcl-2 protein expression.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27588133

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

    PubMed

    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.

  7. The antiproliferative effect of hexadecylphosphocholine toward HL60 cells is prevented by exogenous lysophosphatidylcholine.

    PubMed

    Boggs, K; Rock, C O; Jackowski, S

    1998-01-01

    The mechanisms that account for the anti-proliferative properties of the biologically active lysophospholipid analog hexadecylphosphocholine (HexPC) were investigated in HL60 cells. HexPC inhibited the incorporation of choline into phosphatidylcholine and the pattern of accumulation of soluble choline-derived metabolites pinpointed CTP:phosphocholine cytidylyltransferase (CT) as the inhibited step in vivo. HexPC also inhibited recombinant CT in vitro. HexPC treatment led to accumulation of cells in G2/M phase, triggered DNA fragmentation and caused morphological changes associated with apoptosis. The supplementation of HexPC-treated cells with exogenous lysophosphatidylcholine (LPC) completely reversed the cytotoxic effects of HexPC and restored HL60 cell proliferation in the presence of the drug. LPC provided an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC. This result contrasted with the response of HL60 cells to 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) where LPC overcame the cytotoxic effects but did not support continued cell proliferation. Morphological integrity, DNA stability and cell viability were maintained in cells treated with LPC plus either antineoplastic agent. Thus the inhibition of phosphatidylcholine biosynthesis at the CT step accounts for the cytotoxicity of both HexPC and ET-18-OCH3 which is overridden by providing an alternate pathway for phosphatidylcholine synthesis via the acylation of exogenous LPC.

  8. Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells.

    PubMed Central

    Bokoch, G M; Prossnitz, V

    1992-01-01

    The formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation. Images PMID:1310693

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

    PubMed

    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. PMID:14758720

  10. Z-ajoene induces apoptosis of HL-60 cells: involvement of Bcl-2 cleavage.

    PubMed

    Li, Min; Min, Ji-Mei; Cui, Jing-Rong; Zhang, Li-He; Wang, Kui; Valette, Annie; Davrinche, Christian; Wright, Michel; Leung-Tack, Jeanne

    2002-01-01

    Garlic organosulfur components exhibit antitumor activity, but the molecular mechanisms underlying these effects have not been well characterized. We showed that Z-ajoene, a sulfur-rich compound purified from garlic, induced time- and dose-dependent apoptosis in HL-60 cells. This process implied the activation of caspase-3 and the cleavage of the antiapoptotic protein Bcl-2. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-[OMe]-fluoromethylketone inhibited Bcl-2 cleavage and apoptosis induced by Z-ajoene. This effect was partially prevented by treatment of HL-60 cells with the antioxidant N-acetylcysteine. Hence, the transmission of apoptotic signal induced by Z-ajoene involved a reactive oxygen species-dependent pathway leading to caspase-dependent Bcl-2 cleavage.

  11. Sonodynamic therapy induces apoptosis of human leukemia HL-60 cells in the presence of protoporphyrin IX.

    PubMed

    Su, Xiaomin; Wang, Xiaobing; Zhang, Kun; Yang, Shuang; Liu, Quanhong; Leung, Albert W; Xu, Chuanshan; Wang, Pan

    2016-04-01

    Sonodynamic therapy (SDT) is expected to be a novel therapeutic strategy for tumor. The protoporphyrin IX disodium salt (PpIX), a photosensitizer, can be activated by ultrasound. The present study aims to investigate apoptosis of HL-60 cells induced by PpIX-mediated SDT. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to examine cell toxicity. Apoptosis was detected using Annexin V-PE/7-amino-actinomycin D (7-AAD) double staining. Detection of apoptotic bodies was examined by Hoechst33342 (HO) staining. Western blotting was used to analyze the protein of caspase-3 and poly ADP-ribose polymerase (PARP). Intracellular reactive oxygen species (ROS) was detected by a flow cytometer after exposures. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound were significantly boosted. In addition, as determined by Annexin V-PE/7-AAD staining, SDT significantly induced HL-60 cell apoptosis, the obvious nuclear condensation was also found with HO staining at 4 hours post-SDT treatment. Furthermore, Western blotting showed visible enhancement of caspase-3 and PARP cleavage in this process. Besides, intracellular ROS production was significantly enhanced after SDT. Our findings demonstrate that PpIX-mediated SDT could induce apoptosis on HL-60 cells, suggesting that apoptosis is an important mechanism of cell death induced by PpIX-mediated SDT. PMID:26891272

  12. Lipid Raft Is Required for PSGL-1 Ligation Induced HL-60 Cell Adhesion on ICAM-1

    PubMed Central

    Xu, Tingshuang; Liu, Wenai; Luo, Jixian; Li, Chunfeng; Ba, Xueqing; Ampah, Khamal Kwesi; Wang, Xiaoguang; Jiang, Yong; Zeng, Xianlu

    2013-01-01

    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. PMID:24312591

  13. Epigenetic modification and preliminary investigation of the mechanism of the immune evasion of HL-60 cells.

    PubMed

    Liu, Jin Hong; Bian, Yong Mei; Xie, Yi; Lu, Dao Pei

    2015-07-01

    The aim of the present study was to explore the effect of epigenetic modification of class II transactivator (CIITA) methylation on histocompatibility complex (MHC) class II expression and the immune evasion of leukemia HL-60 cells. HL-60 cells were treated with various concentrations of 5-aza-2'deoxycytidine (5-Aza-CdR) and 0.5 µmol/l suberoylanilide hydroxamic acid (SAHA) for 24 h and then stimulated by interferon γ (IFN-γ) for 48 h. The mRNA levels of MHC class I, II and co-stimulatory molecules were quantified by reverse transcription polymerase chain reaction (RT-PCR). The levels of CIITA protein were determined by western blot analysis, and the CpG island methylation ratios in the CIITA promoter IV (CIITApIV) were analyzed by bisulfite-sequencing PCR (BSP). MHC I as well as the co-stimulatory molecules CD40 and CD80 were significantly increased following treatment with 5-Aza-CdR + SAHA + IFN-γ (epigenetic groups) compared with those in the control group and IFN-γ group (P<0.05). The expression of MHC class II and CIITA was restored and increased in an 5-Aza-CdR concentration-dependent manner in the three epigenetic groups. The results of the BSP assay showed that the methylation rate of CIITApIV CpG sites decreased with the treatment of epigenetic modification and negatively correlated to the 5-Aza-CdR concentration. This demonstrated that the negative expression of CIITA protein was the key reason for the loss of MHC II expression in HL-60 cells. The results of the present study may help to illustrate the mechanism of immune evasion in HL-60 cells. PMID:25815463

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

    PubMed

    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.

  15. Differential cell fates induced by all-trans retinoic acid-treated HL-60 human leukemia cells.

    PubMed

    Ozeki, Munetaka; Shively, John E

    2008-09-01

    HL-60 human leukemia cells, differentiated into a neutrophil lineage by all-trans retinoic acid (ATRA) treatment, express three members of the carcinoembryonic antigen (CEA) gene family, CEA-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM3 (CD66d), and CEACAM6 (CD66c). CD66d is a neutrophil lineage-specific marker, and CD66a and CD66c are found on epithelial and other cells. HL-60 cells continuously treated with ATRA underwent apoptosis, and cells transiently treated for 1 day underwent cell-cycle arrest, entered into senescence, and exhibited reduced apoptosis with CD66-positive cells accounting for the majority of live cells. CD66 antigens were also induced in NB4 leukemic cells upon continuous treatment with ATRA. NB4 cells underwent apoptosis with a higher frequency in transient versus continuous-treated cells (38% vs. 19% at Day 5), in contrast to HL-60 cells that underwent cell-cycle arrest and senescence when transiently treated with ATRA. CD66 antigens were not induced in transient, ATRA-treated NB4 cells compared with HL-60 cells. Cell-cycle arrest in HL-60 cells involved reduction in expression levels of p21, cyclins D and E, while Rb1 exhibited reduction in protein levels without changes in mRNA levels over the time course of ATRA treatment. Analysis of several proapoptotic proteins implicated the activation of calpain and cleavage of Bax in the intrinsic apoptotic pathway, similar to published studies about the apoptosis of neutrophils. CD1d expression was also induced by ATRA in HL-60 cells and ligation with anti-CD1d antibody-induced apoptosis. In contrast, CD1d-positive primary monocytes were protected from spontaneous apoptosis by CD1d ligation. These studies demonstrate distinct cell fates for ATRA-treated HL-60 cells that provide new insights into ATRA-induced cell differentiation.

  16. Modulation of cytokine production by interferential current in differentiated HL-60 cells.

    PubMed

    Sontag, W

    2000-04-01

    The influence of interferential current (IFC) on the release of four cytokines was investigated. IFC is an amplitude-modulated 4 kHz current used in therapeutic applications. Human promyelocytes (HL-60) were differentiated to monocytes/macrophages by treatment with calcitriol. Release of tumor necrosis factor alpha (TNFalpha) and interleukines 1beta, 6, and 8 (IL-1beta, IL-6, and IL-8) into the supernatant was measured after exposure to IFC at different modulation frequencies. TNFalpha release was stimulated about twofold by 4 kHz sine waves alone. The influences of exposure time (5-30 min) and current density (2.5-2500 microA/c m(2)) were tested. A maximum field effect was found at an exposure time of 15 min and a current density of 250 microA/cm(2). With these exposure conditions (15 min and 250 microA/cm(2) ), cells were treated at different modulation frequencies and reacted for TNFalpha, IL-1beta, and IL-8 release in a complex manner. Within the frequencies studied (0-125 Hz), we found stimulation as well as depression of the release. In a second run the cells were activated by pretreatment with 10 microg/ml lipopolysaccharide (LPS) and exposed in the same way as the nonactivated cells. Again the modulation frequency influenced, in a complex way, the induction of TNFalpha, IL-1beta, and IL-8, resulting in a pattern of stimulation and depression of release different from that found in nonactivated cells. For IL-6 production no significant changes were detected in activated or non-activated cells.

  17. Cdc6 is a rate-limiting factor for proliferative capacity during HL60 cell differentiation

    SciTech Connect

    Barkley, Laura R.; Hong, Hye Kyung; Kingsbury, Sarah R.; James, Michelle; Stoeber, Kai Williams, Gareth H.

    2007-10-15

    The DNA replication (or origin) licensing pathway represents a critical step in cell proliferation control downstream of growth signalling pathways. Repression of origin licensing through down-regulation of the MCM licensing factors (Mcm2-7) is emerging as a ubiquitous route for lowering proliferative capacity as metazoan cells exit the cell division cycle into quiescent, terminally differentiated and senescent 'out-of-cycle' states. Using the HL60 monocyte/macrophage differentiation model system and a cell-free DNA replication assay, we have undertaken direct biochemical investigations of the coupling of origin licensing to the differentiation process. Our data show that down-regulation of the MCM loading factor Cdc6 acts as a molecular switch that triggers loss of proliferative capacity during early engagement of the somatic differentiation programme. Consequently, addition of recombinant Cdc6 protein to in vitro replication reactions restores DNA replication competence in nuclei prepared from differentiating cells. Differentiating HL60 cells over-expressing either wild-type Cdc6 or a CDK phosphorylation-resistant Cdc6 mutant protein (Cdc6A4) exhibit an extended period of cell proliferation compared to mock-infected cells. Notably, differentiating HL60 cells over-expressing the Cdc6A4 mutant fail to down-regulate Cdc6 protein levels, suggesting that CDK phosphorylation of Cdc6 is linked to its down-regulation during differentiation and the concomitant decrease in cell proliferation. In this experimental model, Cdc6 therefore plays a key role in the sequential molecular events leading to repression of origin licensing and loss of proliferative capacity during execution of the differentiation programme.

  18. Neutrophilic differentiation modulates the apoptotic response of HL-60 cells to sodium butyrate and sodium valproate.

    PubMed

    Vrba, J; Dolezel, P; Ulrichova, J

    2010-01-01

    Differentiation of myeloid leukemic cells may result in less sensitivity to various apoptotic stimuli. We examined whether human leukemia HL-60 cells differentiating by all-trans retinoic acid (ATRA) acquired resistance to the apoptogenic activity of two histone deacetylase (HDAC) inhibitors, butyrate and valproate. In undifferentiated cells, the cytotoxicity of both butyrate and valproate was associated with activation of the intrinsic apoptotic pathway since we observed dissipation of mitochondrial membrane potential, induction of caspase-9 and caspase-3 activities, appearance of sub-G1 DNA and loss of plasma membrane asymmetry and/or integrity. Both HDAC inhibitors were also able to induce accumulation of undifferentiated cells in the G0/G1 phase of the cell cycle. ATRA was found to enhance the apoptotic effect of both butyrate and valproate in undifferentiated cells. This aside, ATRA appeared to synergize with butyrate in the induction of the G0/G1 cell cycle arrest. In cells pretreated for 72 h with ATRA, butyrate and valproate in combination with ATRA induced lower dissipation of mitochondrial membrane potential and weaker apoptotic and/or necrotic changes in plasma membrane, whereas DNA fragmentation was not diminished compared to undifferentiated cells. Similar results were also obtained when butyrate or valproate were combined with another neutrophilic differentiation inducer, dimethyl sulfoxide. We conclude that neutrophilic differentiation modulates but does not abrogate the apoptotic response of HL-60 cells to butyrate and valproate, and nuclei are preferentially affected during apoptosis in differentiated cells.

  19. Nicotine induces chromatin changes and c-Jun up-regulation in HL-60 leukemia cells.

    PubMed

    Landais, Emilie; El-Khoury, Victoria; Prevost, Alain; Dufer, Jean; Liautaud-Roger, Françoise

    2005-12-01

    Although nicotine has been implicated as a potential factor in the pathogenesis of human cancer, its mechanisms of action regarding cancer development remain largely unknown. HL-60 cells were used to investigate the effects of a short-term treatment with nicotine at concentrations found in the blood of smokers. The findings show that nicotine induces chromatin decondensation, histone H3 acetylation and up-regulation of the c-Jun transcription factor mRNA. This increase is inhibited by mecamylamine, a nicotinic receptor antagonist, suggesting that nicotine alters cellular function directly via nicotinic acetylcholine receptors and may then play a role in cell physiology and tumor promotion.

  20. Activity of cyclin B1 in HL-60 cells treated with etoposide.

    PubMed

    Żuryń, Agnieszka; Krajewski, Adrian; Szulc, Dawid; Litwiniec, Anna; Grzanka, Alina

    2016-06-01

    Cyclin B1 triggers G2/M phase transition phosphorylating with its catalytical partner - Cdc2 many of the molecular targets essential for cell cycle progression. Human leukemia cell line HL-60 were treated with increasing doses of etoposide (ETP) (0.5; 0.75; 1μM) to investigate how the drug affects cell morphology, viability, cell cycle distribution and expression of cyclin B1. To achieve this aim we applied light and transmission electron microscopy to observe morphological and ultra structural changes, image-based cytometry for apoptosis evaluation and cell cycle analysis, and then we conducted immunohistochemical and immunofluorescence staining to visualize cyclin localization and expression. Quantitive data about cyclin B1 expression were obtained from flow cytometry. Etoposide caused decrease in cell viability, induced apoptosis and G2/M arrest accompanied by enhanced expression of cyclin B1. Changes in expression and localization of cyclin B1 may constitute a part of the mechanism responsible for resistance of HL-60 cells to etoposide. Our results may reflect involvement of cyclin B1 in opposite processes - apoptosis induction and maintenance of cell viability in leukemia cells. We hypothesized possible roles and pathways by which cyclin B1 takes part in drug treatment response and chemosensitivity. PMID:27297620

  1. Cytoprotective effect of isoniazid against H2O2 derived injury in HL-60 cells.

    PubMed

    Khan, Saifur R; Aljuhani, Naif; Morgan, Andrew G M; Baghdasarian, Argishti; Fahlman, Richard P; Siraki, Arno G

    2016-01-25

    To combat tuberculosis (TB), host phagocytic cells need to survive against self-generating oxidative stress-induced necrosis. However, the effect of isoniazid (INH) in protecting cells from oxidative stress-induced necrosis has not been previously investigated. In this in vitro study, the cytotoxic effect of H2O2 generation using glucose oxidase (a model of oxidative stress) was found to be abrogated by INH in a concentration-dependent manner in HL-60 cells (a human promyelocytic leukemia cell). In cells treated with glucose oxidase, both ATP and mitochondrial membrane potential were found to be decreased. However, treatment with INH demonstrated small but significant attenuation in decreasing ATP levels, and complete reversal for the decrease in mitochondrial membrane potential. Quantitative proteomics analysis identified up-regulation of 15 proteins and down-regulation of 14 proteins which all together suggest that these proteomic changes signal for increasing cellular replication, structural integrity, ATP synthesis, and inhibiting cell death. In addition, studies demonstrated that myeloperoxidase (MPO) was involved in catalyzing INH-protein adduct formation. Unexpectedly, these covalent protein adducts were correlated with INH-induced cytoprotection in HL-60 cells. Further studies are needed to determine whether the INH-protein adducts were causative in the mechanism of cytoprotection.

  2. Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection.

    PubMed Central

    Müller, A; Hacker, J; Brand, B C

    1996-01-01

    Legionella pneumophila, the causative agent of Legionnaires' disease and Pontiac fever, replicates within and eventually kills human macrophages. In this study, we show that L. pneumophila is cytotoxic to HL-60 cells, a macrophage-like cell line. We demonstrate that cell death mediated by L. pneumophila occurred at least in part through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented host cell DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether potential virulence factors like the metalloprotease and the macrophage infectivity potentiator of L. pneumophila are involved in the induction of apoptosis. None of these factors are essential for the induction of apoptosis in HL-60 cells but may be involved in other cytotoxic mechanisms that lead to accidental cell death (necrosis). The ability of L. pneumophila to promote cell death may be important for the initiation of infection, bacterial survival, and escape from the host immune response. Alternatively, the triggering of apoptosis in response to bacterial infection may have evolved as a means of the host immune system to reduce or inhibit bacterial replication. PMID:8945524

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

    PubMed

    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.

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

    PubMed

    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.

  5. Catalase regulates cell growth in HL60 human promyelocytic cells: evidence for growth regulation by H(2)O(2).

    PubMed

    Hachiya, Misao; Akashi, Makoto

    2005-03-01

    Reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)) are generated constitutively in mammalian cells. Because of its relatively long life and high permeability across membranes, H(2)O(2) is thought to be an important second messenger. Generation of H(2)O(2) is increased in response to external insults, including radiation. Catalase is located at the peroxisome and scavenges H(2)O(2). In this study, we investigated the role of catalase in cell growth using the H(2)O(2)-resistant variant HP100-1 of human promyelocytic HL60 cells. HP100-1 cells had an almost 10-fold higher activity of catalase than HL60 cells without differences in levels of glutathione peroxidase, manganese superoxide dismutase (MnSOD), and copper-zinc SOD (CuZnSOD). HP100-1 cells had higher proliferative activity than HL60 cells. Treatment with catalase or the introduction of catalase cDNA into HL60 cells stimulated cell growth. Exposure of HP100-1 cells to a catalase inhibitor resulted in suppression of cell growth with concomitant increased levels of intracellular H(2)O(2). Moreover, exogenously added H(2)O(2) or depletion of glutathione suppressed cell growth in HL60 cells. Extracellular signal regulated kinase 1/2 (ERK1/2) was constitutively phosphorylated in HP100-1 cells but not in HL60 cells. Inhibition of the ERK1/2 pathway suppressed the growth of HP100-1 cells, but inhibition of p38 mitogen-activated protein kinase (p38MAPK) did not affect growth. Moreover, inhibition of catalase blocked the phosphorylation of ERK1/2 but not of p38MAPK in HP100-1 cells. Thus our results suggest that catalase activates the growth of HL60 cells through dismutation of H(2)O(2), leading to activation of the ERK1/2 pathway; H(2)O(2) is an important regulator of growth in HL60 cells.

  6. Discovery of novel inducers of cellular differentiation using HL-60 promyelocytic cells.

    PubMed

    Mata-Greenwood, E; Ito, A; Westenburg, H; Cui, B; Mehta, R G; Kinghorn, A D; Pezzuto, J M

    2001-01-01

    Non-physiological inducers of terminal differentiation have been used as novel therapies for the prevention and therapy of cancer. We have used cultured HL-60 promyelocytic cells to monitor differentiation, proliferation and cell death events as induced by a large set of extracts derived from plants. Screening of more than 1400 extracts led to the discovery of 34 with potent activity (ED50 <8 mg/ml). Bioassay-guided fractionation led to the isolation of zapotin and 2',5,6-trimethoxyflavone as active principles from Casimiroa edulis, dibenzyltrisulfide and 2-[(phenylmethyl)dithio]ethanol as active principles from Petiveria alliacea, and desmethylrocaglamide from Aglaia ponapensis. Zapotin demonstrated the most favorable biological profile in that induction of differentiation correlated with proliferation arrest, and a lack of cytotoxicity. We conclude that the HL-60 cell model is a useful system for the discovery of novel pharmacophores with potential to suppress the process of carcinogenesis, and that flavonoids may be especially useful in this capacity.

  7. Caffeine induces a second wave of apoptosis after low dose-rate gamma radiation of HL-60 cells.

    PubMed

    Vávrová, Jirina; Mareková-Rezácová, Martina; Vokurková, Doris; Szkanderová, Sylva; Psutka, Jan

    2003-10-01

    Most cell lines that lack functional p53 protein are arrested in the G(2) phase of the cell cycle due to DNA damage. It was previously found that the human promyelocyte leukemia cells HL-60 (TP53 negative) that had been exposed to ionizing radiation at doses up to 10 Gy were arrested in the G(2) phase for a period of 24 h. The radioresistance of HL-60 cells that were exposed to low dose-rate gamma irradiation of 3.9 mGy/min, which resulted in a pronounced accumulation of the cells in the G(2) phase during the exposure period, increased compared with the radioresistance of cells that were exposed to a high dose-rate gamma irradiation of 0.6 Gy/min. The D(0) value (i.e. the radiation dose leading to 37% cell survival) for low dose-rate radiation was 3.7 Gy and for high dose-rate radiation 2.2 Gy. In this study, prevention of G(2) phase arrest by caffeine (2 mM) and irradiation of cells with low dose-rate irradiation in all phases of the cell cycle proved to cause radiosensitization (D(0)=2.2 Gy). The irradiation in the presence of caffeine resulted in a second wave of apoptosis on days 5-7 post-irradiation. Caffeine-induced apoptosis occurring later than day 7 post-irradiation is postulated to be a result of unscheduled DNA replication and cell cycle progress.

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

    SciTech Connect

    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.

  9. Activation of 2',5'-oligoadenylate synthetase activity on induction of HL-60 leukemia cell differentiation.

    PubMed Central

    Schwartz, E L; Nilson, L A

    1989-01-01

    A 27-fold increase in 2',5'-oligoadenylate synthetase activity, an enzyme associated with the antiproliferative actions of interferon (IFN), was observed after treatment of HL-60 human leukemia cells with dimethyl sulfoxide (DMSO), an inducer of granulocytic differentiation of the cells. Enzyme activity was elevated after 24 h of exposure to DMSO, was maximal at 48 hours, and declined thereafter. A comparable increase was observed after treatment with 1 U of alpha interferon (IFN-alpha) per ml or 8 U of beta interferon (IFN-beta) per ml. Elevated levels of expression of other IFN-inducible genes, including type I histocompatibility antigen (HLA-B) mRNA and 2',5'-oligoadenylate phosphodiesterase activity, were also observed with DMSO treatment. DMSO-treated HL-60 cells had an increased amount of a 1.8-kilobase mRNA for oligoadenylate [oligo(A)] synthetase when compared with that of control cells; both DMSO- and IFN-treated HL-60 cells also expressed 1.6-, 3.4-, and 4.3-kilobase mRNA. The increase in both oligo(A) synthetase activity and mRNA levels was inhibited by polyclonal antiserum to human IFN-alpha; however, no IFN-alpha mRNA could be detected in the cells. Antiserum to IFN-beta or gamma interferon (IFN-gamma) had no effect on oligo(A) synthetase expression or activity nor was there any detectable IFN-beta 1 or IFN-beta 2 mRNA in the cells. The anti-IFN-alpha serum did not block the elevation of HLA-B mRNA in DMSO-treated cells. These observations suggest that the increased expression of oligo(A) synthetase in DMSO-treated cells may be mediated by the release of an IFN-alpha-like factor; however, the levels of any IFN-alpha mRNA produced in the cells were extremely low. Images PMID:2476665

  10. Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione.

    PubMed

    Ochi, T; Nakajima, F; Sakurai, T; Kaise, T; Oya-Ohta, Y

    1996-01-01

    Inducibility of apoptosis in cultured human HL-60 cells by arsenic compounds, such as arsenite, arsenate, methylarsonic acid (MAA), and dimethylarsinic acid (DMAA), was investigated, together with the role of glutathione (GSH) in the induction. Among the arsenic compounds DMAA was the most potent in terms of the ability to cause the morphological changes (formation of nuclear fragmentation and apoptotic bodies) characteristic of apoptosis. Furthermore, fragmentation of internucleosomal DNA was also induced by DMAA. Depletion of cell GSH by L-buthionine-SR-sulfoximine, a selective inhibitor of gamma-glutamylcysteine synthetase, enhanced the cytotoxicity of arsenite, arsenate, and MAA, while such depletion suppressed the cytotoxicity of DMAA. The depletion of GSH also suppressed the morphological changes and the fragmentation of internucleosomal DNA caused by DMAA, both of which are characteristic features of apoptosis. The results suggest that the death of cells caused by DMAA is due to apoptosis and that GSH is involved in the induction of apoptosis by this arsenic compound.

  11. Undifferentiated HL-60 cells internalize an antitumor alkyl ether phospholipid more rapidly than resistant K562 cells.

    PubMed

    Tsutsumi, T; Tokumura, A; Kitazawa, S

    1998-02-01

    In this study, we confirmed a previous finding that 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (methyl-PAF) expresses higher antineoplastic activity against the promyelocytic leukemia cell line HL-60, than against the erythroleukemic cell line K562, and intended to clarify the reason for this. Using an albumin back-exchange method, we measured the rates of binding and internalization of [3H]methyl-PAF by HL-60 and K562 cells. We found that methyl-PAF associated very rapidly and to similar extents with the two types of cells at low concentrations of extracellular bovine serum albumin, but that when bound to the cell surface, it was internalized into HL-60 cells faster than into K562 cells. The internalization of methyl-PAF by HL-60 cells was concentration-independent, intracellular ATP-independent and susceptible to thiol group-modifying reagents and cytochalasin B. Thus the inward transbilayer movement of methyl-PAF seems to occur by cytochalasin B-sensitive protein-mediated mechanism based on passive diffusion not requiring energy, in which SH-groups of protein play a critical role. We also found that the internalization of 1-hexadecanoyl-2-(4,4-difluoro-5,7- dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (Bodipy-C5-PC), whose structure resembles that of methyl-PAF, into HL-60 cells was faster than that into K562 cells. Using a combination of an albumin back-exchange method and observation by confocal laser scanning microscopy, we next examined the intracellular distribution of this fluorescent phospholipid probe after its internalization. Intracellular membranes, especially those peripheral to nuclei, were fluorescence-labeled in both HL-60 and K562 cells, but fluorescence of the nuclear membranes was weak, suggesting that this probe seems mainly to accumulate in intracellular granules, and may interact directly with several key enzymes for phospholipid metabolism, leading to cell injury. Because the difference between

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

    PubMed

    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.

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

    PubMed

    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

  14. Association of phorbol ester-induced hyperphosphorylation and reversible regulation of transferrin membrane receptors in HL60 cells.

    PubMed Central

    May, W S; Jacobs, S; Cuatrecasas, P

    1984-01-01

    Phorbol diesters are tumor-promoting agents that cause differentiation of HL60 human leukemic cells and concomitantly alter surface transferrin-receptor expression [Rovera, G., Ferreo, D., Pagliardi, G. L., Vartikar, J., Pessano, S., Bottero, L., Abraham, S. & Lebman, D. (1982) Ann. N.Y. Acad. Sci. 397, 211-220]. Transferrin-receptor regulation is shown here to result from a rapid and reversible internalization process that is temporally associated with reversible increased phosphorylation (hyperphosphorylation) of the transferrin receptor. Such a reversible mechanism involving regulation of these surface proteins could result in the rapid generation of an early signal for HL60 cellular differentiation. Images PMID:6326098

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

    PubMed

    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. PMID:27377174

  16. Citrus flavone tangeretin inhibits leukaemic HL-60 cell growth partially through induction of apoptosis with less cytotoxicity on normal lymphocytes.

    PubMed Central

    Hirano, T.; Abe, K.; Gotoh, M.; Oka, K.

    1995-01-01

    Certain anti-cancer agents are known to induce apoptosis in human tumour cells. However, these agents are intrinsically cytotoxic against cells of normal tissue origin, including myelocytes and immunocytes. Here we show that a naturally occurring flavone of citrus origin, tangeretin (5,6,7,8,4'-pentamethoxyflavone), induces apoptosis in human promyelocytic leukaemia HL-60 cells, whereas the flavone showed no cytotoxicity against human peripheral blood mononuclear cells (PBMCs). The growth of HL-60 cells in vitro assessed by [3H]thymidine incorporation or tetrazolium crystal formation was strongly suppressed in the presence of tangeretin; the IC50 values range between 0.062 and 0.173 microM. Apoptosis of HL-60 cells, assessed by cell morphology and DNA fragmentation, was demonstrated in the presence of > 2.7 microM tangeretin. Flow cytometric analysis of tangeretin-treated HL-60 cells also demonstrated apoptotic cells with low DNA content and showed a decrease of G1 cells and a concomitant increase of S and/or G2/M cells. Apoptosis was evident after 24 h of incubation with tangeretin, and the tangeretin effect as assessed by DNA fragmentation or growth inhibition was significantly attenuated in the presence of Zn2+, which is known to inhibit Ca(2+)-dependent endonuclease activity. Ca2+ and Mg2+, in contrast, promoted the effect of tangeretin. Cycloheximide significantly decreased the tangeretin effect on HL-60 cell growth, suggesting that protein synthesis is required for flavonoid-induced apoptosis. Tangeretin showed no cytotoxicity against either HL-60 cells or mitogen-activated PBMCs even at high concentration (27 microM) as determined by a dye exclusion test. Moreover, the flavonoid was less effective on growth of human T-lymphocytic leukaemia MOLT-4 cells or on blastogenesis of PBMCs. These results suggest that tangeretin inhibits growth of HL-60 cells in vitro, partially through induction of apoptosis, without causing serious side-effects on immune cells

  17. The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells.

    PubMed Central

    Stuart, J A; Anderson, K L; French, P J; Kirk, C J; Michell, R H

    1994-01-01

    1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and < or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form. Images Figure 2 PMID

  18. Protein-bound polysaccharide-K (PSK) induces apoptosis and inhibits proliferation of promyelomonocytic leukemia HL-60 cells.

    PubMed

    Hirahara, Noriyuki; Fujioka, Masaki; Edamatsu, Takeo; Fujieda, Ayako; Sekine, Fujio; Wada, Tsutomu; Tanaka, Tsuneo

    2011-09-01

    Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101), and is clinically used in combination therapy for gastrointestinal cancer and small cell lung carcinoma. PSK is a biological response modifier (BRM), and its mechanism of action is partly mediated, by modulating host immune systems, such as the activation of immune effector cells and the neutralization of transforming growth factor-beta (TGFβ) activity. Direct inhibition of tumor cell proliferation has been reported as another mechanism, but how PSK induces such an effect remains to be elucidated. Here, the anti-proliferative activity of PSK was examined using seven different human malignant cell lines (WiDr, HT29, SW480, KATOIII, AGS, HL60 and U937), and PSK was found to inhibit the proliferation of HL-60 cells most profoundly. Therefore, HL-60 cells were used to clarify the mechanism of anti-proliferative activity. Caspase-3 activation followed by apoptosis are involved at least in part in the PSK-induced anti-proliferative activity against HL-60 cells. PMID:21868514

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

    PubMed Central

    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

  20. 18α-Glycyrrhetinic Acid Induces Apoptosis of HL-60 Human Leukemia Cells through Caspases- and Mitochondria-Dependent Signaling Pathways.

    PubMed

    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

    2016-01-01

    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. PMID:27376261

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

    PubMed Central

    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

  2. Inhibition of NF-kappa B can enhance Fas-mediated apoptosis in leukemia cell line HL-60.

    PubMed

    Wang, Li; Zhao, Shi; Wang, Hong-Xiang; Zou, Ping

    2010-09-01

    This study explored the effects of nuclear factor-kappa B (NF-κB) inhibitor Bay 11-7082 on Fas/FasL system and Fas-mediated apoptosis in cell line HL-60 cells. The mRNA and protein levels of Fas, FasL, and X-linked inhibitor of apoptosis protein (XIAP) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM); the level of sFasL was evaluated by enzyme-linked immunosorbent assay (ELISA); and apoptosis was determined by FCM. After treatment with Bay 11-7082, the mRNA and protein levels of FasL and XIAP in HL-60 cells were significantly lower than in the controls (P<0.05), but the mRNA and protein levels of Fas and sFasL did not change significantly (P>0.05). Apoptotic rate of HL-60 cells treated with Bay 11-7082 was significantly higher than in the controls (P<0.05). Therefore, we conclude that Bay 11-7082 can enhance Fas-mediated apoptosis in HL-60 cells by downregulating FasL and XIAP levels.

  3. Stimulation of hydrogen peroxide production by drinking water contaminants in HL-60 cells sensitized by retinoic acid.

    PubMed

    Yoshida, H; Inoue, S; Yoshida, K; Nakajima, O; Mizuno, S

    1998-07-01

    Chemical carcinogens, such as chloroform and trichloroethylene, are present in drinking water in Japan. As these contaminants are believed to have a role in carcinogenesis, we examined if chloroform and trichloroethylene, as well as methylene chloride, xylene, benzene, and ethanol, have the ability to generate hydrogen peroxide (H(2)O(2)) in human polymorphonuclear leukocytes (PMN) and human leukemia (HL-60) cells. Methylene chloride, benzene, xylene, trichloroethylene, and ethanol did not increase cellular H(2)O(2): production as measured by flow cytometry nor as observed by confocal laser microscopy. In PMN and RAuntreated HL-60 cells chloroform did not significantly affect H(2)O(2) levels. However, in HL-60 cells sensitized by pretreatment of 10 nM retinoic acid (RA) for 12 h, chloroform induced a significant increase in H(2)O(2), but the increase induced by trichloroethylene was not significant. The observed increase in fluorescence was confirmed using a confocal laser microscope. These results indicate that chloroform and trichloroethylene may stimulate H(2)O(2) production in HL60 cells sensitized by pretreatment of RA. Our method may be useful to test if weak stimulants can stimulate intracellular H(2)O(2) production.

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

    PubMed

    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.

  5. Enhancement of the incorporation of 5-fluorodeoxyuridylate into DNA of HL-60 cells by metabolic modulations

    SciTech Connect

    Tanaka, M.; Kimura, K.; Yoshida, S.

    1983-11-01

    The exposure of HL-60 human promyelocytic leukemia cells to 0.5 microM 5-fluoro-2'-(/sup 3/H)deoxyuridine (FdUrd) for 16 hr resulted in the incorporation of 5.14 +/- 0.31 (S.D.) X 10(-7) mol FdUrd into DNA per mol of DNA nucleotide, which corresponds to 0.146 +/- 0.082 pmol FdUrd per 10(7) cells. Pretreatment with 50 microM deoxythymidine for 24 hr led to a 2.7-fold increase in the incorporation of this analogue into newly synthesized DNA during the ensuing 16-hr exposure to 0.5 microM (/sup 3/H)FdUrd. Pretreatment with 0.5 microM methotrexate for 3 hr also increased the (/sup 3/H)FdUrd incorporation into newly synthesized DNA approximately 5-fold. The coexistence of deoxythymidine or methotrexate with (/sup 3/H)FdUrd, however, led to decreased incorporation of FdUrd into DNA. More than 50% of the radioactivity in DNA separated by Cs2SO4 equilibrium density gradient centrifugation was proven to be fluorodeoxyuridylate by means of its binding to Lactobacillus casei deoxythymidine monophosphate synthetase.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Retrotransposon Alu is enriched in the epichromatin of HL-60 cells.

    PubMed

    Olins, Ada L; Ishaque, Naveed; Chotewutmontri, Sasithorn; Langowski, Jörg; Olins, Donald E

    2014-01-01

    Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a "rim" staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells,>95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither "classical" heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus. PMID:24824428

  8. 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.

    PubMed

    Kostrzewa-Nowak, Dorota; Tarasiuk, Jolanta

    2013-12-01

    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. PMID:24076328

  9. Induction of Apoptosis by Functionalized Fullerene-based Sonodynamic Therapy in HL-60 cells.

    PubMed

    Yumita, Nagahiko; Watanabe, Takahiro; Chen, Fu-Shih; Momose, Yasunori; Umemura, Shin-Ichiro

    2016-06-01

    Ultrasound has been widely utilized for medical diagnosis and therapy due to its ability to penetrate deep-seated tissue with less attenuation of energy and minimal undesirable side-effects. Functionalized fullerenes, such as polyhydroxy fullerene (PHF), have attracted particular attention due to their water solubility and potential application in tumor imaging and therapy as carbon nanomaterials. The present study investigated sonodynamically-induced apoptosis using PHF. Cell suspensions were treated with 2-MHz continuous ultrasound in the presence of PHF for 3 min and apoptosis was assessed by cell morphology using confocal microscopy, fragmentation of DNA (ladder pattern after agarose-gel electrophoresis) and caspase-3 activation. Cells were ultrasound-irradiated from the bottom of the culture dishes under the following condition: frequency, 2 MHz; output power, 3 W/cm(2) Electron spin resonance was used to measure reactive oxygen species. The number of apoptotic cells after sonodynamic exposure (ultrasound and PHF) was significantly higher than produced from other treatments, such as ultrasound alone and PHF alone. Furthermore, DNA fragmentation, caspase-3 activation and enhanced 2,2,6,6-tetramethyl-4-piperidinyloxy (4oxoTEMPO) formation were observed in the sonodynamically-treated cells. Histidine, a well-known reactive oxygen scavenger, significantly inhibited sonodynamically-induced apoptosis, caspase-3 activation and 4oxoTEMPO formation. Sonodynamic therapy with PHF induced apoptosis that was characterized by a series of typical morphological features, such as shrinkage of the cell and fragmentation into membrane-bound apoptotic bodies, in HL-60 cells. The significant inhibition of sonodynamically-induced apoptosis, caspase-3 activation, and 4oxoTEMPO formation due to histidine and tryptophan suggests that reactive oxygen species, such as singlet oxygen, are involved in the sonodynamic induction of apoptosis. These findings indicate that PHF

  10. Induction of Apoptosis by Functionalized Fullerene-based Sonodynamic Therapy in HL-60 cells.

    PubMed

    Yumita, Nagahiko; Watanabe, Takahiro; Chen, Fu-Shih; Momose, Yasunori; Umemura, Shin-Ichiro

    2016-06-01

    Ultrasound has been widely utilized for medical diagnosis and therapy due to its ability to penetrate deep-seated tissue with less attenuation of energy and minimal undesirable side-effects. Functionalized fullerenes, such as polyhydroxy fullerene (PHF), have attracted particular attention due to their water solubility and potential application in tumor imaging and therapy as carbon nanomaterials. The present study investigated sonodynamically-induced apoptosis using PHF. Cell suspensions were treated with 2-MHz continuous ultrasound in the presence of PHF for 3 min and apoptosis was assessed by cell morphology using confocal microscopy, fragmentation of DNA (ladder pattern after agarose-gel electrophoresis) and caspase-3 activation. Cells were ultrasound-irradiated from the bottom of the culture dishes under the following condition: frequency, 2 MHz; output power, 3 W/cm(2) Electron spin resonance was used to measure reactive oxygen species. The number of apoptotic cells after sonodynamic exposure (ultrasound and PHF) was significantly higher than produced from other treatments, such as ultrasound alone and PHF alone. Furthermore, DNA fragmentation, caspase-3 activation and enhanced 2,2,6,6-tetramethyl-4-piperidinyloxy (4oxoTEMPO) formation were observed in the sonodynamically-treated cells. Histidine, a well-known reactive oxygen scavenger, significantly inhibited sonodynamically-induced apoptosis, caspase-3 activation and 4oxoTEMPO formation. Sonodynamic therapy with PHF induced apoptosis that was characterized by a series of typical morphological features, such as shrinkage of the cell and fragmentation into membrane-bound apoptotic bodies, in HL-60 cells. The significant inhibition of sonodynamically-induced apoptosis, caspase-3 activation, and 4oxoTEMPO formation due to histidine and tryptophan suggests that reactive oxygen species, such as singlet oxygen, are involved in the sonodynamic induction of apoptosis. These findings indicate that PHF

  11. Correlation between secretion and phospholipase D activation in differentiated HL60 cells.

    PubMed Central

    Stutchfield, J; Cockcroft, S

    1993-01-01

    Receptor-directed agonists including N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), C5a, ATP and UTP all activate phospholipase D (PLD), which is accompanied by secretion in differentiated HL60 cells. Interference in the production of phosphatidase (PA) by the PLD pathway by diverting it towards the production of phosphatidylethanol (PEt) in the presence of ethanol leads to near-total inhibition of the secretion evoked by ATP and UTP and a partial inhibition of that evoked by fMetLeuPhe and C5a. In streptolysin-O-permeabilized cells, fMetLeuPhe is able to activate PLD, and this is dependent on the presence of a low concentration of guanosine 5'-[gamma-thio]-triphosphate (GTP[S]). Ca2+ (10 microM) and GTP[S] individually or in combination are also able to activate PLD and secretion. The stimulation of secretion in permeabilized cells stimulated by Ca2+ alone or fMetLeuPhe or GTP[S] is also abrogated when the production of PA is diverted to PEt by the presence of ethanol. Activation of PLD by GTP[S] or fMetLeuPhe is decreased if the cells are permeabilized first and GTP[S] or fMetLeuPhe is added subsequently. This corresponds well with the loss of the secretory response. We conclude that the ability of GTP[S] or fMetLeuPhe to stimulate secretion from permeabilized cells is dependent on a prior activation of the PLD signalling pathway. PA, generated as a consequence of PLD activation, acts as second messenger that can provide an initiating signal for secretion and is not required for exocytosis itself. PMID:8352731

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

    PubMed

    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.

  13. Global protein expression dataset acquired during isoniazid-induced cytoprotection against H2O2 challenge in HL-60 cells.

    PubMed

    Khan, Saifur R; Baghdasarian, Argishti; Fahlman, Richard P; Siraki, Arno G

    2016-03-01

    Isoniazid (INH) is one of the first-line anti-tuberculosis drugs. Its effect on oxidative stress, however, is unknown. Here we used a model of oxidative stress by employing glucose/glucose oxidase (GOx), which (based on the availability of glucose and oxygen) is known to produce H2O2. This reaction induces oxidative stress culminating in necrotic cell death in HL-60 cells (a human promyelocytic leukemia cell line). The changes in protein levels have been quantified using global proteome expression changes through stable isotope labeling by amino acids in cell culture (SILAC) followed by LC-MS/MS analysis. A total of 1459 and 1712 proteins were identified in forward and reverse experiments, respectively. However, only 390 proteins were reproducibly identified in both samples. These 390 proteins were taken into account for further analysis which has been described in "Cytoprotective effect of isoniazid against H2O2 derived injury in HL-60 cells" [1].

  14. The effect of aqueous cinnamon extract on the apoptotic process in acute myeloid leukemia HL-60 cells

    PubMed Central

    Assadollahi, Vahideh; Parivar, Kazem; Roudbari, Nasim Hayati; Khalatbary, Ali Reza; Motamedi, Masoumeh; Ezatpour, Behrouz; Dashti, Gholam Reza

    2013-01-01

    Background: Acute promyelocytic leukemia (APL) is an acute leukemia diagnosed by translocation of chromosomes 15 and 17 [T (15,17)] and aggregation of neoplastic promyelocytes which are incapable of being converted into mature cells. Today, many tend to use medicinal herbs in studies and clinical applications for treatment of cancers. Cinnamon with scientific name “cinnamomumzelanicum” is a shrub of Laurales order, lauraceae family with cinnamomum genus. It is a medicinal shrub with anti-proliferation effect on tumor cells. This study was conducted to determine the effects of aqueous cinnamon extract on HL-60 cells as a model for APL. Materials and Methods: In this in vitro experimental study, HL-60 cell line was cultured under the influence of cinnamon extract's concentrations of 0.01, 0.1, 1, and 2 mg/ml in with intervals of 24, 48, and 72 h. Growth inhibition and toxic effects of cinnamon extract were evaluated through tetrazolium salt reduction. The effect of this herb on the cell cycle was studied by flow cytometry. The Hoechst stain was used to detect apoptotic cell nuclei. Results: Cinnamon extract inhibited the growth of HL-60 cells as correlated with concentration and time. After 72 h of treating HL-60 cells with 0.01 mg/l cinnamon extract, the growth of cells was inhibited by 90.1%. Cinnamon extract stopped the cell cycle in G1 phase and the Hoechst staining verified the apoptotic process in those cells. Conclusion: Considering the inhibitory property of cinnamon extract, we recommend it as a single drug or besides other medications for treating promyelocytic leukemia. PMID:23977653

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

    PubMed

    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.

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

    SciTech Connect

    Sato, Tomonobu; Okumura, Fumihiko; Iguchi, Akihiro; Ariga, Tadashi; Hatakeyama, Shigetsugu

    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.

  17. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

    PubMed

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-29

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.

  18. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells

    PubMed Central

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

  19. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

    PubMed

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    Huang, Albert C; Hu, Limei; Kauffman, Stuart A; Zhang, Wei; Shmulevich, Ilya

    2009-01-01

    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 genes, of transcription

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

    SciTech Connect

    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.

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

    PubMed

    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.

  4. Phenotypic and functional analysis of HL-60 cells used in opsonophagocytic-killing assay for Streptococcus pneumoniae.

    PubMed

    Kim, Kyung-Hyo; Seoh, Ju Young; Cho, Su Jin

    2015-02-01

    Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1α, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

  5. Oxidative stress-mediated intrinsic apoptosis in human promyelocytic leukemia HL-60 cells induced by organic arsenicals

    PubMed Central

    Fan, Xiao-Yang; Chen, Xin-You; Liu, Yu-Jiao; Zhong, Hui-Min; Jiang, Feng-Lei; Liu, Yi

    2016-01-01

    Arsenic trioxide has shown the excellent therapeutic efficiency for acute promyelocytic leukemia. Nowadays, more and more research focuses on the design of the arsenic drugs, especially organic arsenicals, and on the mechanism of the inducing cell death. Here we have synthesized some organic arsenicals with Schiff base structure, which showed a better antitumor activity for three different kinds of cancer cell lines, namely HL-60, SGC 7901 and MCF-7. Compound 2a (2-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol) and 2b (2-methoxy-4-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol) were chosen for further mechanism study due to their best inhibitory activities for HL-60 cells, of which the half inhibitory concentration (IC50) were 0.77 μM and 0.51 μM, respectively. It was illustrated that 2a or 2b primarily induced the elevation of reactive oxygen species, decrease of glutathione level, collapse of mitochondrial membrane potential, release of cytochrome c, activation of Caspase-3 and apoptosis, whereas all of the phenomena can be eliminated by the addition of antioxidants. Therefore, we concluded that compound 2a and 2b can induce the oxidative stress-mediated intrinsic apoptosis in HL-60 cells. Both the simplicity of structure with Schiff base group and the better anticancer efficiency demonstrate that organic arsenicals are worthy of further exploration as a class of potent antitumor drugs. PMID:27432798

  6. Characterization of high affinity neurotensin receptor NTR1 in HL-60 cells and its down regulation during granulocytic differentiation

    PubMed Central

    Choi, Se-Young; Chae, Hee-Don; Park, Tae-Ju; Ha, Hyunjung; Kim, Kyong-Tai

    1999-01-01

    We investigated responses to neurotensin in human promyelocytic leukaemia HL-60 cells. Neurotensin increased the cytosolic calcium concentration ([Ca2+]i) in a concentration-dependent manner and also produced inositol 1,4,5-trisphosphate (InsP3). Among the tested neurotensin analogues, neurotensin 8-13, neuromedin-N, and xenopsin also increased [Ca2+]i, whereas neurotensin 1–11 and neurotensin 1–8 did not elicit detectable responses. SR48692, an antagonist of NTR1 neurotensin receptors, blocked the neurotensin-induced [Ca2+]i increase, whereas levocabastine, which is known as an NTR2 neurotensin receptor antagonist, did not attenuate the neurotensin-evoked effect. The expression of NTR1 neurotensin receptors was confirmed by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT–PCR). During 1.25% dimethylsulfoxide (DMSO)-triggered granulocytic differentiation of HL-60 cells, the neurotensin-induced [Ca2+]i rise became gradually smaller and completely disappeared 4 days after treatment with DMSO. The mRNA level for neurotensin receptors was also decreased after differentiation. The results show that HL-60 cells express NTR1 neurotensin receptors and suggest that granulocytic differentiation involves transcriptional regulation of the receptors resulting in down-regulation of the neurotensin-induced signalling. PMID:10193787

  7. The effect of 5-azacytidine (5-aza-CR) and its analogue on cell differentiation and DNA methylation of HL-60 cells.

    PubMed

    He, Z X; Rao, H

    1993-04-01

    The effect of 5-aza-CR and 5-aza-2'-deoxycytidine (5-aza-CdR) on cell differentiation and DNA methylation of HL-60 cells was studied. The differentiation index of HL-60 cells was measured after being treated with drugs by using the NBT stain method. DNA methylase activities of HL-60 cells treated with the drugs were assayed by using 3H-methyl-S-adenosylmethionine (3H-SAM) as a methyl donor. The DNA methylation level of HL-60 cells treated with the drugs was measured by HPLC. The results showed that the HL-60 cell differentiation index was increased after being treated with 5-aza-CR or 5-aza-CdR at a certain concentration for 4 days. But, at the same time, DNA methylase activity and the DNA methylation level were decreased. And all these changes were related to the concentration of the drugs. 5-Aza-CdR was more efficient than 5-aza-CR. We also assayed the E. coli RNA polymerase activity in vitro by using different DNA templets different in DNA methylation level. We found that the transcriptional activity of RNA polymerase was increased with the decrease of the DNA methylation level of HL-60 cells.

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

    SciTech Connect

    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.

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

    PubMed

    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.

  10. 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

    SciTech Connect

    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.

  11. Arsenic trioxide induces oxidative stress, DNA damage, and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells

    PubMed Central

    2014-01-01

    Background Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of all acute myloid leukemia cases. It is a blood cancer that is formed by chromosomal mutation. Each year in the United States, APL affects about 1,500 patients of all age groups and causes approximately 1.2% of cancer deaths. Arsenic trioxide (ATO) has been used successfully for treatment of APL patients, and both induction and consolidated therapy have resulted in complete remission. Recently published studies from our laboratory have demonstrated that ATO pharmacology as an anti-leukemic drug is associated with cytotoxic and genotoxic effects in leukemia cells. Methods In the present study, we further investigated the detailed molecular mechanism of ATO-mediated intrinsic pathway of apoptosis; using HL-60 cells as a test model. Oxidative stress was assessed by spectrophotometric measurements of MDA and GSH levels while genotoxicity was determined by single cell gel electrophoresis (Comet assay). Apoptosis pathway was analyzed by Western blot analysis of Bax, Bcl2 and caspase 3 expression, as well as immunocytochemistry and confocal imaging of Bax and Cyt c translocation and mitochondrial membrane potential depolarization. Results ATO significantly (p < 0.05) induces oxidative stress, DNA damage, and caspase 3 activityin HL-60 cells in a dose-dependent manner. It also activated the intrinsic pathway of apoptosis by significantly modulating (p < 0.05) the expression and translocation of apoptotic molecules and decreasing the mitochondrial membrane potential in leukemia cells. Conclusion Taken together, our research demonstrated that ATO induces mitochondrial pathway of apoptosis in HL-60 cells. This apoptotic signaling is modulated via oxidative stress, DNA damage, and change in mitochondrial membrane potential, translocation and upregulation of apoptotic proteins leading programmed cell death. PMID:24887205

  12. In Vitro Antioxidant and Antiproliferative Activities of Novel Orange Peel Extract and It's Fractions on Leukemia HL-60 Cells.

    PubMed

    Diab, Kawthar A E; Shafik, Reham Ezzat; Yasuda, Shin

    2015-01-01

    In the present work, novel orange peel was extracted with 100%EtOH (ethanol) and fractionated into four fractions namely F1, F2, F3, F4 which were eluted from paper chromatographs using 100%EtOH, 80%EtOH, 50%EtOH and pure water respectively. The crude extract and its four fractions were evaluated for their total polyphenol content (TPC), total flavonoid content (TFC) and radical scavenging activity using DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. Their cytotoxic activity using WST assay and DNA damage by agarose gel electrophoresis were also evaluated in a human leukemia HL-60 cell line. The findings revealed that F4 had the highest TPC followed by crude extract, F2, F3 and F1. However, the crude extract had the highest TFC followed by F4, F3, F2, and F1. Depending on the values of EC50 and trolox equivalent antioxidant capacity, F4 possessed the strongest antioxidant activity while F1 and F2 displayed weak antioxidant activity. Further, incubation HL-60 cells with extract/fractions for 24h caused an inhibition of cell viability in a concentration- dependent manner. F3 and F4 exhibited a high antiproliferative activity with a narrow range of IC50 values (45.9 - 48.9 μg/ml). Crude extract exhibited the weakest antiproliferative activity with an IC50 value of 314.89 μg/ml. Analysis of DNA fragmentation displayed DNA degradation in the form of a smear-type pattern upon agarose gel after incubation of HL-60 cells with F3 and F4 for 6 h. Overall, F3 and F4 appear to be good sources of phytochemicals with antioxidant and potential anticancer activities. PMID:26514490

  13. Induction of mitochondrial dependent apoptosis and cell cycle arrest in human promyelocytic leukemia HL-60 cells by an extract from Dorstenia psilurus: a spice from Cameroon

    PubMed Central

    2013-01-01

    Background The use of edible plants is an integral part of dietary behavior in the West region of Cameroon. Dorstenia psilurus (Moraceae) is widely used as spice and as medicinal plant for the treatment of several diseases in Cameroon. The aim of this study is to investigate the cytotoxic and apoptotic potential of methanol extract of D. psilurus in human promyelocytic leukemia (HL-60) cells and prostate cancer (PC-3) cells. Methods Cytotoxicity of D. psilurus extract was tested in HL-60 and PC-3 cells using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and flow cytometric methods Results The methanol extract of D. psilurus have significant in vitro cytotoxic activity in HL-60 cells and PC-3 cells with IC50 value of 12 ±1.54 μg/ml and 18 ± 0.45 μg/ml respectively after 48 h. The mechanism of antiproliferative activity showed that after 24 h, D. psilurus extract induces apoptosis on HL-60 cells by the generation of reactive oxygen species (ROS) along with concurrent loss of mitochondrial membrane potential, modification in the DNA distribution and enhance of G2/M phase cell cycle. Conclusion The extract induces apoptosis of HL-60 cells associated with ROS production, loss of mitochondrial membrane potential and apoptotic DNA fragmentation. PMID:24016040

  14. The role of p38 MAPK activation in auranofin-induced apoptosis of human promyelocytic leukaemia HL-60 cells

    PubMed Central

    Park, Seon-Joo; Kim, In-Sook

    2005-01-01

    In a previous study, we reported an antileukaemic activity of auranofin (AF), demonstrating its dual effects: on the induction of apoptotic cell death and its synergistic action with retinoic acid on cell differentiation. In this study, we investigated the downstream signalling events of AF-induced apoptosis to determine the molecular mechanisms of AF activity. Treatment of HL-60 cells with AF induced apoptosis in a concentration- and time-dependent manner. Western blot analysis showed that AF-induced apoptosis was accompanied by the activation of caspase-8, caspase-9, and caspase-3, and the release of cytochrome c from the mitochondria. The phosphorylation and kinase activities of p38 mitogen-activated protein kinase (p38 MAPK) increased gradually until 12 h after AF (2 μM) treatment, and p38 MAPK was also activated concentration-dependently. Pretreatment with SB203580, a specific inhibitor of p38 MAPK, significantly blocked DNA fragmentation and the cleavage of procaspase-8, procaspase-3, and poly-ADP-ribose polymerase (PARP), whereas SB203580 alone had no effect. Reactive oxygen species (ROS) were also detected within 1 h after AF treatment, and the antioxidant N-acetyl-L-cysteine (NAC) effectively protected the cells from apoptosis by inhibiting the phosphorylation of p38 MAPK and the activation of caspases. These results suggest that ROS generation and the subsequent activation of p38 MAPK are essential for the proapoptotic effects of AF in human promyelocytic leukaemia HL-60 cells. PMID:16086031

  15. Global protein expression dataset acquired during isoniazid-induced cytoprotection against H2O2 challenge in HL-60 cells

    PubMed Central

    Khan, Saifur R.; Baghdasarian, Argishti; Fahlman, Richard P.; Siraki, Arno G.

    2016-01-01

    Isoniazid (INH) is one of the first-line anti-tuberculosis drugs. Its effect on oxidative stress, however, is unknown. Here we used a model of oxidative stress by employing glucose/glucose oxidase (GOx), which (based on the availability of glucose and oxygen) is known to produce H2O2. This reaction induces oxidative stress culminating in necrotic cell death in HL-60 cells (a human promyelocytic leukemia cell line). The changes in protein levels have been quantified using global proteome expression changes through stable isotope labeling by amino acids in cell culture (SILAC) followed by LC–MS/MS analysis. A total of 1459 and 1712 proteins were identified in forward and reverse experiments, respectively. However, only 390 proteins were reproducibly identified in both samples. These 390 proteins were taken into account for further analysis which has been described in “Cytoprotective effect of isoniazid against H2O2 derived injury in HL-60 cells” [1]. PMID:26937455

  16. Genotoxic effects of exposure to radiofrequency electromagnetic fields (RF-EMF) in HL-60 cells are not reproducible.

    PubMed

    Speit, Günter; Gminski, Richard; Tauber, Rudolf

    2013-08-15

    Conflicting results have been published regarding the induction of genotoxic effects by exposure to radiofrequency electromagnetic fields (RF-EMF). Various results indicating a genotoxic potential of RF-EMF were reported by the collaborative EU-funded REFLEX (Risk Evaluation of Potential Environmental Hazards From Low Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods) project. There has been a long-lasting scientific debate about the reliability of the reported results and an attempt to reproduce parts of the results obtained with human fibroblasts failed. Another part of the REFLEX study was performed in Berlin with the human lymphoblastoid cell line HL-60; genotoxic effects of RF-EMF were measured by means of the comet assay and the micronucleus test. The plausibility and reliability of these results were also questioned. In order to contribute to a clarification of the biological significance of the reported findings, a repeat study was performed, involving scientists of the original study. Comet-assay experiments and micronucleus tests were performed under the same experimental conditions that had led to genotoxic effects in the REFLEX study. Here we report that the attempts to reproduce the induction of genotoxic effects by RF-EMF in HL-60 cells failed. No genotoxic effects of RF-EMF were measured in the repeat experiments. We could not find an explanation for the conflicting results. However, the negative repeat experiments suggest that the biological significance of genotoxic effects of RF-EMF reported by the REFLEX study should be re-assessed.

  17. Effect of a 2.45-GHz radiofrequency electromagnetic field on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells.

    PubMed

    Koyama, Shin; Narita, Eijiro; Suzuki, Yoshihisa; Taki, Masao; Shinohara, Naoki; Miyakoshi, Junji

    2015-01-01

    The potential public health risks of radiofrequency (RF) fields have been discussed at length, especially with the use of mobile phones spreading extensively throughout the world. In order to investigate the properties of RF fields, we examined the effect of 2.45-GHz RF fields at the specific absorption rate (SAR) of 2 and 10 W/kg for 4 and 24 h on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells. Neutrophil chemotaxis was not affected by RF-field exposure, and subsequent phagocytosis was not affected either compared with that under sham exposure conditions. These studies demonstrated an initial immune response in the human body exposed to 2.45-GHz RF fields at the SAR of 2 W/kg, which is the maximum value recommended by the International Commission for Non-Ionizing Radiation Protection (ICNIRP) guidelines. The results of our experiments for RF-field exposure at an SAR under 10 W/kg showed very little or no effects on either chemotaxis or phagocytosis in neutrophil-like human HL-60 cells.

  18. Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells.

    PubMed

    Wen, Xi; Jin, Tian; Xu, Xuehua

    2016-01-01

    Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells. PMID:27684322

  19. Effect of ozone on platelet-activating factor production in phorbol-differentiated HL60 cells, a human bronchial epithelial cell line (BEAS S6), and primary human bronchial epithelial cells.

    PubMed

    Samet, J M; Noah, T L; Devlin, R B; Yankaskas, J R; McKinnon, K; Dailey, L A; Friedman, M

    1992-11-01

    Platelet-activating factor (PAF) is a phospholipid with a wide spectrum of pro-inflammatory properties. In the lung, PAF induces airway hyperresponsiveness, neutrophil sequestration, and increased vascular permeability. The alveolar macrophage and the bronchial epithelium are tissues that are exposed to inhaled ozone (O3). We studied the effect of an in vitro O3 exposure on PAF production in a macrophage-like HL60 human cell line (dHL60), a human bronchial epithelial cell line (BEAS S6), and also in primary human bronchial epithelial cells. PAF was quantified by thin-layer chromatographic separation of lipid extracts from cells radiolabeled with [3H]lysoPAF and by radioimmunoassay. In vitro exposure of dHL60 cells to 0.05 to 1.0 ppm O3 for 15 to 120 min was found to significantly increase PAF levels above air control values at all exposure levels and time points (average increase of 92%). Similarly, BEAS S6 cells grown on collagen-coated filter supports and exposed to 0.05 to 1.0 ppm O3 for 60 min released an average increase in PAF of 626% above control values. Primary human bronchial epithelial cells also demonstrated significant increases in [3H]PAF release (average increase of 289% after exposure to 1.0 ppm O3 for 60 min) compared with paired air controls. These findings suggest that some of the effects of O3 inhalation may be mediated by PAF.

  20. Synthesis of a fluorescently labeled compound for the detection of arsenic-induced apoptotic HL60 cells.

    PubMed

    Femia, A Lis; Temprana, C Facundo; Amor, M Silvia; Grasselli, Mariano; Alonso, Silvia Del V

    2012-03-01

    Arsenic compounds have shown medical usefulness since they proved to be effective in causing complete remission of acute promyelocytic leukemia. In this work we obtained a fluorescently labeled arsenic compound that can be used with current fluorescence techniques for basic and applied research, focused on arsenic-induced apoptosis studies. This compound is an arsanilic acid bearing a covalently linked FITC that was chemically synthesized and characterized by fluorescence, UV-Vis, mass and FTIR spectrometry. In addition, we assessed its apoptotic activity as well as its fluorescent labeling properties in HL60 cell line as a leukemia cell model through flow cytometry. We obtained a compound with a 1:1 FITC:arsenic ratio and a 595 m/z, confirming its structure by FTIR. This compound proved to be useful at inducing apoptosis in the leukemia cell model and labeling this apoptotic cell population, in such a way that the highest FITC fluorescence correlated with the highest arsenic amount.

  1. Antitumor triptycene analogs induce a rapid collapse of mitochondrial transmembrane potential in HL-60 cells and isolated mitochondria.

    PubMed

    Wang, Yang; Perchellet, Elisabeth M; Ward, Mary M; Lou, Kaiyan; Zhao, Huiping; Battina, Srinivas K; Wiredu, Bernard; Hua, Duy H; Perchellet, Jean-Pierre H

    2006-01-01

    Since synthetic analogs of triptycene (TT code number), such as bisquinones TT2 and TT13, can trigger cytochrome c release without caspase activation and retain their ability to induce apoptosis in multidrug-resistant (MDR) tumor cells, fluorescent probes of transmembrane potential have been used to determine whether these antitumor compounds might directly target mitochondria in cell and cell-free systems to cause the collapse of mitochondrial membrane potential ( downward arrow Deltapsim) that is linked to permeability transition pore (PTP) opening. Using JC-1 dye, the abilities of various TT analogs to induce the downward arrow Deltapsim in wild-type and MDR HL-60 cells are rapid (within 5-20 min), irreversible after drug removal, concentration dependent in the 0.64-25 microM range, and generally related to their antitumor activities in vitro. The downward arrow Deltapsim caused by TT2 and TT13, which are more potent than mitoxantrone, staurosporine and the reference depolarizing agent, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in HL-60 cells, are not prevented by caspase-2 or -8 inhibitors, suggesting that activation of these apical caspases upstream of mitochondria is not involved in this process. Antitumor TT analogs (0.64-25 microM) also mimic the abilities of the known depolarizing agents, CCCP, alamethicin, gramicidin A and 100 microM CaCl(2), to directly induce within 20 min the downward arrow Deltapsim in isolated mitochondria prepared from mouse liver and loaded with rhodamine 123 dye. The fact that 20 microM Ca(2+), which is insufficient to trigger depolarization on its own, is required to reveal the depolarizing effect of TT2 in isolated mitochondria suggests that antitumor TT analogs might interact with the PTP to alter its conformation and increase its Ca(2+) sensitivity. Indeed, such Ca(2+)-dependent downward arrowDeltapsim of isolated mitochondria treated with 25 microM TT2 or 100 microM Ca(2+) are blocked by ruthenium red. Daunorubicin

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

    PubMed Central

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

    2014-01-01

    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 (R2 = 0.899) with the degree of nuclear fragmentation and other three parameters showed a very good correlations (R2 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. PMID:25071957

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

    PubMed

    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.

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

    EPA Science Inventory

    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...

  5. Differential regulation of leukocyte function-associated antigen-1/ intercellular adhesion molecules-1-dependent adhesion and aggregation in HL-60 cells.

    PubMed

    Katagiri, K; Kinashi, T; Irie, S; Katagiri, T

    1996-05-15

    Activation of integrin and organization of cytoskeletal proteins are highly regulated in cell adhesion and aggregation. The interaction of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecules-1 (ICAM-1) mediates cell adhesion and aggregation, which facilitate leukocyte trafficking to inflamed tissues and augment effector functions. We investigated how LFA-1/ICAM-1-mediated adhesion and aggregation are regulated in HL-60 cells induced to differentiate into neutrophils by retinoic acid (RA). Uninduced HL-60 cells did not bind to ICAM-1 even with stimulation by 12-0-tetradecanoyl phorbol-13-acetate, although they express LFA-1 on the cell surface. When cultured with RA for 24 hours, HL-60 cells were able to adhere to ICAM-1 constitutively. The induction of adhesion did not accompany any change in surface density of LFA-1, indicating that the avidity of LFA-1 was increased. The change in its avidity required de novo synthesis of proteins. Although ICAM-1 was intensely expressed on RA-induced HL-60 cells, these cells did not show any cellular aggregation. The HL-60 cells transfected with the active form of Ras (Val12) exhibited LFA-1/ICAM-1-dependent aggregation by RA stimulation without change in the avidity of LFA-1. In these Ras-transfectants, a cytoskeletal protein, paxillin, was tyrosine-phosphorylated, and the level of F-actin increased. Transforming growth factor (TGF) beta, as well as cytochalasin D, prevented both the tyrosine phosphorylation of paxillin and the aggregation without any effects on the avidity of LFA-1. Thus, an increase in the avidity of LFA-1 was not sufficient for the induction of aggregation, which required activation of Ras and reorganization of cytoskeletal proteins. These results suggest that distinct regulatory mechanisms control LFA-1/ICAM-1-dependent adhesion and aggregation in HL-60 cells differentiating into neutrophils.

  6. Enhanced expression of extracellular calcium sensing receptor in monocyte-differentiated versus undifferentiated HL-60 cells: potential role in regulation of a nonselective cation channel

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Ye, C.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    Human promyelocytic leukemia cells (HL-60) have been used widely as a model for studying the differentiation of hematopoietic progenitor cells in vitro. After treatment with phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], HL-60 cells differentiate into cells with the phenotype of monocytes/macrophages. We previously showed that peripheral blood monocytes and the murine J774 monocytic cell line express the CaR, and myeloid progenitors in the bone marrow and myeloid cells in peripheral blood other than monocytes express lower levels of the CaR. Therefore, we investigated whether undifferentiated HL-60 cells express a functional G protein-coupled, extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) and if the expression of the CaR increases as these cells differentiate along the monocytic lineage. The use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by sequencing of the amplified products, identified an authentic CaR transcript in undifferentiated HL-60 cells. Both immunocytochemistry and Western blot analysis using a CaR-specific antiserum detected low levels of CaR protein expression in undifferentiated HL-60 cells. The levels of CaR protein increased considerably following treatment of the cells with PMA (50 nM) or 1,25(OH)(2)D(3) (100 nM) for 5 days. Northern analysis using a CaR-specific riboprobe identified CaR transcripts in undifferentiated HL-60 cells, but CaR mRNA levels did not change appreciably after treatment with either agent, suggesting that upregulation of CaR protein occurs at a translational level. PMA-treated HL-60 cells expressed a nonselective cation channel (NCC), and the calcimimetic CaR activator, NPS R-467, but not its less active stereoisomer, NPS S-467, as well as the polycationic CaR agonist, neomycin, activated this NCC, demonstrating that the CaR expressed in these cells is functionally active. Therefore, HL-60 cells exhibit an increase in Ca

  7. Eugenol isolated from the essential oil of Eugenia caryophyllata induces a reactive oxygen species-mediated apoptosis in HL-60 human promyelocytic leukemia cells.

    PubMed

    Yoo, Chae-Bin; Han, Ki-Tae; Cho, Kyu-Seok; Ha, Joohun; Park, Hee-Juhn; Nam, Jung-Hwan; Kil, Uk-Hyun; Lee, Kyung-Tae

    2005-07-01

    Eugenol is a major component of essential oil isolated from the Eugenia caryophyllata (Myrtaceae), which has been widely used as a herbal drug. In this study, we investigated the effects of eugenol on the cytotoxicity, induction of apoptosis, and the putative pathways of its actions in human promyelocytic leukemia cells (HL-60) under the standard laboratory illumination. Eugenol-treated HL-60 cells displayed features of apoptosis including DNA fragmentation and formation of DNA ladders in agarose gel electrophoresis. We observed that eugenol transduced the apoptotic signal via ROS generation, thereby inducing mitochondrial permeability transition (MPT), reducing anti-apoptotic protein bcl-2 level, inducing cytochrome c release to the cytosol, and subsequent apoptotic cell death. Taken together, the present study demonstrated that ROS plays a critical role in eugenol-induced apoptosis in HL-60, and this is the first report on the mechanism of the anticancer effect of eugenol. PMID:15922856

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

    SciTech Connect

    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 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.

  9. Flow cytometric analysis of pentakis(aziridino)thiatriazadiphosphorine oxide (SOAz)-induced changes in cell cycle progression of HeLa and HL-60 cells.

    PubMed

    Hecquet, C; Nafziger, J; Ronot, X; Marie, J P; Adolphe, M

    1985-02-01

    The treatment of HeLa and HL-60 cells with various concentrations of pentakis(arizidino)thiatriazadiphosphorine oxide results in inhibition of growth and modification of cell cycle distribution. These phenomena were observed at 10(-4) M and 5 X 10(-5) M for HeLa cells and 10(-5) M and 5 X 10(-6) M for HL-60 cells. The estimation of DNA content by flow cytometry showed an important shift in the distribution of cycling cells with a striking arrest in G2 for both cell lines with a concomitant late S-phase accumulation for HeLa cells. Incubation of cells in drug-free medium 3 days after treatment did not show any change in DNA distribution, suggesting the irreversibility of drug action.

  10. Genistein decreases cellular redox potential, partially suppresses cell growth in HL-60 leukemia cells and sensitizes cells to γ-radiation-induced cell death

    PubMed Central

    KIM, IN GYU; KIM, JIN SIK; LEE, JAE HA; CHO, EUN WIE

    2014-01-01

    Various mechanisms have been proposed to underlie the cellular activity of genistein, based on biological experiments and epidemiological studies. The present study demonstrated that genistein inhibited the expression of cytoplasmic nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate dehydrogenase (cICDH), thus increasing levels of intracellular reactive oxygen species (ROS) in human promyeloid leukemia HL-60 cells. In genistein-treated cells, the cellular redox potential (GSH/GSSG) was significantly decreased. This decrease in redox potential was caused by significant downregulation of the cICDH gene, generating the reducing equivalents (NADPH) for maintenance of cellular redox potential and cellular ROS level, which may regulate cell growth and cell death. Genistein-induced ROS partially induced rapid transition into the G2/M phase by upregulation of p21wap1/cip1 and apoptotic cell death. Treatment of cells with N-acetylcysteine, a well-known antioxidant (ROS scavenger), not only partially restored cell growth and inhibited cell cycle arrest in G2/M, but also prevented apoptotic cell death. By contrast, normal lymphocytes did not significantly progress into the G2/M phase and radiation-induced cell death was inhibited by genistein treatment. Therefore, genistein and γ-irradiation together synergistically cause cell death in leukemia cells, however, genistein has a radioprotective effect in normal human lymphocytes. In conclusion, it was suggested that genistein selectively functions, not as an antioxidant, but as a pro-oxidant in HL-60 cells. This property can increase ionizing radiation-induced cell cycle arrest and sensitivity to apoptotic cell death in human promyeloid leukemia HL-60 cells, but does not cause significant damage to normal cells. PMID:25310747

  11. The effect of IFN-gamma and TNF-alpha on the eosinophilic differentiation and NADPH oxidase activation of human HL-60 clone 15 cells.

    PubMed

    Lopez, Juan A; Newburger, Peter E; Condino-Neto, Antonio

    2003-12-01

    The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.

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

    PubMed

    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.

  13. Induction of apoptosis by the plant alkaloid sampangine in human HL-60 leukemia cells is mediated by reactive oxygen species.

    PubMed

    Kluza, Jérôme; Mazinghien, Romain; Degardin, Klara; Lansiaux, Amélie; Bailly, Christian

    2005-11-21

    Sampangine is a plant-derived copyrine alkaloid extracted from the stem bark of Cananga odorata. This azaoxoaporphine alkaloid primarily exhibits antifungal and antimycobacterial activities but also displays in vitro antimalarial activity against Plasmodium falciparum and it is cytotoxic to human malignant melanoma cells. Recently, sampangine was described as a pro-apoptotic agent, but the biochemical pathway leading to cell death remained unclear. Considering that sampangine possesses an iminoquinone moiety, potentially functioning as an oxidizing agent, we have investigated the implication of an oxidant stress on sampangine-induced cytotoxicity. We show that the treatment of human HL-60 leukemia cells for 48 h with sampangine induced an important oxidative burst. Real time flow cytometry measurements indicated that the production of oxidative species is very rapid, within minutes following the drug addition. Quenching of reactive oxygen species by the antioxidants N-acetyl cystein, vitamin C and vitamin E abolishes the pro-apoptotic activity of sampangine. The drug-induced production of reactive oxygen species is associated with cell cycle perturbations and mitochondrial alterations. This study shed light on the mechanism of action of sampangine and provides novel opportunities to use azaoxoaporphine alkaloids as lead compounds for the design of pro-apoptotic anticancer agents. PMID:16289142

  14. Protein-bound polysaccharide-K induces apoptosis via mitochondria and p38 mitogen-activated protein kinase-dependent pathways in HL-60 promyelomonocytic leukemia cells.

    PubMed

    Hirahara, Noriyuki; Edamatsu, Takeo; Fujieda, Ayako; Fujioka, Masaki; Wada, Tsutomu; Tajima, Yoshitsugu

    2013-07-01

    Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101). PSK is a biological response modifier (BRM), and its mechanism of action is partly mediated by modulating host immune systems; however, recent studies showed antiproliferative activity of PSK. Therefore, we examined the mechanism underlying the antiproliferative activity of PSK using seven different human malignant cell lines (WiDr, HT29, SW480, KATOIII, AGS, HL-60 and U937), and PSK was found to inhibit the proliferation of HL-60 cells most profoundly. Therefore, HL-60 cells were used to elucidate the mechanism of the antiproliferative activity. Western blotting was performed to detect phosphorylated p38 mitogen-activated protein kinase (MAPK). A p38 MAPK inhibitor, SB203580, was used to examine the roles in PSK-induced apoptosis and growth inhibition. Flow cytometry was performed for mitochondrial membrane potential detection. PSK activated caspase-3 and induced p38 MAPK phosphorylation. Co-treatment with SB203580 blocked PSK-induced apoptosis, caspase-3 activation and growth inhibition. PSK induced apoptosis via the mitochondrial pathway. The depolarization of mitochondria induced by PSK was reversed by co-treatment with SB203580. The present study revealed that PSK induced apoptosis in HL-60 cells via a mitochondrial and p38 MAPK-dependent pathway. PMID:23604455

  15. Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid.

    PubMed

    Scheibe, R J; Kuehl, H; Krautwald, S; Meissner, J D; Mueller, W H

    2000-01-01

    The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p-nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL-60 cells was 120 microM. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 microM; K(i) HL-60 = 50 microM). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells. PMID:10649440

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

    PubMed

    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.

  17. Ethyl Gallate Induces Apoptosis of HL-60 Cells by Promoting the Expression of Caspases-8, -9, -3, Apoptosis-Inducing Factor and Endonuclease G

    PubMed Central

    Kim, Woong-Hyun; Song, Hyun-Ok; Choi, Hwa-Jung; Bang, Ho-Il; Choi, Du-Young; Park, Hyun

    2012-01-01

    Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways. PMID:23109891

  18. Quercus Suber L. Cork Extracts Induce Apoptosis in Human Myeloid Leukaemia HL-60 Cells.

    PubMed

    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.

  19. α-Tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells.

    PubMed

    Huang, Huarong; Chen, Shaohua; Van Doren, Jeremiah; Li, Dongli; Farichon, Chelsea; He, Yan; Zhang, Qiuyan; Zhang, Kun; Conney, Allan H; Goodin, Susan; Du, Zhiyun; Zheng, Xi

    2015-06-01

    α‑Tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α‑tomatine on human myeloid leukemia HL‑60 cells were investigated. Treatment of HL‑60 cells with α‑tomatine resulted in growth inhibition and apoptosis in a concentration‑dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL‑60 cells. Growth inhibition and apoptosis induced by α‑tomatine in HL‑60 cells was partially abrogated by addition of cholesterol indicating that interactions between α‑tomatine and cell membrane‑associated cholesterol may be important in mediating the effect of α‑tomatine. Activation of nuclear factor‑κB by the phorbol ester, 12‑O‑tetradecanoylphorbol‑13‑acetate failed to prevent apoptosis in HL‑60 cells treated with α‑tomatine. In animal experiments, it was found that treatment of mice with α‑tomatine inhibited the growth of HL‑60 xenografts in vivo. Results from the present study indicated that α‑tomatine may have useful anti‑leukemia activities. PMID:25625536

  20. Structure-activity relationship studies of 5,7-dihydroxyflavones as naturally occurring inhibitors of cell proliferation in human leukemia HL-60 cells.

    PubMed

    Ninomiya, Masayuki; Nishida, Kyohei; Tanaka, Kaori; Watanabe, Kunitomo; Koketsu, Mamoru

    2013-07-01

    Flavonoids are widely occurring polyphenols that are found in plants. The aim of this study was to investigate the structure-activity relationships of 5,7-dihydroxyflavones, with a focus on the effect of B ring structure substitution on the antiproliferative effects of the compounds in human leukemia HL-60 cells. We prepared a series of 5,7-dihydroxyflavones and evaluated their ability to inhibit the proliferation of HL-60 cells by using the MTT assay. The apoptosis- and cell differentiation-inducing ability of the most potent flavones were investigated using staining and morphological analyses. This study explored the antileukemic and chemopreventive potency of 5,7-dihydroxyflavones, particularly diosmetin and chrysoeriol, which have both hydroxy and methoxy groups on the B ring.

  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.

    PubMed

    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. PMID:26717974

  2. Biochemical and ultrastructural effects of monensin on myeloperoxidase (MPO) processing in human leukemic HL-60 cells

    SciTech Connect

    Akin, D.T.; Kinkade J.M. Jr.; Parmley, R.T.

    1986-05-01

    Pulse-chase experiments using /sup 35/S-methionine, immunoprecipitation, and SDS-PAGE fluorography were used to study the effects of monensin (1 ..mu..M) on the post-translational processing and packaging of MPO into azurophil granules (AG). After 20 hr, maturation of MPO was inhibited by 80% and a large intermediate accumulated. Electron microscopy of treated cells revealed striking changes in morphology (formation of large vacuoles with small electron dense cores in the Golgi region) and staining patterns for complex glycoconjugates (PA-TCH-SP) and sulfate (HID), indicating qualitative changes in granule and Golgi characteristics. The distribution of DAB-reactive peroxidase was relatively unchanged. Subcellular fractionation using Percoll density gradient centrifugation showed labeled MPO remained in a lower density region and did not chase into higher density AG as seen with untreated cells. These data indicate that monensin inhibits both the maturation of MPO and the AG. Further studies are required to determine how closely these two processes are related.

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25088254

  5. Arsenic trioxide and radiation enhance apoptotic effects in HL-60 cells through increased ROS generation and regulation of JNK and p38 MAPK signaling pathways.

    PubMed

    Ho, Sheng-Yow; Wu, Wei-Jr; Chiu, Hui-Wen; Chen, Yi-An; Ho, Yuan-Soon; Guo, How-Ran; Wang, Ying-Jan

    2011-09-01

    The induction of apoptotic cell death is a significant mechanism of tumor cells under the influence of radio-/chemotherapy, and resistance to these treatments has been linked to some cancer cell lines with a low propensity for apoptosis. The present study aimed to investigate the enhanced effects and mechanisms in apoptosis and the cycle distribution of HL-60 cells, a human leukemia cell line lacking a functional p53 protein, after combination treatment with arsenic trioxide (ATO) and irradiation (IR). Our results indicated that combined treatment led to increased cytotoxicity and apoptotic cell death in HL-60 cells, which was correlated with the activation of cdc-2 and increased expression of cyclin B, the induction of intracellular reactive oxygen species (ROS) generation, the loss of mitochondria membrane potential, and the activation of caspase-3. The combined treatment of HL-60 cells pre-treated with Z-VAD or NAC resulted in a significant reduction in apoptotic cells. In addition, activation of JNK and p38 MAPK may be involved in combined treatment-mediated apoptosis. The data suggest that a combination of IR and ATO could be a potential therapeutic strategy against p53-deficient leukemia cells.

  6. Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

    PubMed Central

    French, B T; Patrick, D E; Grever, M R; Trewyn, R W

    1991-01-01

    6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8)-deficient HL-60 cells is characterized by 2 days of growth, after which morphological differentiation proceeds. Addition of the tRNA wobble base queuine, in the presence of 6-TG, maintains the proliferative capability of the cells. The ability of 6-TG to induce differentiation correlates with c-myc mRNA down-regulation, but queuine has no effect on this parameter. Treatment with 6-TG for 2-3 days commits HL-60 cells to granulocytic differentiation, and, once committed, these cells do not respond to the monocytic inducer phorbol 12-myristate 13-acetate. Nonetheless, when cells are treated with queuine and 6-TG, they maintain the promyelocytic morphology and are capable of being induced down the monocytic pathway by phorbol 12-myristate 13-acetate as indicated by stabilization of c-fms mRNA and cell adherence. In the absence of queuine, phorbol 12-myristate 13-acetate is incapable of inducing monocytic markers in the 6-TG-treated cells. The data presented indicate that 6-TG-induced differentiation of HL-60 cells is a tRNA-facilitated event and that the tRNA wobble base queuine is capable of maintaining both the proliferative and pluripotent potential of the cells. Images PMID:1988936

  7. Ethanolic extract of fermented Thunb induces human leukemic HL-60 and Molt-4 cell apoptosis via oxidative stress and a mitochondrial pathway.

    PubMed

    Banjerdpongchai, Ratana; Kongtawelert, Prachya

    2011-01-01

    Houttuynia cordata Thunb (HCT) is a medicinal plant of the Saururaceae family which features antimutagenic and antiviral properties. For extraction, the whole plants were fermented or non-fermented with yeast and ethanol then the whole plants were dried, ground and extracted with 95% ethanol or water. The aims of this study were to compare cytotoxic effects, apoptosis induction, and mechanism(s) with the ethanolic and water extracts of fermented and non-fermented HCT. Cytotoxicity was assessed using the MTT assay in human leukemic HL-60, Molt-4 and peripheral blood mononuclear cells (PBMCs). Apoptotic death was characterized by staining with propidium iodide and examined under a fluorescence microscope. Peroxide radical production and reduction of mitochondrial transmembrane potential (MTP) were determined using 2',7'-dichlorohydrofluorescein diacetate and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. The expression of caspase-9 was identified by immunoblotting. The ethanolic extract of fermented HCT was cytotoxic to HL-60 >Molt- 4 > PBMCs, to a greater extent than the non-fermented preparation and the number of apoptotic cells was higher. The alcoholic (fermented) extract produced more radicals than the non-fermented in HL-60 cells but the converse was observed in Molt-4 cells. Reduction of MTP was found in HL-60 and Molt-4 cells treated with the alcoholic (fermented) extract and caspase-9 was cleaved dose-dependently in both cells. In conclusion, the alcoholic extract of fermented HCT was more toxic to human leukemic cells than the non-fermented and both cell lines underwent apoptosis via oxidative stress and a mitochondrial pathway. PMID:22393956

  8. Studies of the effect of 1,25-dihydroxycholecalciferol on the proliferation and differentiation of the human promyelocytic leukaemia cell line HL-60.

    PubMed

    Djulbegović, B; Christmas, S E; Evans, G; Moore, M

    1986-01-01

    Treatment of the human promyelocytic leukaemia cell line HL-60 with 1,25(OH)2D3, the active metabolite of vitamin D3, led to a dose- and time-dependent inhibition of growth and 3H-TdR incorporation at the population level. A similar effect was noted at the single cell level in clonogenic assays and autoradiographic experiments. Flow cytometry indicated that there was an arrest of cells in the G0/G1 phase of the cell cycle. Parallel to the loss of proliferative capacity 1,25(OH)2D3 induced differentiation of HL-60 into monocyte/macrophages as measured by the enzyme NSE and the macrophage membrane antigen recognised by the monoclonal antibody EB11 as well as by morphological changes. These findings reinforce the concept of concordant induction of differentiation and loss of proliferative capacity and demonstrate that the latter occurs not only at the population level but also at the single cell level in this system. In limiting dilution assays in liquid culture there was evidence for positive interactions between HL-60 cells as untreated cells gave less colonies at low dilutions than would have been expected by Poisson statistical analysis. In the presence of 10(-8) M 1,25(OH)2D3 more complex growth parameters were noted indicating the involvement of both positive and negative cellular interactions.

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

    PubMed

    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.

  10. Disruption of the PI3K/AKT/mTOR signaling cascade and induction of apoptosis in HL-60 cells by an essential oil from Monarda citriodora.

    PubMed

    Pathania, Anup Singh; Guru, Santosh Kumar; Verma, M K; Sharma, Chetna; Abdullah, Sheikh Tasduq; Malik, Fayaz; Chandra, Suresh; Katoch, Meenu; Bhushan, Shashi

    2013-12-01

    We have isolated an essential oil from Monarda citriodora (MC) and characterized its 22 chemical constituents with thymol (82%), carvacrol (4.82%), β-myrcene (3.45%), terpinen-4-ol (2.78%) and p-cymene (1.53%) representing the major constituents. We have reported for the first time the chemotherapeutic potential of MC in human promyelocytic leukemia HL-60 cells by means of apoptosis and disruption of the PI3K/AKT/mTOR signaling cascade. MC and its major constituent, thymol, inhibit the cell proliferation in different types of cancer cell lines like HL-60, MCF-7, PC-3, A-549 and MDAMB-231. MC was found to be more cytotoxic than thymol in HL-60 cells with an IC50 value of 22 μg/ml versus 45 μg/ml for thymol. Both MC and thymol induce apoptosis in HL-60 cells, which is evident by Hoechst staining, cell cycle analysis and immuno-expression of Bcl-xL, caspase-3,-8,-9 and PARP-1 cleavage. Both induce apoptosis by extrinsic and intrinsic apoptotic pathways that were confirmed by enhanced expression of death receptors (TNF-R1, Fas), caspase-9, loss of mitochondrial membrane potential and regression of Bcl-2/Bax ratio. Interestingly, both MC and thymol inhibit the downstream and upstream signaling of PI3K/AKT/mTOR pathway. The degree of apoptosis induction and disruption of the PI3K signaling cascade by MC was significantly higher when compared to thymol. PMID:23994707

  11. Disruption of the PI3K/AKT/mTOR signaling cascade and induction of apoptosis in HL-60 cells by an essential oil from Monarda citriodora.

    PubMed

    Pathania, Anup Singh; Guru, Santosh Kumar; Verma, M K; Sharma, Chetna; Abdullah, Sheikh Tasduq; Malik, Fayaz; Chandra, Suresh; Katoch, Meenu; Bhushan, Shashi

    2013-12-01

    We have isolated an essential oil from Monarda citriodora (MC) and characterized its 22 chemical constituents with thymol (82%), carvacrol (4.82%), β-myrcene (3.45%), terpinen-4-ol (2.78%) and p-cymene (1.53%) representing the major constituents. We have reported for the first time the chemotherapeutic potential of MC in human promyelocytic leukemia HL-60 cells by means of apoptosis and disruption of the PI3K/AKT/mTOR signaling cascade. MC and its major constituent, thymol, inhibit the cell proliferation in different types of cancer cell lines like HL-60, MCF-7, PC-3, A-549 and MDAMB-231. MC was found to be more cytotoxic than thymol in HL-60 cells with an IC50 value of 22 μg/ml versus 45 μg/ml for thymol. Both MC and thymol induce apoptosis in HL-60 cells, which is evident by Hoechst staining, cell cycle analysis and immuno-expression of Bcl-xL, caspase-3,-8,-9 and PARP-1 cleavage. Both induce apoptosis by extrinsic and intrinsic apoptotic pathways that were confirmed by enhanced expression of death receptors (TNF-R1, Fas), caspase-9, loss of mitochondrial membrane potential and regression of Bcl-2/Bax ratio. Interestingly, both MC and thymol inhibit the downstream and upstream signaling of PI3K/AKT/mTOR pathway. The degree of apoptosis induction and disruption of the PI3K signaling cascade by MC was significantly higher when compared to thymol.

  12. Induction of poly(ADP-ribose) polymerase-1 cleavage by antitumor triptycene bisquinones in wild-type and daunorubicin-resistant HL-60 cell lines.

    PubMed

    Wang, Yang; Perchellet, Elisabeth M; Tamura, Masafumi; Hua, Duy H; Perchellet, Jean Pierre

    2002-12-15

    In contrast to their inactive parent compound triptycene (code name TT0), new synthetic analogs (TT code number) mimic the antitumor effects of the anthracycline quinone antibiotic daunorubicin (DAU) in the nM range in vitro but have the additional advantage of also blocking nucleoside transport and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones may induce DNA fragmentation at 24 h by an active mechanism that requires RNA and protein syntheses and protease activities, the most cytotoxic of them, TT24, was tested for its ability to induce poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early marker of apoptosis. PARP-1 cleavage starts at 2-3 h and is maximally induced at 6 h by 1.6 microM concentrations of TT24 and DAU in wild-type drug-sensitive HL-60-S cells. However, in MDR HL-60-RV cells, PARP-1 cleavage is still induced by 4 microM TT24 but not by 4-10 microM DAU. The magnitude of PARP-1 cleavage may increase with the number of quinoid rings in the triptych structure and, in contrast to TT0, all lead antitumor TT bisquinones share the ability to fully induce PARP-1 cleavage in HL-60-S cells. A 1 h pulse treatment is sufficient for TT24 and DAU to induce PARP-1 cleavage at 6 h. Since the abilities of TT24 and DAU to induce PARP-1 cleavage are inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone but not by N-tosyl-L-phenylalanine chloromethyl ketone, caspase-mediated apoptosis may be involved in the mechanism by which these quinone antitumor drugs induce the proteolytic cleavage of PARP-1 at 6 h and the internucleosomal fragmentation of DNA at 24 h in the HL-60 tumor cell system. PMID:12406551

  13. Apoptosis induced by the alkaloid sampangine in HL-60 leukemia cells: correlation between the effects on the cell cycle progression and changes of mitochondrial potential.

    PubMed

    Kluza, Jérôme; Clark, Alice M; Bailly, Christian

    2003-12-01

    Sampangine, a plant-derived copyrine alkaloid extracted from the stem bark of Cananga odorata, primarily exhibits antifungal and antimycobacterial activities, but it also displays in vitro antimalarial activity against Plasmodium falciparum and is cytotoxic to human malignant melanoma cells. It inhibits cell aggregation, but no molecular target has yet been identified. We investigated the biochemical pathway involved in sampangine-induced cytotoxicity toward HL-60 cells. These leukemia cells are prone to enter apoptosis after treatment with various stimuli, including genotoxic compounds structurally close to sampangine, such as ascididemin. PMID:15033745

  14. A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell

    PubMed Central

    1992-01-01

    We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions. PMID:1474373

  15. Human myeloblastic leukemia cells (HL-60) express a membrane receptor for estrogen that signals and modulates retinoic acid-induced cell differentiation

    SciTech Connect

    Kauss, M. Ariel; Reiterer, Gudrun; Bunaciu, Rodica P.; Yen, Andrew

    2008-10-01

    Estrogen receptors are historically perceived as nuclear ligand activated transcription factors. An estrogen receptor has now been found localized to the plasma membrane of human myeloblastic leukemia cells (HL-60). Its expression occurs throughout the cell cycle, progressively increasing as cells mature from G{sub 1} to S to G{sub 2}/M. To ascertain that the receptor functioned, the effect of ligands, including a non-internalizable estradiol-BSA conjugate and tamoxifen, an antagonist of nuclear estrogen receptor function, were tested. The ligands caused activation of the ERK MAPK pathway. They also modulated the effect of retinoic acid, an inducer of MAPK dependent terminal differentiation along the myeloid lineage in these cells. In particular the ligands inhibited retinoic acid-induced inducible oxidative metabolism, a functional marker of terminal myeloid cell differentiation. To a lesser degree they also diminished retinoic acid-induced earlier markers of cell differentiation, namely CD38 and CD11b. However, they did not regulate retinoic acid-induced G{sub 0} cell cycle arrest. There is thus a membrane localized estrogen receptor in HL-60 myeloblastic leukemia cells that can cause ERK activation and modulates the response of these cells to retinoic acid, indicating crosstalk between the membrane estrogen and retinoic acid evoked pathways relevant to propulsion of cell differentiation.

  16. Concise synthesis of carbazole-1,4-quinones and evaluation of their antiproliferative activity against HCT-116 and HL-60 cells.

    PubMed

    Nishiyama, Takashi; Hatae, Noriyuki; Yoshimura, Teruki; Takaki, Sawa; Abe, Takumi; Ishikura, Minoru; Hibino, Satoshi; Choshi, Tominari

    2016-10-01

    We report a convenient synthesis of carbazole-1,4-quinone alkaloid koeniginequinones A and B using a tandem ring-closing metathesis with the dehydrogenation reaction sequence under an O2 atmosphere as an important step. Using this method, carbazole-1,4-quinones substituted at the 5-, 6-, 7-, and/or 8-positions have been synthesized. Moreover, 24 compounds, including koeniginequinones A and B, have been evaluated for their antiproliferative activity against HCT-116 and HL-60 cells, and the 6-nitro analog exhibited the most potent activity against both tumor cell types. PMID:27318980

  17. Redox regulation of cAMP levels by ascorbate in 1,25-dihydroxy- vitamin D3-induced differentiation of HL-60 cells.

    PubMed Central

    López-Lluch, G; Burón, M I; Alcaín, F J; Quesada, J M; Navas, P

    1998-01-01

    1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation to monocyte-macrophage lineage of several leukaemic cell lines such as HL-60, U937, M1 and Mono Mac 6. Ascorbate also modulates growth and differentiation of different animal cells in culture. We have previously reported the stimulating effect of ascorbate on 1, 25-(OH)2D3-induced HL-60 cell differentiation. We show here that 1, 25-(OH)2D3 induces a transient increase in cAMP levels in these cells, and ascorbate significantly increases these cAMP levels. Ascorbate alone does not have any effect. Other cAMP-increasing agents such as isobutylmethylxanthine, forskolin and prostaglandin E2 maintain high levels of cAMP at 48 h of incubation and also enhance differentiation along the monocytic pathway induced by 1, 25-(OH)2D3, as revealed by specific differentiation markers, demonstrating the importance of cAMP in the differentiation process. It is also shown that the presence of ascorbate and its free radical (AFR) during 1,25-(OH)2D3-induced differentiation significantly decreases cytoplasmic NADH levels compared with those induced by 1,25-(OH)2D3 in HL-60 cells. The results indicate that NADH is an inhibitor of adenylate cyclase in these cells. AFR is an electron acceptor of the trans-plasma-membrane electron-transport system, and NADH is the electron donor. Through this system, ascorbate and AFR keep levels of NADH low, thereby decreasing its inhibitory effect on adenylate cyclase activity and so increasing cAMP synthesis. We also demonstrate that other ascorbate derivatives, such as ascorbate 2-phosphate and dehydroascorbate, both of which are unable to produce AFR, do not alter intracellular NADH levels during 1, 25-(OH)2D3-induced differentiation. Also, ascorbate and AFR increase specific differentiation markers (CD14 and NitroBlue Tetrazolium reduction) but neither ascorbate 2-phosphate nor dehydroascorbate show this enhancing activity. In summary, we propose that the effect of ascorbate on 1

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

    PubMed

    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.

  19. Cytotoxic and apoptotic effects of extracts of Artemisia ciniformis Krasch. and Popov ex Poljakov on K562 and HL-60 cell lines.

    PubMed

    Tayarani-Najaran, Zahra; Hajian, Zahra; Mojarrab, Mahdi; Emami, Seyed Ahmad

    2014-01-01

    Artemisia, as one of the largest genera in the tribe Anthemideae of the Asteraceae comprises an important part of Iranian flora. While cytotoxic and apoptotic properties have already been reported for some species of the genus there is not any report on cytotoxic effects of A. ciniformis. Petroleum ether (40-60), dichloromethane, ethyl acetate, ethanol and ethanol-water (50:50) extracts of the aerial parts of A. cinformis were subjected to cytotoxic and apoptotic evaluations on two cancer human cell lines (K562 and HL-60) and on J774 normal cells. Among multiple extracts evaluated for cytotoxicity, dichloromethane (CH2Cl2) and petroleum ether (PE) extracts were shown to possess the highest anti-proliferative effects on HL-60 and K562 cells with IC50 values of 31.3 and 25.5 μg/ml respectively. Apoptosis induction verified by sub-G1 peaks was seen in flow cytometry histograms. Increase in the amount of Bax protein, formation of DNA fragments, and cleavage of PARP to 24 and 89 kDa sub units all confirmed induction of apoptosis by A. cinformis extracts. Taken together according to the result of the present study some extracts of A. cinformis could be considered as sources for natural cytotoxic compounds and further mechanistic and phytochemical studies are recommended to fully understand the underlying mechanisms of cancer cell death as well as identification of responsible phytochemicals. PMID:25227790

  20. Cyclic AMP-dependent protein kinase interferes with GTP. gamma. S stimulated IP sub 3 formation in differentiated HL-60 cell membranes

    SciTech Connect

    Misaki, Naoyuki; Imaizumi, Taro; Watanabe, Yashuiro )

    1989-01-01

    The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of {sup 3}H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA in the presence of MgATP for 15 min. at 37{degree}C decreased GTP {gamma}S-stimulated inositol trisphosphate (IP{sub 3}) formation by about 30%, but had no influence on Ca{sup 2+}-stimulated IP{sub 3} formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some {sup 32}P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation.

  1. IGF-I stimulates IL-8 production in the promyelocytic cell line HL-60 through activation of extracellular signal-regulated protein kinase.

    PubMed

    Kooijman, Ron; Coppens, Astrid; Hooghe-Peters, Elisabeth

    2003-12-01

    Interleukin (IL)-8 serves as a major chemoattractant for neutrophils and has also been proposed to affect cancer progression. In the present study, we show that IGF-I stimulates IL-8 mRNA expression and IL-8 secretion in the leukemic cell line HL-60. Stimulation of IL-8 expression was completely attenuated by two inhibitors of mitogen-activated protein kinase (MAPK) kinase (MEK), which phosphorylates the MAPKs extracellular-regulated kinase (ERK)1 and ERK2, and by the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. In contrast, inhibitors of p38 MAPK and phosphatidylinositol-3 kinase (PI3K) did not abrogate the effect of IGF-I. We also show that IGF-I stimulates the activation of ERK1 and ERK2, but we could not detect any effect of IGF-I on the phosphorylation of p38, JNK(p46) or JNK(p54). Collectively, our results suggest that basal JNK activity and activation of the MEK-ERK pathway are required for upregulation of IL-8 by IGF-I in HL-60 cells.

  2. Lipase-catalyzed preparation of optically active 1'-acetoxychavicol acetates and their structure-activity relationships in apoptotic activity against human leukemia HL-60 cells.

    PubMed

    Azuma, Hideki; Miyasaka, Keita; Yokotani, Tsuyoshi; Tachibana, Taro; Kojima-Yuasa, Akiko; Matsui-Yuasa, Isao; Ogino, Kenji

    2006-03-15

    Structure-activity relationships of 1'-acetoxychavicol acetate (ACA) for apoptotic activity against human leukemia HL-60 cells were investigated using optically active ACA and various racemic ACA analogues. Natural-type (or with different acyl group) ACA showed a high apoptotic activity, but the ortho or meta isomers, 4-deacetoxy analogue, and the 2'-3' dehydrogenated derivative had no effect, or a weak activity. Optically active (R)- and (S)-ACA were prepared by a lipase-catalyzed esterification. Using a mixture of vinyl acetate-tetrahydrofuran (1:1 v/v) as a solvent at refluxing temperature, optically pure (R)- and (S)-ACA were obtained (99.7% ee and 99.1% ee, respectively). The apoptosis-inducing effects of both enantiomers were compared by means of an MTT assay and the detection of typical apoptotic phenomena (DNA fragmentation, caspase-3 activation, and PARP cleavage) and these two activities were almost equal. These results indicate that the essential moieties of ACA for apoptotic activity against HL-60 cells are both the presence of a 4-acetoxyl group and an unsaturated double bond between C-2' and C-3', and that the configuration at the 1'-position is unrelated to activity.

  3. Mechanism study of PEGylated polyester and β-cyclodextrin integrated micelles on drug resistance reversal in MRP1-overexpressed HL60/ADR cells.

    PubMed

    Ji, Qian; Qiu, Liyan

    2016-08-01

    Chemotherapy is one of the main strategies for cancer treatment, but its effective application is seriously limited by the development of drug resistance. In this study, we designed micellar vectors for doxorubicin based on amphiphilic copolymers sequentially linking β-cyclodextrin (β-CD), polylacticacid (PLA) or polycaprolactone (PCL) block, and polyethylene glycol (PEG) block to overcome drug resistance in human acute myeloid leukemia cells (HL60/ADR) overexpressing multidrug resistance protein 1 (MRP1). The significant enhancement in cytotoxicity and inhibited HL60/ADR tumor growth in mouse was achieved. More importantly, several analyses were performed to understand the interactions between various polymers and MRP1 at the cellular level. The results showed that the polymers did not show remarkable correlation of MRP1 gene and protein expression, but could decrease intracellular ATP, mitochondrial membrane potential and glutathione levels, which was greatly dependent on the molecular structure of polymers. In conclusion, these novel micelles can be considered as a kind of promising drug delivery system for tumor therapy to reverse drug resistance related to MRP1 overexpression. PMID:27088190

  4. Kayeassamin A Isolated from the Flower of Mammea siamensis Triggers Apoptosis by Activating Caspase-3/-8 in HL-60 Human Leukemia Cells

    PubMed Central

    Uto, Takuhiro; Tung, Nguyen Huu; Thongjankaew, Pinjutha; Lhieochaiphant, Sorasak; Shoyama, Yukihiro

    2016-01-01

    Background: Mammea siamensis (Miq.) T. Anders. is used as a medicinal plant in Thailand and has several traditional therapeutic properties. In a previous study, we isolated eight compounds from the flower of M. siamensis and demonstrated that kayeassamin A (KA) exhibited potent antiproliferative activity against human leukemia and stomach cancer cell lines. Objective: In this study, we investigated the effect of KA on cell viability and apoptotic mechanisms in HL-60 human leukemia cells. Materials and Methods: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Nuclear morphology and DNA fragmentation were observed using Hoechst 33258 staining and agarose gel electrophoresis, respectively. The sub-G1 phase of cells was analyzed by flow cytometry after the cellular DNA had been stained with propidium iodide. The protein levels of poly (ADP-ribose) polymerase (PARP) and caspases were determined by Western blotting. Results: KA exhibited a significant cytotoxic effect in a dose- and time-dependent manner, and induced chromatin condensation, DNA fragmentation, and sub-G1 phase DNA content, known as molecular events associated with the induction of apoptosis. In addition, KA strongly induced the activation of PARP and caspase-3 and -8, with weak caspase-9 activation. Furthermore, KA-induced DNA fragmentation was abolished by pretreatment with z-VAD-FMK (a broad caspase inhibitor), z-DEVD-FMK (a caspase-3 inhibitor), and z-IETD-FMK (a caspase-8 inhibitor), but not by z-LEHD-FMK (a caspase-9 inhibitor) pretreatment. Conclusion: These results indicate that KA triggers apoptotic cell death by activation of caspase-3 and -8 in HL-60 cells. SUMMARY Kayeassamin A (KA) isolated from the flower of Mammea siamensis exhibited a significant cytotoxic effect in HL-60 human leukemia cells. KA triggers apoptotic cell death by activating caspase-3/-8. Abbreviations Used: KA: Kayeassamin A; MTT: 3-(4,5-dimethylthiazol-2-yl)-2

  5. Kayeassamin A Isolated from the Flower of Mammea siamensis Triggers Apoptosis by Activating Caspase-3/-8 in HL-60 Human Leukemia Cells

    PubMed Central

    Uto, Takuhiro; Tung, Nguyen Huu; Thongjankaew, Pinjutha; Lhieochaiphant, Sorasak; Shoyama, Yukihiro

    2016-01-01

    Background: Mammea siamensis (Miq.) T. Anders. is used as a medicinal plant in Thailand and has several traditional therapeutic properties. In a previous study, we isolated eight compounds from the flower of M. siamensis and demonstrated that kayeassamin A (KA) exhibited potent antiproliferative activity against human leukemia and stomach cancer cell lines. Objective: In this study, we investigated the effect of KA on cell viability and apoptotic mechanisms in HL-60 human leukemia cells. Materials and Methods: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Nuclear morphology and DNA fragmentation were observed using Hoechst 33258 staining and agarose gel electrophoresis, respectively. The sub-G1 phase of cells was analyzed by flow cytometry after the cellular DNA had been stained with propidium iodide. The protein levels of poly (ADP-ribose) polymerase (PARP) and caspases were determined by Western blotting. Results: KA exhibited a significant cytotoxic effect in a dose- and time-dependent manner, and induced chromatin condensation, DNA fragmentation, and sub-G1 phase DNA content, known as molecular events associated with the induction of apoptosis. In addition, KA strongly induced the activation of PARP and caspase-3 and -8, with weak caspase-9 activation. Furthermore, KA-induced DNA fragmentation was abolished by pretreatment with z-VAD-FMK (a broad caspase inhibitor), z-DEVD-FMK (a caspase-3 inhibitor), and z-IETD-FMK (a caspase-8 inhibitor), but not by z-LEHD-FMK (a caspase-9 inhibitor) pretreatment. Conclusion: These results indicate that KA triggers apoptotic cell death by activation of caspase-3 and -8 in HL-60 cells. SUMMARY Kayeassamin A (KA) isolated from the flower of Mammea siamensis exhibited a significant cytotoxic effect in HL-60 human leukemia cells. KA triggers apoptotic cell death by activating caspase-3/-8. Abbreviations Used: KA: Kayeassamin A; MTT: 3-(4,5-dimethylthiazol-2-yl)-2

  6. 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.

    PubMed

    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.

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

    SciTech Connect

    Lu, M.L.; Sato, Mitsuhiro; Cao, Boliang; Richie, J.P.

    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.

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

    PubMed

    Husain, Islam; Sharma, Anjana; Kumar, Suresh; Malik, Fayaz

    2016-01-01

    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. PMID:26891220

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

    PubMed Central

    Husain, Islam; Sharma, Anjana; Kumar, Suresh; Malik, Fayaz

    2016-01-01

    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. PMID:26891220

  10. 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.

    PubMed

    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.

  11. Five novel naphthylisoquinoline alkaloids with growth inhibitory activities against human leukemia cells HL-60, K562 and U937 from stems and leaves of Ancistrocladus tectorius.

    PubMed

    Jiang, Chao; Li, Zhan-Lin; Gong, Ping; Kang, Sheng-Li; Liu, Ming-Sheng; Pei, Yue-Hu; Jing, Yong-Kui; Hua, Hui-Ming

    2013-12-01

    Two new 7,6'-coupled naphthylisoquinolines, namely ancistrotectorines A (1) and B (2), two new 5,3'-coupled naphthylisoquinolines, namely ancistrotectorines C (3) and D (4), and one new 7,8-coupled naphthylisoquinoline, namely ancistrotectorine E (5), together with 9 known naphthylisoquinoline alkaloids, hamatine (6), ancistrobertsonine B (7), ancistrocladinine (8), hamatinine (9), ancistrotanzanine A (10), ancistrotanzanine B (11), ancistrotectoriline B (12), 7-epi-ancistrobrevine D (13), and ancistrotectorine (14), were isolated from the 70% EtOH extract of Ancistrocladus tectorius. Their structures were elucidated based on the extensive analysis of spectroscopic data (1D, 2D NMR and MS). Compound 5 exhibited inhibitory activities against HL-60, K562 and U937 cell lines with IC50 values of 1.70, 4.18 and 2.56 μM respectively.

  12. Copper(II) and uranyl(II) complexes with acylthiosemicarbazide: synthesis, characterization, antibacterial activity and effects on the growth of promyelocytic leukemia cells HL-60.

    PubMed

    Angelusiu, Madalina Veronica; Almajan, Gabriela Laura; Rosu, Tudor; Negoiu, Maria; Almajan, Eva-Ruxandra; Roy, Jenny

    2009-08-01

    New chelates of N(1)-[4-(4-X-phenylsulfonyl)benzoyl]-N(4)-butyl-thiosemicarbazide (X=H, Cl, Br) with Cu(2+) and UO(2)(2+) have been prepared and characterized by analytical and physico-chemical techniques such as magnetic susceptibility measurements, elemental and thermal analyses, electronic, ESR and IR spectral studies. Room temperature ESR spectra of Cu(II) complexes yield {g} values characteristic of distorted octahedral and pseudo-tetrahedral geometry. Infrared spectra indicate that complexes contain six-coordinate uranium atom with the ligand atoms arranged in an equatorial plane around the linear uranyl group. Effects of these complexes on the growth of human promyelocytic leukemia cells HL-60 and their antibacterial activity (against Staphylococcus epidermidis ATCC 14990, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 14579, Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 11775 strains) were studied comparatively with that of free ligands. PMID:19356828

  13. 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.

    PubMed

    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.

  14. 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.

    PubMed

    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.

  15. Effect of the orthoquinone moiety in 9,10-phenanthrenequinone on its ability to induce apoptosis in HCT-116 and HL-60 cells.

    PubMed

    Hatae, Noriyuki; Nakamura, Jun; Okujima, Tetsuo; Ishikura, Minoru; Abe, Takumi; Hibino, Satoshi; Choshi, Tominari; Okada, Chiaki; Yamada, Hiroko; Uno, Hidemitsu; Toyota, Eiko

    2013-08-15

    9,10-Phenanthrenequinone (9,10-PQ) is one of the most abundant quinones among diesel exhaust particulates. Recent data have suggested that quinones induce apoptosis in immune, epithelial and tumor cells, leading to respirator illness; however, the mechanisms by which quinones induce apoptosis and the structure required for this remain unknown. We studied the antitumor activity of 9,10-PQ analogs against two human tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. The loss of the cis-orthoquinone unit in 9,10-PQ abrogated its ability to induce apoptosis in the two tumor cell lines, and the LC50 values of these analogs were indicated over 10 μM. An analog of 9,10-PQ in which the biaryl unit had been deleted displayed a reduced ability to induce tumor cell apoptosis, while the analogs 1,10-phenanthroline-5,6-dione (9) and pyrene-4,5-dione (10), which also had modified biaryl units, exhibited increased tumor cell apoptotic activity. The cis-orthoquinone unit in 9,10-PQ was identified as essential for its ability to induce apoptosis in tumor cells, and its biaryl unit is also considered to influence orthoquinone-mediated apoptotic activity.

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

    SciTech Connect

    Park, Yoon Shin; Lim, Goh-Woon; Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung; Yoo, Eun-Sun; Chan Ra, Jeong; Ryu, Kyung-Ha

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

    PubMed Central

    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

  18. Daunorubicin activates NFkappaB and induces kappaB-dependent gene expression in HL-60 promyelocytic and Jurkat T lymphoma cells.

    PubMed

    Boland, M P; Foster, S J; O'Neill, L A

    1997-05-16

    The anthracycline antibiotic, daunorubicin, can induce programmed cell death (apoptosis) in cells. Recent work suggests that this event is mediated by ceramide via enhanced ceramide synthase activity. Since the generation of ceramide has been directly linked with the activation of the transcription factor, NFkappaB, this was investigated as a novel target for the action of daunorubicin. Here we describe how treatment of HL-60 promyelocytes and Jurkat T lymphoma cells with daunorubicin results in the activation of the transcription factor NFkappaB. The effect of daunorubicin was evident following 1-2 h treatment, which was in contrast to the time course of activation obtained with the cytokine, tumor necrosis factor, where NFkappaB activation was detected within minutes of cellular stimulation. Activated complexes were shown to contain predominantly p50 and p65/RelA subunit components. Daunorubicin also induced IkappaB degradation and increased the expression of an NFkappaB-linked reporter gene. In addition, the drug was found to strongly potentiate the ability of tumor necrosis factor to induce an NFkappaB-linked reporter gene, suggesting a synergy between these two agents in this response. These events were sensitive to the iron chelator, deferoxamine mesylate (desferal), and the anti-oxidant and metal chelator pyrrolidine dithiocarbamate. A structurally related compound, mitoxantrone, which, unlike daunorubicin, is unable to undergo redox cycling in cells, also activated NFkappaB in a pyrrolidine dithiocarbamate-sensitive manner. A specific inhibitor of ceramide synthase, fumonisin B1, had no effect on daunorubicin induced NFkappaB activation at a range of concentrations previously reported to block apoptosis induced by this drug. However, this agent could inhibit increases in ceramide induced by daunorubicin, in addition to blocking ceramide synthase activity from HL-60 cells which was activated in response to daunorubicin treatment. These data therefore suggest

  19. Interactions of dimethyl sulfoxide and granulocyte-macrophage colony-stimulating factor on the cell cycle kinetics and phosphoproteins of G1-enriched HL-60 cells: evidence of early effects on lamin B phosphorylation.

    PubMed

    Brennan, J K; Lee, K S; Frazel, M A; Keng, P C; Young, D A

    1991-03-01

    We have found that GM-CSF and DMSO have antagonistic effects on the proliferation but not maturation of asynchronously growing HL-60 cells such that growth in the presence of both more closely resembles normal hematopoiesis (Brennan et al., J. Cell Physiol. 132:246, 1987). Studies were undertaken to determine whether or not the agents affected the same mitogenic pathway and locus in the cell cycle. HL-60 populations containing at least 90% G1 cells were obtained by centrifugal elutriation, exposed to 100 u/ml recombinant human GM-CSF and/or 0-1.25% DMSO, and phosphoprotein changes quantified on autoradiograms of [32P]-orthophosphate-labeled cell proteins separated by giant 2-D gel electrophoresis. Results were correlated with 1) intracellular pH, determined by measurement of BCECF fluorescence; 2) [32P]-orthophosphate uptake; 3) cell cycle progression, determined by flow quantitation of DNA content in mithramycin or propidium iodide-stained cells; and 4) growth, determined by cell volume and concentration. GM-CSF stimulated and DMSO inhibited the GM-CSF-stimulated phosphorylation of 1 protein (approximately 65 kDa, p.i. 5.6) within 2 min of exposure. These effects were sustained through G1, not associated with changes in intracellular pH, and preceded similar antagonistic effects on phosphate uptake (15-30 minutes), cell volume change (16-24 hr), and cell concentration increase (28-32 hr). GM-CSF accelerated and DMSO inhibited G1 to S transit with the most marked antagonism observed in the second cycle following synchronization (28 to 40 hrs). Cell maturation (morphology, NBT reduction) was dominated by DMSO and not antagonized by GM-CSF. We have identified p65 as the nuclear intermediate filament protein, lamin B, on the basis of its locus on gels and its binding of a monoclonal antibody to intermediate filaments and antiserum to human lamin B on immunoblots. These studies suggest that at least part of the GM-CSF-DMSO antagonism is exerted through the same

  20. Effect of ajoene, a natural antitumor small molecule, on human 20S proteasome activity in vitro and in human leukemic HL60 cells.

    PubMed

    Xu, Bo; Monsarrat, Bernard; Gairin, Jean Edouard; Girbal-Neuhauser, Elisabeth

    2004-04-01

    The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.

  1. 2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells

    SciTech Connect

    Hsu, Mei-Hua; Liu, Chin-Yu; Lin, Chiao-Min; Chen, Yen-Jung; Chen, Chun-Jen; Lin, Yu-Fu; Huang, Li-Jiau; Lee, Kuo-Hsiung; Kuo, Sheng-Chu

    2012-03-01

    2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1 appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe. Highlights: ► HKL-1 is a potential antimitotic agent against HL-60 cells. ► HKL-1 induces spindle disruption and sustained resulted in mitotic catastrophe. ► CENP-E and aurora B protein expressions significantly reduced. ► Bcl-2 family protein expressions altered and caspase-9/-3 activation. ► HKL-1 is an attractive candidate for possible use as a novel antimitotic agent.

  2. 2,2',4,4'-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-{kappa}B

    SciTech Connect

    Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E. . E-mail: ganey@msu.edu

    2007-06-15

    Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2',4,4'-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A{sub 2} with subsequent release of arachidonic acid (AA) and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 min. The increase in COX-2 mRNA was associated with release of {sup 3}H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-{kappa}B and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A{sub 2}, reduced {sup 3}H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter {sup 3}H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-{kappa}B. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-{kappa}B prevented the 2244-TCB

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

    SciTech Connect

    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.

  4. Seed dormancy breaking diterpenoids, including novel brassicicenes J and K, from fungus Alternaria brassicicola, and their necrotic/apoptotic activities in HL-60 cells.

    PubMed

    Kenmoku, Hiromichi; Takeue, Sayaka; Oogushi, Megumi; Yagi, Yasuyuki; Sassa, Takeshi; Toyota, Masao; Asakawa, Yoshinori

    2014-03-01

    To find new metabolites similar to cotylenins and fusicoccins from the fungus Alternaria brassicicola, screening tests were carried out using the lettuce seed dormancy breaking assay. Activity-guided fractionation of the EtOAc extract from the culture using the assay afforded the isolation of two novel fusicoccane diterpenoids named brassicicenes J (1) and K (2), along with three known brassicicenes A (3), B (4), and F (5). Their structures were elucidated from extensive NMR spectral data and by comparison of these with those reported in the literature. Brassicicenes (1-5) exhibited weak to moderate seed dormancy breaking activities against lettuce seeds in the presence of abscisic acid. In addition, the necrotic/apoptotic activities of the brassicicenes (1-5), fusicoccin A (6) and cotylenin A (7) were evaluated by determining their cytotoxicity, cell viability and caspase-3/7 activation on the HL-60 cell line. Brassicicene K (2) exhibited similar cytostatic profiles to that of cotylenin A (7), and brassicicenes J (1), A (3), B (4), and F (5) exhibited necrotic activity. This is the first report of the seed dormancy breaking activity of brassicicenes in plants, and of necrotic/apoptotic activity in mammalian cells. PMID:24689212

  5. Enhancement of caffeic acid phenethyl ester on all-trans retinoic acid-induced differentiation in human leukemia HL-60 cells

    SciTech Connect

    Kuo, H.-C.; Kuo, W.-H.; Lee, Y.-J.; Wang, C.-J.; Tseng, T.-H. . E-mail: tht@csmu.edu.tw

    2006-10-01

    All-trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL); however, the response is sometimes very slow. Furthermore, relapse and resistance to treatment often occur despite continued treatment with ATRA. Thereafter, combination treatment strategies have been suggested to circumvent these problems. The present study demonstrates that caffeic acid phenethyl ester (CAPE), a major component of honeybee propolis, enhanced ATRA-induced granulocytic differentiation in HL-60, a human promyelocytic cell line. The differentiation was assessed by Wright-Giemsa stain, nitroblue tetrazolium reduction, and membrane differentiation marker CD11b. In addition, CAPE enhanced ATRA-induced cell cycle arrest at the G1 phase by decreasing the association of cdk2-cyclin E complex. Finally, it was demonstrated that CAPE promoted the ATRA-mediated nuclear transcription activation of RAR{alpha} assessed by EMSA assay and enhanced the expression of target genes including RAR{alpha}, C/EBP{epsilon}, and p21 protein resulting in the differentiation development of leukemia. It is suggested that CAPE possesses the potential to enhance the efficiency of ATRA in the differentiation therapy of APL.

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

    PubMed

    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.

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

    PubMed

    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. PMID:25262027

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

    PubMed

    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.

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

    SciTech Connect

    Tang, G.H.; Shen, Y.; Shen, H.M.

    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.

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

    PubMed

    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.

  11. Seed dormancy breaking diterpenoids from the liverwort Plagiochila sciophila and their differentiation inducing activity in human promyelocytic leukemia HL-60 cells.

    PubMed

    Kenmoku, Hiromichi; Tada, Hiroyuki; Oogushi, Megumi; Esumi, Tomoyuki; Takahashi, Hironobu; Noji, Masaaki; Sassa, Takeshi; Toyota, Masao; Asakawa, Yoshinori

    2014-07-01

    To obtain the structural diversity of bioactive compounds similar to cotylenins and fusicoccins that modulate 14-3-3 protein-protein interactions in eukaryotes, screening tests were carried out using the lettuce seed dormancy breaking-assay. An acetone extract of the liverwort Plagiochila sciophila exhibited significant activity against the seeds in the presence of the plant hormone abscisic acid. Activity-guided fractionation of the extract afforded the isolation of seven novel fusicoccane-type diterpenoids, named fusicosciophins A-E (1-5), 8-deacetyl (6) and 9-deacetyl fusicosciophin E (7). Their structures were determined by spectroscopic methods and X-ray crystallographic analyses. All the pure isolated compounds (1-7) exhibited moderate lettuce seed dormancy breaking activity. In addition, the differentiation-inducing activity and cytotoxicity of these isolates, together with fusicoccin A (FC-A) and all-trans retinoic acid (ATRA), were evaluated in human promyelocytic leukemia HL-60 cells and human mouth epidermal carcinoma KB cells, respectively. Fusicosciophins (2 and 4) and FC-A exhibited moderate differentiation-inducing activity (EC50 31.2-59.1 microM) compared with ATRA (EC50 0.3 microM), while 2, 4 and ATRA exhibited higher selectivity indices (IC50/EC50 >3.38-667) than FC-A (IC50/EC50 1.05). This is the first report on the isolation of fusicoccane-type diterpenoids from liverworts having seed dormancy breaking activity and differentiation-inducing activity in mammal cells. PMID:25230492

  12. Seed dormancy breaking diterpenoids from the liverwort Plagiochila sciophila and their differentiation inducing activity in human promyelocytic leukemia HL-60 cells.

    PubMed

    Kenmoku, Hiromichi; Tada, Hiroyuki; Oogushi, Megumi; Esumi, Tomoyuki; Takahashi, Hironobu; Noji, Masaaki; Sassa, Takeshi; Toyota, Masao; Asakawa, Yoshinori

    2014-07-01

    To obtain the structural diversity of bioactive compounds similar to cotylenins and fusicoccins that modulate 14-3-3 protein-protein interactions in eukaryotes, screening tests were carried out using the lettuce seed dormancy breaking-assay. An acetone extract of the liverwort Plagiochila sciophila exhibited significant activity against the seeds in the presence of the plant hormone abscisic acid. Activity-guided fractionation of the extract afforded the isolation of seven novel fusicoccane-type diterpenoids, named fusicosciophins A-E (1-5), 8-deacetyl (6) and 9-deacetyl fusicosciophin E (7). Their structures were determined by spectroscopic methods and X-ray crystallographic analyses. All the pure isolated compounds (1-7) exhibited moderate lettuce seed dormancy breaking activity. In addition, the differentiation-inducing activity and cytotoxicity of these isolates, together with fusicoccin A (FC-A) and all-trans retinoic acid (ATRA), were evaluated in human promyelocytic leukemia HL-60 cells and human mouth epidermal carcinoma KB cells, respectively. Fusicosciophins (2 and 4) and FC-A exhibited moderate differentiation-inducing activity (EC50 31.2-59.1 microM) compared with ATRA (EC50 0.3 microM), while 2, 4 and ATRA exhibited higher selectivity indices (IC50/EC50 >3.38-667) than FC-A (IC50/EC50 1.05). This is the first report on the isolation of fusicoccane-type diterpenoids from liverworts having seed dormancy breaking activity and differentiation-inducing activity in mammal cells.

  13. Induction of apoptosis by penta-O-galloyl-beta-D-glucose through activation of caspase-3 in human leukemia HL-60 cells.

    PubMed

    Pan, M H; Lin, J H; Lin-Shiau, S Y; Lin, J K

    1999-09-24

    Penta-O-galloyl-beta-D-glucose is structurally related to (-)-epigallocatechin gallate and is isolated from hydrolyzed tannin. Penta-O-galloyl-beta-D-glucose can inhibit tumor promotion by teleocidin. We investigated the effects of penta-O-galloyl-beta-D-glucose and various tea polyphenols on cell viability in human leukemia HL-60 cells. In this study, we demonstrated that penta-O-galloyl-beta-D-glucose was able to induce apoptosis in a concentration- and time-dependent manner; however, other polyphenols were less effective. We further investigated the molecular mechanisms of penta-O-galloyl-beta-D-glucose-induced apoptosis. Treatment with penta-O-galloyl-beta-D-glucose caused induction of caspase-3/CPP32 activity in dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly-(ADP-ribose) polymerase. Pretreatment with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and Z-Val-Ala-Asp-fluoromethyl-ketone (Z-VAD-FMK) inhibited penta-O-galloyl-beta-D-glucose-induced DNA fragmentation. Furthermore, treatment with penta-O-galloyl-beta-D-glucose (50 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Our results indicate that penta-O-galloyl-beta-D-glucose allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA, and induces DFF-45 (DNA fragmentation factor) degradation. These results lead to a working hypothesis that penta-O-galloyl-beta-D-glucose-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3, degradation of poly-(ADP-ribose) polymerase, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by penta-O-galloyl-beta-D-glucose may provide a pivotal mechanism for its cancer chemopreventive action.

  14. Raf-1 signaling is required for the later stages of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells but is not mediated by the MEK/ERK module.

    PubMed

    Wang, Xuening; Studzinski, George P

    2006-11-01

    We are interested in determining the signaling pathways for 1,25-dihydroxyvitamin D3 (1,25D)-induced differentiation of HL60 leukemic cells. One possible candidate is Raf-1, which is known to signal cell proliferation and neoplastic transformation through MEK, ERK, and downstream targets. It can also participate in the regulation of cell survival and various forms of cell differentiation, though the precise pathways are less well delineated. Here we report that Raf-1 has a role in monocytic differentiation of human myeloid leukemia HL60, which is not mediated by MEK and ERK, but likely by direct interaction with p90RSK. Specifically, we show that Raf-1 and p90RSK are increasingly activated in the later stages of differentiation of HL60 cells, at the same time as activation of MEK and ERK is decreasing. Transfection of a wild-type Raf-1 construct enhances 1,25D-induced differentiation, while antisense Raf-1 or short interfering (si) Raf-1 reduces 1,25D-induced differentiation. In contrast, antisense oligodeoxynucleotides (ODN) and siRNAs to MEK or ERK have no detectable effect on differentiation. In late stage differentiating cells Raf-1 and p90RSK are found as a complex, and inhibition of Raf-1, but not MEK or ERK expression reduces the levels of phosphorylated p90 RSK. These findings support the thesis that Raf-1 signals cell proliferation and cell differentiation through different intermediary proteins.

  15. 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}

    SciTech Connect

    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.

  16. GSH depletion, protein S-glutathionylation and mitochondrial transmembrane potential hyperpolarization are early events in initiation of cell death induced by a mixture of isothiazolinones in HL60 cells.

    PubMed

    Di Stefano, Anna; Frosali, Simona; Leonini, Alessandra; Ettorre, Anna; Priora, Raffaella; Di Simplicio, Francesca Cherubini; Di Simplicio, Paolo

    2006-02-01

    We recently described that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) induces apoptosis at low concentrations (0.001-0.01%) and necrosis at higher concentrations (0.05-0.1%). In this study, we show that glutathione (GSH) depletion, reactive oxygen species generation, hyperpolarization of mitochondrial transmembrane potential (DeltaPsim) and formation of protein-GSH mixed disulphides (S-glutathionylation) are early molecular events that precede the induction of cell death by CMI/MI. When the cells exhibit common signs of apoptosis, they show activation of caspase-9, reduction of DeltaPsim and, more importantly, decreased protein S-glutathionylation. In contrast, necrosis is associated with severe mitochondrial damage and maximal protein S-glutathionylation. CMI/MI-induced cytotoxicity is also accompanied by decreased activity of GSH-related enzymes. Pre-incubation with L-buthionine-(S,R)-sulfoximine (BSO) clearly switches the mode of cell death from apoptosis to necrosis at 0.01% CMI/MI. Collectively, these results demonstrate that CMI/MI alters the redox status of HL60 cells, and the extent and kinetics of GSH depletion and S-glutathionylation appear to determine whether cells undergo apoptosis or necrosis. We hypothesize that S-glutathionylation of certain thiol groups accompanied by GSH depletion plays a critical role in the molecular mechanism of CMI/MI cytotoxicity.

  17. Vildagliptin and its metabolite M20.7 induce the expression of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells

    PubMed Central

    Asakura, Mitsutoshi; Karaki, Fumika; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

    2016-01-01

    Vildagliptin is a potent, orally active inhibitor of dipeptidyl peptidase-4 (DPP-4) for the treatment of type 2 diabetes mellitus. It has been reported that vildagliptin can cause hepatic dysfunction in patients. However, the molecular-mechanism of vildagliptin-induced liver dysfunction has not been elucidated. In this study, we employed an expression microarray to determine hepatic genes that were highly regulated by vildagliptin in mice. We found that pro-inflammatory S100 calcium-binding protein (S100) a8 and S100a9 were induced more than 5-fold by vildagliptin in the mouse liver. We further examined the effects of vildagliptin and its major metabolite M20.7 on the mRNA expression levels of S100A8 and S100A9 in human hepatoma HepG2 and leukemia HL-60 cells. In HepG2 cells, vildagliptin, M20.7, and sitagliptin – another DPP-4 inhibitor – induced S100A9 mRNA. In HL-60 cells, in contrast, S100A8 and S100A9 mRNAs were significantly induced by vildagliptin and M20.7, but not by sitagliptin. The release of S100A8/A9 complex in the cell culturing medium was observed in the HL-60 cells treated with vildagliptin and M20.7. Therefore, the parental vildagliptin- and M20.7-induced release of S100A8/A9 complex from immune cells, such as neutrophils, might be a contributing factor of vildagliptin-associated liver dysfunction in humans. PMID:27759084

  18. Libraries of 2β-(N-substituted piperazino)-5α-androstane-3α, 17β-diols: chemical synthesis and cytotoxic effects on human leukemia HL-60 cells and on normal lymphocytes.

    PubMed

    Roy, Jenny; Maltais, René; Jegham, Hajer; Poirier, Donald

    2011-05-01

    Libraries of steroid derivatives with two levels of molecular diversity were prepared to optimize the antiproliferative activity on leukemia HL-60 cells by first varying the amino acid (AA) at R(1) (libraries A, B, C, and D: with 45, 45, 20, and 20 members, respectively) and, subsequently, the capping group at R(2) (library E: 168 members). The screening of these aminosteroids revealed interesting structure-activity relationships. In library A, the compounds bearing a tetrahydroisoquinolone residue as the first element of diversity showed potent cytotoxicity, principally when isovaleric or cyclohexyl acetic acid was used as a capping group (>40% of cell growth inhibition at 1 μM). In library B, the phenylalanine (Phe) derivatives bearing a cyano group induced a higher growth inhibition than the other Phe derivatives. The screening of library C indicated the increase of hydrophobicity of proline (Pro) seems to preserve the cytotoxic effect achieved by the lead compound. However, the synthesis of structural Pro variants (library D) clearly shows weaker activities when compared to L-Pro building blocks. Finally, by incorporating some of the most active AA of libraries A-D in library E, we observed that the amide coupling functionality gave stronger cytotoxic activity compared to the corresponding sulfonamides or benzylamines. Six of the most active amide derivatives (E-37P, E-41P, E-42P, E-46P, E-48F, and E-12T) were selected and IC(50) determined on HL-60 cells as well as on normal human lymphocytes. Among this series of new anticancer agents, good to high selectivity indices (SI = IC(50) (lymphocytes)/IC(50) (HL-60 cells) = 5 - 55) were obtained.

  19. 'Attached cell' antigen 28.3.7 mapping to human chromosome 15 characterises TPA-induced differentiation of the promyelocytic HL-60 cell line to give macrophage/monocyte populations.

    PubMed Central

    Blaineau, C; Avner, P; Tunnacliffe, A; Goodfellow, P

    1983-01-01

    Human cells growing in vitro attached to the substratum express a cell antigen called 28.3.7 identified by a species-specific monoclonal antibody. This antigen is not expressed on human cells growing in suspension. The antigen has a mol. wt. in reduced SDS-polyacrylamide gel electrophoresis gels of 95 000 and in human-mouse somatic cell hybrids, expression of the antigen is controlled by a gene, MIC7, mapping to human chromosome 15. The antigen functions as a marker for macrophage differentiation. In vitro differentiation of the 28.3.7 antigen-negative human promyelocytic leukaemia line HL-60 induced by phorbol ester, results in the formation of a macrophage/monocyte population and the concomitant expression of the 28.3.7 antigen on this adherent cell population. Images Fig. 1. PMID:6641710

  20. 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.

    PubMed

    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.

  1. Expression of human kinase suppressor of Ras 2 (hKSR-2) gene in HL60 leukemia cells is directly upregulated by 1,25-dihydroxyvitamin D{sub 3} and is required for optimal cell differentiation

    SciTech Connect

    Wang Xuening; Wang, T.-T.; White, John H.; Studzinski, George P. . E-mail: studzins@umdnj.edu

    2007-08-15

    Induction of terminal differentiation of neoplastic cells offers potential for a novel approach to cancer therapy. One of the agents being investigated for this purpose in preclinical studies is 1,25-dihydroxyvitamin D{sub 3} (1,25D), which can convert myeloid leukemia cells into normal monocyte-like cells, but the molecular mechanisms underlying this process are not fully understood. Here, we report that 1,25D upregulates the expression of hKSR-2, a new member of a small family of proteins that exhibit evolutionarily conserved function of potentiating ras signaling. The upregulation of hKSR-2 is direct, as it occurs in the presence of cycloheximide, and occurs primarily at the transcriptional level, via activation of vitamin D receptor, which acts as a ligand-activated transcription factor. Two VDRE-type motifs identified in the hKSR-2 gene bind VDR-RXR alpha heterodimers present in nuclear extracts of 1,25D-treated HL60 cells, and chromatin immunoprecipitation assays show that these VDRE motifs bind VDR in 1,25D-dependent manner in intact cells, coincident with the recruitment of RNA polymerase II to these motifs. Treatment of the cells with siRNA to hKSR-2 reduced the proportion of the most highly differentiated cells in 1,25D-treated cultures. These results demonstrate that hKSR-2 is a direct target of 1,25D in HL60 cells, and is required for optimal monocytic differentiation.

  2. Differentiation and apoptosis induction by lovastatin and γ-tocotrienol in HL-60 cells via Ras/ERK/NF-κB and Ras/Akt/NF-κB signaling dependent down-regulation of glyoxalase 1 and HMG-CoA reductase.

    PubMed

    Chen, Chun-Chia; Liu, Tzu-Yu; Huang, Shih-Pin; Ho, Chi-Tang; Huang, Tzou-Chi

    2015-11-01

    Glyoxalase 1 (GLO1) and HMG-CoA reductase (HMGCR) are highly expressed in most tumor cells and little in normal cells. In this study, treatment of HL-60 cells with lovastatin induced characteristic apoptosis in a dose-dependent manner. We demonstrated that lovastatin treatment inhibited Ras and Raf protein translocation to cell membrane and eliminated the phosphorylation of the downstream effectors Akt and ERK, and the subsequent NF-κB translocation into nucleus. Specific inhibitors and γ-tocotrienol confirmed the Ras/Raf/ERK/NF-κB/GLO1 and Ras/Akt/NF-κB/GLO1 pathways. Data revealed that lovastatin induced HL-60 cell death was attenuated by mevalonate treatment. We demonstrated also that γ-tocotrienol showed its apoptotic effect on the HL-60 cell through the same pathway. γ-Tocotrienol enhanced the apoptotic effect of lovastatin through the down-regulation of GLO1 and HMGCR resulting in an increase of methylglyoxal and a decrease of cholesterol and led to the apoptosis of HL-60 cells. Data also revealed that both lovastatin and gamma-tocotrienol induced significant HL-60 cell differentiation. These results suggest that both lovastatin and gamma-tocotrienol could induce differentiation and followed by apoptosis.

  3. Antitumor triptycene bisquinones induce a caspase-independent release of mitochondrial cytochrome c and a caspase-2-mediated activation of initiator caspase-8 and -9 in HL-60 cells by a mechanism which does not involve Fas signaling.

    PubMed

    Perchellet, Elisabeth M; Wang, Yang; Weber, Rebeka L; Lou, Kaiyan; Hua, Duy H; Perchellet, Jean-Pierre H

    2004-11-01

    Synthetic triptycene analogs (TT code number) mimic the antitumor effects of daunorubicin (DAU) in vitro, but have the advantage of blocking nucleoside transport, inhibiting both DNA topoisomerase I and II activities, and retaining their efficacy in multidrug-resistant (MDR) tumor cells. Since TT bisquinones induce poly(ADP-ribose) polymerase-1 (PARP-1) cleavage at 6 h and internucleosomal DNA fragmentation at 24 h, which are, respectively, early and late markers of apoptosis, these antitumor drugs were tested for their ability to trigger the release of mitochondrial cytochrome c (Cyt c) and the caspase activation cascade in the HL-60 cell system. Based on their ability to reduce the viability of wild-type, drug-sensitive HL-60-S cells in the nanomolar range, six lead antitumor TT bisquinones have been identified so far: TT2, TT13, TT16, TT19, TT24 and TT26. In accord with the fact that effector caspase-3 is responsible for PARP-1 cleavage, 4 microM concentrations of DAU and these TT bisquinones all maximally induce caspase-3 activity at 6 h in HL-60-S cells, an effect which persists when the drugs are removed after a 1-h pulse treatment. Since caspase-3 may be activated by initiator caspase-9 and -8, it is significant to show that such caspase activation cascade is induced by 4 microM DAU and TT bisquinones at 6 h in HL-60-S cells. Although the relationship is not perfect, the ability of TT analogs to induce caspase-3, -8 and -9 activities may be linked to their quinone functionality and cytotoxicity. Interestingly, 4 microM concentrations of TT bisquinones retain their ability to induce caspase-3, -8 and -9 activities at 6 h in the MDR HL-60-RV cell line where 4 microM DAU becomes totally ineffective. The release of mitochondrial Cyt c is also detected within 6 h in HL-60-S cells treated with 4 microM DAU or TT bisquinones, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Caspase-2 and -8 may both act upstream of

  4. Levels of phospho-Smad2/3 are sensors of the interplay between effects of TGF-beta and retinoic acid on monocytic and granulocytic differentiation of HL-60 cells.

    PubMed

    Cao, Zhouhong; Flanders, Kathleen C; Bertolette, Daniel; Lyakh, Lyudmila A; Wurthner, Jens U; Parks, W Tony; Letterio, John J; Ruscetti, Francis W; Roberts, Anita B

    2003-01-15

    We have investigated the role of Smad family proteins, known to be important cytoplasmic mediators of signals from the transforming growth factor-beta (TGF-beta) receptor serine/threonine kinases, in TGF-beta-dependent differentiation of hematopoietic cells, using as a model the human promyelocytic leukemia cell line, HL-60. TGF-beta-dependent differentiation of these cells to monocytes, but not retinoic acid-dependent differentiation to granulocytes, was accompanied by rapid phosphorylation and nuclear translocation of Smad2 and Smad3. Vitamin D(3) also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-beta1. Simultaneous treatment of these cells with TGF-beta1 and all-trans-retinoic acid (ATRA), which leads to almost equal numbers of granulocytes and monocytes, significantly reduced the level of phospho-Smad2/3 and its nuclear accumulation, compared with that in cells treated with TGF-beta1 alone. TGF-beta1 and ATRA activate P42/44 mitogen-activated protein (MAP) kinase with nearly identical kinetics, ruling out its involvement in these effects on Smad phosphorylation. Addition of the inhibitor-of-protein serine/threonine phosphatases, okadaic acid, blocks the ATRA-mediated reduction in TGF-beta-induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R mutant cells, which harbor a defective retinoic acid receptor-alpha (RAR-alpha), ATRA is unable to reduce levels of TGF-beta-induced phospho-Smad2/3, coincident with its inability to differentiate these cells along granulocytic pathways. Together, these data suggest a new level of cross-talk between ATRA and TGF-beta, whereby a putative RAR-alpha-dependent phosphatase activity limits the levels of phospho-Smad2/3 induced by TGF-beta, ultimately reducing the levels of nuclear Smad complexes mediating the TGF-beta-dependent differentiation of the cells to monocytic end points.

  5. 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

    PubMed Central

    Daga, Martina; Cetrangolo, Giovanni Paolo; Ciamporcero, Eric Stefano; Petrella, Claudia; Graf, Maria; Uchida, Koji; Mamone, Gianfranco; Ferranti, Pasquale; Ames, Paul R. J.

    2015-01-01

    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. PMID:26078803

  6. (4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G{sub 2}/M arrest, and triggers apoptosis in human leukemia HL-60 cells

    SciTech Connect

    Magalhães, Hemerson I.F.; Wilke, Diego V.; Bezerra, Daniel P.; Cavalcanti, Bruno C.; Rotta, Rodrigo; Lima, Dênis P. de; Beatriz, Adilson; Moraes, Manoel O.; Diniz-Filho, Jairo; Pessoa, Claudia

    2013-10-01

    (4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC{sub 50} values in the nanomolar range. Cell cycle arrest in G{sub 2}/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G{sub 2}/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential. - Highlights: • PHT inhibits tubulin polymerization. • PHT arrests cancer cells in G{sub 2}/M phase of the cell cycle. • PHT induces caspase-dependent apoptosis.

  7. Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells.

    PubMed

    Shen, Miaoqing; Bunaciu, Rodica P; Congleton, Johanna; Jensen, Holly A; Sayam, Lavanya G; Varner, Jeffrey D; Yen, Andrew

    2011-12-01

    All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.

  8. Evaluation of Antiradical and Anti-Inflammatory Activities of Ethyl Acetate and Butanolic Subfractions of Agelanthus dodoneifolius (DC.) Polhill & Wiens (Loranthaceae) Using Equine Myeloperoxidase and Both PMA-Activated Neutrophils and HL-60 Cells

    PubMed Central

    Boly, Rainatou; Franck, Thierry; Kohnen, Stephan; Lompo, Marius; Guissou, Innocent Pierre; Dubois, Jacques; Serteyn, Didier; Mouithys-Mickalad, Ange

    2015-01-01

    The ethyl acetate and n-butanolic subfractions of Agelanthus dodoneifolius were investigated for their antioxidant and antimyeloperoxidase (MPO) activities. The reactive oxygen species (ROS) generation was assessed by lucigenin-enhanced chemiluminescence (CL) and dichlorofluorescein- (DCF-) induced fluorescence techniques from phorbol myristate acetate- (PMA-) stimulated equine neutrophils and human myeloid cell line HL-60, respectively. In parallel, the effects of the tested subfractions were evaluated on the total MPO release by stimulated neutrophils and on the specific MPO activity by means of immunological assays. The results showed the potent activity of the butanolic subfraction, at least in respect of the chemiluminescence test (IC50 = 0.3 ± 0.1 µg/mL) and the ELISA and SIEFED assays (IC50 = 2.8 ± 1.2 µg/mL and 1.3 ± 1.0 µg/mL), respectively. However, the ethyl acetate subfraction was found to be the most potent in the DCF assay as at the highest concentration, DCF fluorescence intensity decreases of about 50%. Moreover, we demonstrated that the ethyl acetate subfraction was rich in catechin (16.51%) while it was not easy to identify the main compounds in the butanolic subfraction using the UPLC-MS/MS technique. Nevertheless, taken together, our results provide evidence that Agelanthus dodoneifolius subfractions may represent potential sources of natural antioxidants and of antimyeloperoxidase compounds. PMID:25821497

  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.

    PubMed

    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. Synthesis, structure activity relationship and mode of action of 3-substitutedphenyl-1-(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenones as novel anticancer agents in human leukaemia HL-60 cells.

    PubMed

    Murthy, Y L N; Suhasini, K P; Pathania, A S; Bhushan, S; Nagendra Sastry, Y

    2013-04-01

    A novel class of 3-substitutedphenyl-1-(2,2,8,8-tetramethyl-3,4,9,10-tetrahydro-2H,8H-pyrano[2,3-f]chromen-6-yl)-propenones were designed, synthesized and evaluated for their antiproliferative activity against the human cancer cell lines of diverse origin. Structure activity relationship was elucidated with various substitutions on the benzene ring and these variations significantly affected the potency. Most of the twelve tested compounds inhibited the growth of aggressive cancer cell lines. Moreover, three compounds 4j, 4k and 4l displayed excellent cytotoxic profile by inhibiting >90% cell proliferation in HL-60 and Caco-2 cells at 50 μM concentration. Further studies to elucidate the mode of action revealed that these three compounds induced G0/G1 cell cycle arrest and apoptosis, which was accompanied by loss of mitochondrial membrane potential, DNA fragmentation and nuclear morphology in HL-60 cells.

  11. Phospholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes

    SciTech Connect

    Pai, J.K.; Siegel, M.I.; Egan, R.W.; Billah, M.M.

    1988-09-05

    There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-(32P) lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-(32P)PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-(32P)phosphatidic acid (alkyl-(32P)PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-(32P)PA must be formed via PLD-catalyzed hydrolysis of alkyl-(32P)PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-(32P)phosphatidylethanol (alkyl-(32P)PEt). Formation of alkyl-(32P)PEt parallels that of alkyl-(32P)PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration.

  12. Selective induction of G2/M arrest and apoptosis in HL-60 by a potent anticancer agent, HMJ-38.

    PubMed

    Yang, Jai-Sing; Hour, Mann-Jen; Kuo, Sheng-Chu; Huang, Li-Jiau; Lee, Miau-Rong

    2004-01-01

    We previously reported that HMJ-38 was the most potent 2-phenyl-4-quinozolinone derivative in inhibiting tubulin polymerization and showed significant cytotoxicity against several human tumor cell lines. In this work, we studied its cytotoxic effect on HL-60 leukemia cells and the underlying mechanisms. We first investigated the effects of HMJ-38 on viability, cell cycle and induction of apoptosis in HL-60 and normal human peripheral blood mononuclear cells (PBMC). After 24-hour treatment with HMJ-38, a dose- and time-dependent decrease in the viability of HL-60 cells was observed and the approximate IC50 was 4.48 microM. The cytotoxic effect of HMJ-38 on PBMC was less significant than that on HL-60 cells, either with 24 or 48 hours of treatment. Cell cycle analysis showed that HMJ-38 induced significant G2/M arrest and apoptosis in HL-60 cells. The HMJ-38-induced G2/M arrest occurred before the onset of apoptosis. Within 24 hours of treatment, HMJ-38 influenced the CDK/cyclin B activity by increasing Chk1, Wee1 and p21 and decreasing Cdc25C protein levels. The HMJ-38-induced apoptosis was further confirmed by morphological assessment and DNA fragmentation assay. Induction of apoptosis in HMJ-38-treated HL-60 cells was accompanied by an apparent increase of cytosolic cytochrome c, down-regulation of Bcl-2, up-regulation of Bax and cleavage of pro-caspase-9, -3 and poly(ADP)ribosylpolymerase (PARP). The results of the significant reduction of caspase activities and apoptosis by caspase inhibitors indicated that the HMJ-38-induced apoptosis was mainly mediated by activation of caspases-9 and -3. HMJ-38 also activated ERK in HL-60 cells. Pre-incubating cells with ERK inhibitors (U0126 and PD98059) attenuated the HMJ-38-induced ERK activation and apoptosis. Nevertheless, cells remained arrested in G2/M. These results suggest that HMJ-38 is a potent anticancer drug and it shows a remarkable action on cell cycle before commitment for apoptosis is reached. PMID:15274354

  13. Loss of irreversibility of granulocytic differentiation induced by dimethyl sulfoxide in HL-60 sublines with a homogeneously staining region.

    PubMed

    Kitajima, K; Haque, M; Nakamura, H; Hirano, T; Utiyama, H

    2001-11-16

    The human HL-60 acute leukemia cell line harbors double minutes (dmins) during early passages. During its continuous culture for a long term, a single marker chromosome with a homogeneously staining region (HSR) replaces the dmins. The both structures harbor amplified c-MYC sequences. Here we ask how the cellular phenotype is altered by the c-MYC integration into a HSR. Treatment with dimethyl sulfoxide induces granulocytic differentiation in the both types of cells. In contrast to HL-60/dmin cells, however, no apoptosis followed differentiation and the differentiation phenotype was reverted upon withdrawal of the drug in HL-60/HSR cells. Terminal differentiation and loss of DNase I hypersensitivity sites at c-MYC P2 promoter appeared to be unlinked in the both types of cells. By comparison with HL-60/dmin cells, we conclude that the integration into a HSR of an extrachromosomal gene(s) but not c-MYC likely leads to the loss of irreversibility of the differentiation phenotype.

  14. Nano-hole induction by nanodiamond and nanoplatinum liquid, DPV576, reverses multidrug resistance in human myeloid leukemia (HL60/AR)

    PubMed Central

    Ghoneum, Alia; Sharma, Shivani; Gimzewski, James

    2013-01-01

    Recently nanoparticles have been extensively studied and have proven to be a promising candidate for cancer treatment and diagnosis. In the current study, we examined the chemo-sensitizing activity of a mixture of nanodiamond (ND) and nanoplatinum (NP) solution known as DPV576, against multidrug-resistant (MDR) human myeloid leukemia (HL60/AR) and MDR-sensitive cells (HL60). Cancer cells were cultured with different concentrations of daunorubicin (DNR) (1 × 10 −9−1 × 10 −6 M) in the presence of selected concentrations of DPV576 (2.5%–10% v/v). Cancer cell survival was determined by MTT assay, drug accumulation by flow cytometry and confocal laser scanning microscopy (CLSM), and holes and structural changes by atomic force microscopy (AFM). Co-treatment of HL60/AR cells with DNR plus DPV576 resulted in the reduction of the IC50 to 1/4th. This was associated with increased incidences of holes inside the cells as compared with control untreated cells. On the other hand, HL60 cells did not show changes in their drug accumulation post-treatment with DPV576 and DNR. We conclude that DPV576 is an effective chemo-sensitizer as indicated by the reversal of HL60/AR cells to DNR and may represent a potential novel adjuvant for the treatment of chemo-resistant human myeloid leukemia. PMID:23888112

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

    PubMed

    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. PMID:26832215

  16. Simvastatin Inhibits IL-5-Induced Chemotaxis and CCR3 Expression of HL-60-Derived and Human Primary Eosinophils

    PubMed Central

    Fu, Chia-Hsiang; Tsai, Wan-Chun; Lee, Ta-Jen; Huang, Chi-Che; Chang, Po-Hung; Su Pang, Jong-Hwei

    2016-01-01

    IL-5-induced chemotaxis of eosinophils is an important feature of allergic airway inflammatory diseases. Simvastatin, a lipid lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Our aim was to investigate the effect of simvastatin on IL-5-induced eosinophil chemotaxis and its regulatory mechanisms. Eosinophils were derived by treating HL-60 clone 15 (HC15) cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. The expressions of CC chemokine receptor 3 (CCR3) and interleukin (IL)-5 receptors (IL5Rα and β) were analyzed using RT/real-time PCR. The granular proteins were stained using fast green. Eotaxin-induced chemotaxis was measured using a transwell migration assay. CCR3 protein expression was revealed by immunocytochemistry. An animal model of allergic rhinitis was established by challenging Sprague–Dawley® rats repeatedly with ovalbumin. Butyric acid significantly increased the expression of IL5Rα and IL5Rβ, CCR3 and granular proteins in HC15 cells, indicating the maturation of eosinophils (BA-E cells). IL-5 further enhanced the CCR3 expression at both the mRNA and protein levels and the eotaxin-induced chemotaxis of BA-E cells. Simvastatin inhibited the effects of IL-5 on BA-E cells, but not in the presence of mevalonate. Similar results were also exhibited in human primary eosinophils. In vivo animal studies further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues of allergic rats. Therefore, simvastatin was demonstrated to inhibit IL-5-induced CCR3 expression and chemotaxis of eosinophils mediated via the mevalonate pathway. We confirmed that simvastatin also reduced eosinophilic infiltration in allergic rhinitis. PMID:27275740

  17. Targeting SLUG sensitizes leukemia cells to ADR-induced apoptosis

    PubMed Central

    Wei, Chang-Rong; Liu, Jun; Yu, Xiao-Jun

    2015-01-01

    Background and Aims: Slug is an E-cadherin repressor and a suppressor of PUMA (p53 upregulated modulator of apoptosis) and it has recently been demonstrated that Slug plays an important role in controlling apoptosis. In this study, we examined whether Slug’s ability to silence expression suppresses the growth of leukemia HL-60 cells and/or sensitizes leukemia HL-60 cells to adriamycin (ADR) through induction of apoptosis. Methods: SLUG siRNA was transfected into the HL-60 and HL-60ADR cell lines (an adriamycin resistant cell line). The stably SLUG siRNA transfected HL-60 and HL-60ADR cells was transiently transfected with PUMA siRNA. The mRNA and protein expression of SLUG and PUMA were determined by Quantitative real-time RT-PCR and Western blot assay. The effects of SLUG siRNA alone or combined with ADR or PUMA siRNA on growth and apoptosis in HL-60 and HL-60ADR cells was detected by MTT, ELISA and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Results: The results showed that SLUG was less expressed in the HL-60 cells, and high expressed in the HL-60ADR cells. Obvious down-regulation of SLUG mRNA and protein levels and up-regulation of PUMA mRNA and protein levels after SLUG siRNA transfection was showed in the HL-60ADR cells. Treatment with ADR induced SLUG mRNA and protein in the HL-60 cells. Significant positive correlation was observed between basal SLUG mRNA and protein and ADR sensitivity. SLUG gene silencing by SLUG siRNA transfection inhibited growth and induced apoptosis, and increased ADR killing of the HL-60 and HL-60ADR cell lines. After the SLUG siRNA transfected HL-60 and HL-60ADR cells was transiently transfected with PUMA siRNA, did not increase ADR killing of the HL-60 and HL-60ADR cell lines. Conclusion: SLUG level positively correlated with sensitivity to ADR. SLUG siRNA could effectively reduce SLUG expression and induce PUMA expression and restore the drug sensitivity of resistant leukemic cells to

  18. Ozone exposed epithelial cells modify cocultured natural killer cells

    PubMed Central

    Müller, Loretta; Brighton, Luisa E.

    2013-01-01

    Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs. PMID:23241529

  19. Cytokinesis-block micronucleus assay in WIL2-NS cells: a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils.

    PubMed

    Umegaki, K; Fenech, M

    2000-05-01

    We have developed a method that can detect the DNA-damaging and cytotoxic effects of physiological levels of reactive oxygen species (ROS) and activated human neutrophils. This was achieved using WIL2-NS cells, a human B lymphoblastoid cell line, as target cells and the cytokinesis-block micronucleus (CBMN) assay. With this method, we observed a 4- and a 30-fold increase in the frequency of micronucleated binucleated cells (MNed BNC) when cells were exposed to 10 and 30 microM hydrogen peroxide, for 1 h, respectively. A dose-dependent increase in the frequency of MNed BNC was also detected when cells were exposed to hypoxanthine (HX)/xanthine oxidase (XO), a superoxide generating system: a 50-fold increase in the frequency of MNed BNC was observed at the highest XO dose (12.5 mU/ml). In this CBMN assay, nucleoplasmic bridges (NPB) in BNC and necrotic cells were also readily detected, especially at the higher exposure doses of hydrogen peroxide or HX/XO. When WIL2-NS cells were exposed to neutrophils stimulated with phorbol 12-myristate acetate (PMA) for 1 h, the frequencies of MNed BNC in WIL2-NS cells increased in a dose-dependent manner (30-fold increase at 100 nM PMA) and with an increasing neutrophil:WIL2-NS co-culture ratio. The frequencies of MNed BNC were closely related to the production of ROS, especially hydrogen peroxide, by the neutrophils. Differentiated HL60 cells (DMSO-treated HL60) also produced ROS in response to PMA. In this case, we used a 'Transwell' system to expose WIL2-NS cells to DMSO-treated HL60 cells, because direct contact with DMSO-treated HL60 cells impaired cell division in WIL2-NS target cells. Exposure to PMA-stimulated DMSO-treated HL60 cells resulted in a PMA dose-dependent increase in the frequency of MNed BNC in WIL2-NS cells. MNed BNC frequencies were positively correlated with NPB (r = 0.61-0.93) and necrosis (r = 0.55-0.86) and negatively correlated with nuclear division index (r = -0.72 to -0. 91) in all of the above

  20. Dose- and schedule-dependent activation and drug synergism between thymidine and 5-aza-2'-deoxycytidine in a human promyelocytic leukemia cell line.

    PubMed

    Grant, S; Rauscher, F; Margolin, J; Cadman, E

    1982-02-01

    The ability of thymidine (dThd) to enhance the metabolism and cytotoxicity of subsequent administered 5-aza-2'-deoxycytidine (5-aza-dCyd) was studied in L1210 cells and in the human promyelocytic leukemic cell line, HL-60. Exposure of L1210 cells to 0.1 mM dThd for 5 h resulted in an increase in the total intracellular and acid-precipitable accumulation of 5-aza-dCyd. Higher dThd concentrations and longer exposure intervals resulted in smaller increments in 5-aza-dCyd accumulation. In contrast, in HL-60 cells, a 24-hr exposure in 1 mM dThd resulted in the greatest intracellular accumulation of 5-aza-dCyd, 3.3 times more accumulation than in control cells. There was also a 4-fold increase in the acid-precipitable accumulation and nearly a 3-fold increase in DNA incorporation of 5-aza-dCyd in HL-60 cells exposed to the same dThd schedule. High-pressure liquid chromatographic analysis demonstrated a greater than 3-fold increase in the intracellular amounts of 5-aza-dCyd metabolites eluting in the triphosphate region in these human cells under identical conditions. Shorter dThd incubation exposure intervals (6 hr) and lower dThd concentration (0.1 mM) produced smaller increments in these studies. Both growth and clonogenic assays of HL-60 cells demonstrated a dose- and schedule sequence-dependent synergism between dThd and 5-aza-dCyd.

  1. Neutrophil elastase activity in differentiating HL-60 promyelocytes is decreased by culture with ethanol and elastase deficient neutrophils are produced in alcoholics

    SciTech Connect

    Sachs, C.; Christianson, R.; Pratt, P.; Lynn, W.

    1987-05-01

    Serum-free culture of HL-60 in the presence of recombinant Granulocyte-Macrophage Colony Stimulating Factor in four days elicits a five-fold increase in esterolytic neutrophil elastase (NE) like activity measured with methoxy-succinyl-ala-ala-pro-val p-nitroanilide and purified NE standard but does not cause terminal differentiation. Simultaneous exposure to 0.2, 0.4, or 0.6% (vol./vol.) ethanol blocks this increase in NE activity. Exposure to 0.85% ethanol promotes terminal differentiation to elastase-deficient granulocytes which as been described using DMSO. To ascertain if ethanol may have similar effects on granulocytic differentiation in vivo, they compared oxidase and elastase activities of PMN's in male alcoholics on a binge (ethanol > 200 mg/dl.). In 29 patients an average of 872 (+/- 237) (SD) ng./10/sup 6/ PMN's of active NE was found compared to 1571 (+/- 177) in 13 controls. Patients admitted for treatment of alcoholism had similar NE activity in 3-4 days, showed a slight increase in activity within one week and had NE activity comparable to controls within 2-3 weeks. These findings support the previous observation that smoking related emphysema is less prevalent and severe in patients who regularly consume alcohol. They conclude that ethanol may visibly alter responsiveness of promyelocytic precursors to regulatory differentiating factors.

  2. Protodioscin isolated from fenugreek (Trigonella foenumgraecum L.) induces cell death and morphological change indicative of apoptosis in leukemic cell line H-60, but not in gastric cancer cell line KATO III.

    PubMed

    Hibasami, Hiroshige; Moteki, Hiroyuki; Ishikawa, Kengo; Katsuzaki, Hirotaka; Imai, Kunio; Yoshioka, Kazumi; Ishii, Yaeko; Komiya, Takashi

    2003-01-01

    Protodioscin (PD) was purified from fenugreek (Trigonella foenumgraecum L.) and identified by Mass, and 1H- and 13C-NMR. The effects of PD on cell viability in human leukemia HL-60 and human stomach cancer KATO III cells were investigated. PD displayed strong growth inhibitory effect against HL-60 cells, but weak growth inhibitory effect on KATO III cells. Morphological change showing apoptotic bodies was observed in the HL-60 cells treated with PD, but not in KATO III cells treated with PD. Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 75.2, 96.3, and 100% after a 3-day treatment with 2.5, 5, and 10 microM PD, respectively. The fragmentation by PD of DNA to oligonucleosomal-sized fragments, that is a characteristic of apoptosis, was observed to be both concentration- and time-dependent in the HL-60 cells. These findings suggest that growth inhibition by PD of HL-60 cells results from the induction of apoptosis by this compound in HL-60 cells. PMID:12469212

  3. Protodioscin isolated from fenugreek (Trigonella foenumgraecum L.) induces cell death and morphological change indicative of apoptosis in leukemic cell line H-60, but not in gastric cancer cell line KATO III.

    PubMed

    Hibasami, Hiroshige; Moteki, Hiroyuki; Ishikawa, Kengo; Katsuzaki, Hirotaka; Imai, Kunio; Yoshioka, Kazumi; Ishii, Yaeko; Komiya, Takashi

    2003-01-01

    Protodioscin (PD) was purified from fenugreek (Trigonella foenumgraecum L.) and identified by Mass, and 1H- and 13C-NMR. The effects of PD on cell viability in human leukemia HL-60 and human stomach cancer KATO III cells were investigated. PD displayed strong growth inhibitory effect against HL-60 cells, but weak growth inhibitory effect on KATO III cells. Morphological change showing apoptotic bodies was observed in the HL-60 cells treated with PD, but not in KATO III cells treated with PD. Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 75.2, 96.3, and 100% after a 3-day treatment with 2.5, 5, and 10 microM PD, respectively. The fragmentation by PD of DNA to oligonucleosomal-sized fragments, that is a characteristic of apoptosis, was observed to be both concentration- and time-dependent in the HL-60 cells. These findings suggest that growth inhibition by PD of HL-60 cells results from the induction of apoptosis by this compound in HL-60 cells.

  4. Characterization of the phosphatidylserine-exposing subpopulation of sickle cells.

    PubMed

    de Jong, K; Larkin, S K; Styles, L A; Bookchin, R M; Kuypers, F A

    2001-08-01

    Phosphatidylserine (PS), exclusively present in the inner monolayer of the normal red blood cell (RBC) membrane, is exposed in subpopulations of sickle cells. PS-exposing RBCs were found predominantly among the densest and the very light sickle cells. Within the light RBC fraction, PS exposure was found on reticulocytes, transferrin receptor-expressing reticulocytes, and mature RBCs. The last subset contained low-density valinomycin-resistant RBCs, previously shown to have high Na(+) and low K(+) content. This subpopulation contained the highest percentage of PS-exposing cells. The PS-exposing sickle cells did not show the sustained high cytosolic Ca(++) levels that have been shown to activate scramblase activity. Data from this study indicate that PS exposure can occur at different stages in the life of the sickle RBC and that it correlates with the loss of aminophospholipid translocase activity, the only common denominator of the PS-exposing cells. The additional requirement of scramblase activation may occur during transient increases in cytosolic Ca(++). (Blood. 2001;98:860-867)

  5. Unleashing Cancer Cells on Surfaces Exposing Motogenic IGDQ Peptides.

    PubMed

    Corvaglia, Valentina; Marega, Riccardo; De Leo, Federica; Michiels, Carine; Bonifazi, Davide

    2016-01-20

    Thiolated peptides bearing the Ile-Gly-Asp (IGD) motif, a highly conserved sequence of fibronectin, are used for the preparation of anisotropic self-assembled monolayers (SAM gradients) to study the whole-population migratory behavior of metastatic breast cancer cells (MDA-MB-231 cells). Ile-Gly-Asp-Gln-(IGDQ)-exposing SAMs sustain the adhesion of MDA-MB-231 cells by triggering focal adhesion kinase phosphorylation, similarly to the analogous Gly-Arg-Gly-Asp-(GRGD)-terminating surfaces. However, the biological responses of different cell lines interfaced with the SAM gradients show that only those exposing the IGDQ sequence induce significant migration of MDA-MB-231 cells. In particular, the observed migratory behavior suggests the presence of cell subpopulations associated with a "stationary" or a "migratory" phenotype, the latter determining a considerable cell migration at the sub-cm length scale. These findings are of great importance as they suggest for the first time an active role of biological surfaces exposing the IGD motif in the multicomponent orchestration of cellular signaling involved in the metastatic progression.

  6. Oncogene Regulation during the Growth and Differentiation of a Human Promyelocytic Leukemia Cell Line.

    NASA Astrophysics Data System (ADS)

    Ely, Constance Marie

    To determine the significance of the regulation of the cellular oncogenes c-myc and c-myb during myeloid and monocytic differentiation, we analyzed oncogene expression concurrent with functional and morphological differences in HL-60 cells and in a partial differentiation resistant HL-60 clone (HL-60-1E3). Although HL-60-1E3 cells are unable to develop mature terminally differentiated features with PDBu or DMSO stimulation, they do exhibit partial differentiation features with these conditions. Treatments of HL-60-1E3 cells with PDBu preceded by treatment with dimethylsulfoxide (DMSO), results in complete maturation to macrophage-like cells. Using parallel PDBu-induction studies, we analyzed the kinetics of expression of c-myc, c-myb, c-fms, c-fos, c-raf, and histone H4, together with cell cycle frequency distribution, cytotoxic effector activity and clonogenic potential in HL-60 and HL-60-1E3 cells. The results of these studies revealed altered c-myc and c-myb regulation in resistant cells corresponding to a lack of terminal commitment as assessed by an increase in clonogenic potential and the inability to acquire cytotoxic function. These data suggest that maintenance of the suppressed state of c-myc and c-myb gene expression may be an important component of the regulatory mechanisms which allow HL-60 cells to complete macrophage-like terminal differentiation. A similar series of experiments examining the DMSO-induced granulocyte pathway revealed that differentiation resistance of HL-60-1E3 cells corresponded to altered regulation of both c-myc and c-myb, strengthening the hypothesis that regulation of both of these genes is integral to HL-60 differentiation. Biphasic c-myb expression was observed in both cell populations in the presence of DMSO where maximal expression took place at approximately 72 hours post-induction and was not linked to proliferation. Introduction of SV40:c-myc recombinant plasmids into HL-60 cells resulted in altered nuclear morphology

  7. Retinoic acid synergizes ATO-mediated cytotoxicity by precluding Nrf2 activity in AML cells

    PubMed Central

    Valenzuela, M; Glorieux, C; Stockis, J; Sid, B; Sandoval, J M; Felipe, K B; Kviecinski, M R; Verrax, J; Calderon, P Buc

    2014-01-01

    Background: Standard therapy for acute promyelocytic leukaemia (APL) includes retinoic acid (all-trans retinoic acid (ATRA)), which promotes differentiation of promyelocytic blasts. Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear. Methods: Four leukaemia cancer human models (HL60, THP-1, NBR4 and NBR4-R2 cells) were treated either with ATO alone or ATO plus ATRA. Cancer cell survival was monitored by trypan blue exclusion and DEVDase activity assays. Gene and protein expression changes were assessed by RT-PCR and western blot. Results: ATO induced an antioxidant response characterised by Nrf2 nuclear translocation and enhanced transcription of downstream target genes (that is, HO-1, NQO1, GCLM, ferritin). In cells exposed to ATO plus ATRA, the Nrf2 nuclear translocation was prevented and cytotoxicity was enhanced. HO-1 overexpression reversed partially the cytotoxicity by ATRA-ATO in HL60 cells. The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RARα antagonist Ro-41-52-53. Conclusions: The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy. PMID:25003661

  8. Ethanol extract of Hedyotis diffusa willd upregulates G0/G1 phase arrest and induces apoptosis in human leukemia cells by modulating caspase cascade signaling and altering associated genes expression was assayed by cDNA microarray.

    PubMed

    Kuo, Yu-Jui; Yang, Jai-Sing; Lu, Chi-Cheng; Chiang, Su-Yin; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-09-01

    The authors' previous study has shown that water extract of Hedyotis diffusa Willd (HDW) promoted immune response and exhibited anti-leukemic activity in BALB/c leukemic mice in vivo. In this study, the anti-proliferation effects of ethanol extract of H. diffusa Willd (EEHDW) on lung cancer cell lines (A549, H1355, and LLC), leukemia cell lines (HL-60, WEHI-3), and a mouse melanoma cell line (B16F10) in vitro were investigated. The results demonstrated that EEHDW suppressed the cell proliferation of A549, H1355, HL-60, WEHI-3, and B16F10 cells as well as reduced cell viability in a concentration-dependent manner. We found that EEHDW inhibited the cell proliferation of HL-60 cells in concentration-dependent manner. In addition, EEHDW triggered an arrest of HL-60 cells at G0/G1 phase and sub-G1 population (apoptotic cells). EEHDW provoked DNA condensation and DNA damage in HL-60 cells. The activities of caspase-3, caspase-8, and caspase-9 were elevated in EEHDW-treated HL-60 cells. DNA microarray to investigate and display the gene levels related to cell growth, signal transduction, apoptosis, cell adhesion, cell cycle, DNA damage and repair, transcription and translation was also used. These findings suggest that EEHDW may be a potential herbal medicine and therapeutic agent for the treatment of leukemia.

  9. Comparative genomic hybridization study of arsenic-exposed and non-arsenic-exposed urinary transitional cell carcinoma

    SciTech Connect

    Hsu, L.-I; Chiu, Allen W.; Pu, Y.-S.; Wang, Y.-H.; Huan, Steven K.; Hsiao, C.-H.; Hsieh, F.-I; Chen, C.-J.

    2008-03-01

    To compare the differences in DNA aberrations between arsenic-exposed and non-arsenic-exposed transitional cell carcinoma (TCC), we analyzed 19 arsenic-exposed and 29 non-arsenic-exposed urinary TCCs from Chi-Mei Hospital using comparative genomic hybridization. DNA aberrations were detected in 42 TCCs including 19 arsenic-exposed and 23 non-arsenic-exposed TCCs. Arsenic-exposed TCCs had more changes than unexposed TCCs (mean {+-} SD, 6.6 {+-} 2.9 vs. 2.9 {+-} 2.2). Arsenic exposure was significantly associated with the number of DNA aberrations after adjustment for tumor stage, tumor grade and cigarette smoking in multiple regression analysis. The most frequent DNA gains, which were strikingly different between arsenic-exposed and non-arsenic-exposed TCCs, included those at 1p, 4p, 4q and 8q. A much higher frequency of DNA losses in arsenic-exposed TCCs compared with non-arsenic-exposed TCCs was observed in 10q, 11p and 17p. Chromosomal loss in 17p13 was associated not only with arsenic exposure, but also with tumor stage and grade. The p53 immunohistochemistry staining showed that chromosome 17p13 loss was associated with either p53 no expression (25%) or p53 overexpression (75%). The findings suggest that long-term arsenic exposure may increase the chromosome abnormality in TCC, and 17p loss plays an important role in arsenic-induced urinary carcinogenesis.

  10. Chromosome aberrations in ataxia telangiectasia cells exposed to heavy ions

    NASA Astrophysics Data System (ADS)

    Kawata, T.; Cucinotta, F.; George, K.; Wu, H.; Shigematsu, N.; Furusawa, Y.; Uno, T.; Isobe, K.; Ito, H.

    Understanding of biological effects of heavy ions is important to assess healt h risk in space. One of the most important issues may be to take into account individual susceptibility. Ataxia telangiectasia (A-T) cells are known to exhibit abnormal responses to radiations but the mechanism of hyper radiosensitivity of A-T still remains unknown. We report chromosome aberrations in normal human fibroblasts and AT fibroblasts exposed to low- and high-LET radiations. A chemical-induced premature chromosome condensation (PCC) technique combined with chromosome- painting technique was applied to score chromosome aberrations in G2/M-phase cells. Following gamma irradiation, GM02052 cells were approximately 5 times more sensitive to g-rays than AG1522 cells. GM02052 cells had a much higher frequency of deletions and misrejoining than AG1522 cells. When the frequency of complex type aberrations was compared, GM02052 cells showed more than 10 times higher frequency than AG1522 cells. The results will be compared with those obtained from high-LET irradiations.

  11. The efficiency of photovoltaic cells exposed to pulsed laser light

    NASA Technical Reports Server (NTRS)

    Lowe, R. A.; Landis, G. A.; Jenkins, P.

    1993-01-01

    Future space missions may use laser power beaming systems with a free electron laser (FEL) to transmit light to a photovoltaic array receiver. To investigate the efficiency of solar cells with pulsed laser light, several types of GaAs, Si, CuInSe2, and GaSb cells were tested with the simulated pulse format of the induction and radio frequency (RF) FEL. The induction pulse format was simulated with an 800-watt average power copper vapor laser and the RF format with a frequency-doubled mode-locked Nd:YAG laser. Averaged current vs bias voltage measurements for each cell were taken at various optical power levels and the efficiency measured at the maximum power point. Experimental results show that the conversion efficiency for the cells tested is highly dependent on cell minority carrier lifetime, the width and frequency of the pulses, load impedance, and the average incident power. Three main effects were found to decrease the efficiency of solar cells exposed to simulated FEL illumination: cell series resistance, LC 'ringing', and output inductance. Improvements in efficiency were achieved by modifying the frequency response of the cell to match the spectral energy content of the laser pulse with external passive components.

  12. Cloning assay thresholds on cells exposed to ultrafast laser pulses

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Riemann, Iris; Fischer, Peter; Becker, Thomas P.; Oehring, Hartmut; Halbhuber, Karl-Juergen

    1999-06-01

    The influence of the peak power, laser wavelength and the pulse duration of near infrared (NIR) ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm2 and TW/cm2. A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of-cavity pulse- stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P2/(tau) relation which is typical for a two-photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P2/(tau) relation for the efficiency of fluorescence excitation, two- photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond layers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.

  13. Cloning assay thresholds on cells exposed to ultrafast laser pulses

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Riemann, Iris; Fischer, Peter; Becker, Thomas P.; Oehring, Hartmut; Halbhuber, Karl-Juergen

    1999-06-01

    The influence of the peak power, laser wavelength and the pulse duration of near infrared ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular, we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm2 and TW/cm2. A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond(s) pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of- cavity pulse-stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P2/(tau) relation which is typical for a two- photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P2/(tau) relation for the efficiency of fluorescence excitation, two-photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond lasers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.

  14. Synthesis of protein in intestinal cells exposed to cholera toxin

    SciTech Connect

    Peterson, J.W.; Berg, W.D. Jr.; Coppenhaver, D.H.

    1987-11-01

    The mechanism by which cyclic adenosine monophosphate (AMP), formed by intestinal epithelial cells in response to cholera toxin, ultimately results in alterations in water and electrolyte transport is poorly understood. Several studies have indicated that inhibitors of transcription or translation block much of the transport of ions and water in the intestine and edema formation in tissue elicited by cholera toxin. Data presented in this study confirmed the inhibitory effects of cycloheximide on cholera toxin-induced fluid accumulation in the rabbit intestinal loop model. Neither cycloheximide nor actinomycin D altered the amount of cyclic AMP that accumulated in intestinal cells and Chinese hamster ovary cells exposed to cholera toxin. An increase in (/sup 3/H) leucine incorporation was readily demonstrable in intestinal epithelial cells from rabbits challenged with Vibrio cholerae. Similarly, intestinal epithelial cells incubated with cholera toxin for 4 hr synthesized substantially more protein than controls as determined by relative incorporation of (/sup 35/S) methionine. Most of the new protein synthesized in response to cholera toxin was membrane associated and of high molecular weight. The possible significance of the toxin-induced protein relative to cholera pathogenesis was discussed.

  15. Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia

    PubMed Central

    Ohayon, Delphine; De Chiara, Alessia; Chapuis, Nicolas; Candalh, Céline; Mocek, Julie; Ribeil, Jean-Antoine; Haddaoui, Lamya; Ifrah, Norbert; Hermine, Olivier; Bouillaud, Frédéric; Frachet, Philippe; Bouscary, Didier; Witko-Sarsat, Véronique

    2016-01-01

    Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNA replication, has been described as a key element in survival of mature neutrophil granulocytes, which are non-proliferating cells. Herein, we demonstrated an active export of PCNA involved in cell survival and chemotherapy resistance. Notably, daunorubicin-resistant HL-60 cells (HL-60R) have a prominent cytosolic PCNA localization due to increased nuclear export compared to daunorubicin-sensitive HL-60 cells (HL-60S). By interacting with nicotinamide phosphoribosyltransferase (NAMPT), a protein involved in NAD biosynthesis, PCNA coordinates glycolysis and survival, especially in HL-60R cells. These cells showed a dramatic increase in intracellular NAD+ concentration as well as glycolysis including increased expression and activity of hexokinase 1 and increased lactate production. Furthermore, this functional activity of cytoplasmic PCNA was also demonstrated in patients with acute myeloid leukemia (AML). Our data uncover a novel pathway of nuclear export of PCNA that drives cell survival by increasing metabolism flux. PMID:27759041

  16. Polyamines and polyamine biosynthesis in cells exposed to hyperthermia

    SciTech Connect

    Gerner, E.W.; Stickney, D.G.; Herman, T.S.; Fuller, D.J.

    1983-02-01

    The issue of how polyamines act to sensitize cultured cells to the lethal effects of hyperthermia was investigated using Chinese hamster cells which were induced to express thermotolerance. Intracellular levels of these naturally occurring polycations were manipulated in certain situations by treating whole cells with methylglyoxal bis-(guanylhydrazone), an inhibitor of the S-adenosyl-L-methionine decarboxylases. Exogenous spermine as low as 100 ..mu..M in the culture media dramatically sensitized cells expressing thermotolerance to the lethal effects of subsequent 42/sup 0/C exposures. When thermotolerance was differentially induced in cultures exposed to 42.4/sup 0/C by varying the rate of heating from 37 to 42.4/sup 0/C, the most resistant cells and the highest levels of intracellular spermidine and spermine. This finding was explainable in part by the observation that the putrescine-dependent S-adenosyl-L-methionine decarboxylase activity was minimally affected in cells expressng the greatest degree of thermotolerance. When this enzyme activity was inhibited by drug, lowered intracellular polyamine levels did not correspond with subsequent survival responses to heat. Interestingly, cultures treated with methylglyoxal bis-(guanylhydrazone) 24 hr previous to heat exposure showed a reduced capacity to express rate of heating-induced thermotolerance. Together, these results demonstrate that the polyamines, especially spermidine and spermine, enhance hyperthermia-induced cell killing by some mechanism involving the plasma membrane. Further, our data suggest that methylglyoxal bis-(guanylhydrazone) can act to affect thermal responses by a mechanism(s) other than modification of intracellular polyamine levels.

  17. Dynamic effects and applications for nanosecond pulsed electric fields in cells and tissues

    NASA Astrophysics Data System (ADS)

    Beebe, Stephen J.; Blackmore, Peter F.; Hall, Emily; White, Jody A.; Willis, Lauren K.; Fauntleroy, Laura; Kolb, Juergen F.; Schoenbach, Karl H.

    2005-04-01

    Nanosecond, high intensity pulsed electric fields [nsPEFs] that are below the plasma membrane [PM] charging time constant have decreasing effects on the PM and increasing effects on intracellular structures and functions as the pulse duration decreases. When human cell suspensions were exposed to nsPEFs where the electric fields were sufficiently intense [10-300ns, <=300 kV/cm.], apoptosis signaling pathways could be activated in several cell models. Multiple apoptosis markers were observed in Jurkat, HL-60, 3T3L1-preadipocytes, and isolated rat adipocytes including decreased cell size and number, caspase activation, DNA fragmentation, and/or cytochrome c release into the cytoplasm. Phosphatidylserine externalization was observed as a biological response to nsPEFs in 3T3-L1 preadipocytes and p53-wildtype and -null human colon carcinoma cells. B10.2 mouse fibrosarcoma tumors that were exposed to nsPEFs ex vivo and in vivo exhibited DNA fragmentation, elevated caspase activity, and reduced size and weight compared to contralateral sham-treated control tumors. When nsPEF conditions were below thresholds for apoptosis and classical PM electroporation, non-apoptotic responses were observed similar to those initiated through PM purinergic receptors in HL-60 cells and thrombin in human platelets. These included Ca2+ mobilization from intracellular stores [endoplasmic reticulum] and subsequently through store-operated Ca2+ channels in the PM. In addition, platelet activation measured as aggregation responses were observed in human platelets. Finally, when nsPEF conditions followed classical electroporation-mediated transfection, the expression intensity and number of GFP-expressing cells were enhanced above cells exposed to electroporation conditions alone. These studies demonstrate that application of nsPEFs to cells or tissues can modulate cell-signaling mechanisms with possible applications as a new basic science tool, cancer treatment, wound healing, and gene therapy.

  18. Mangiferin activates Nrf2-antioxidant response element signaling without reducing the sensitivity to etoposide of human myeloid leukemia cells in vitro

    PubMed Central

    Zhang, Ben-ping; Zhao, Jie; Li, Shan-shan; Yang, Li-jing; Zeng, Ling-lan; Chen, Yan; Fang, Jun

    2014-01-01

    Aim: Mangiferin is glucosylxanthone extracted from plants of the Anacardiaceae and Gentianaceae families. The aim of this study was to investigate the effects of mangiferin on Nrf2-antioxidant response element (ARE) signaling and the sensitivity to etoposide of human myeloid leukemia cells in vitro. Methods: Human HL-60 myeloid leukemia cells and mononuclear human umbilical cord blood cells (MNCs) were examined. Nrf2 protein was detected using immunofluorescence staining and Western blotting. Binding of Nrf2 to ARE was examined with electrophoretic mobility shift assay. The level of NQO1 was assessed with real-time RT-PCR and Western blotting. DCFH-DA was used to evaluate intracellular ROS level. Cell proliferation and apoptosis were analyzed using MTT and flow cytometry, respectively. Results: Mangiferin (50 μmol/L) significantly increased Nrf2 protein accumulation in HL-60 cells, particularly in the nucleus. Mangiferin also enhanced the binding of Nrf2 to an ARE, significantly up-regulated NQO1 expression and reduced intracellular ROS in HL60 cells. Mangiferin alone dose-dependently inhibited the proliferation of HL-60 cells. Mangiferin (50 mol/L) did not attenuate etoposide-induced cytotoxicity in HL-60 cells, and combined treatment of mangiferin with low concentration of etoposide (0.8 μg/mL) even increased the cell inhibition rate. Nor did mangiferin change the rate of etoposide-induced apoptosis in HL-60 cells. In MNCs, mangiferin significantly relieved oxidative stress, but attenuated etoposide-induced cytotoxicity. Conclusion: Mangiferin is a novel Nrf2 activator that reduces oxidative stress and protects normal cells without reducing the sensitivity to etoposide of HL-60 leukemia cells in vitro. Mangiferin may be a potential chemotherapy adjuvant. PMID:24374812

  19. Carbon Nanotubes and Human Cells?

    ERIC Educational Resources Information Center

    King, G. Angela

    2005-01-01

    Single-walled carbon nanotubes that were chemically altered to be water soluble are shown to enter fibroblasts, T cells, and HL60 cells. Nanoparticles adversely affect immortalized HaCaT human keratinocyte cultures, indicating that they may enter cells.

  20. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1

    PubMed Central

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M.; Nyström, Sofia; Hinkula, Jorma

    2015-01-01

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection. PMID:26157174

  1. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1.

    PubMed

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M; Nyström, Sofia; Hinkula, Jorma; Larsson, Marie

    2015-08-15

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection. PMID:26157174

  2. Hibiscus anthocyanins rich extract-induced apoptotic cell death in human promyelocytic leukemia cells

    SciTech Connect

    Chang, Y.-C.; Huang, H.-P.; Hsu, J.-D.; Yang, S.-F.; Wang, C.-J. . E-mail: wcj@csmu.edu.tw

    2005-06-15

    Hibiscus sabdariffa Linne (Malvaceae), an attractive plant believed to be native to Africa, is cultivated in the Sudan and Eastern Taiwan. Anthocyanins exist widely in many vegetables and fruits. Some reports demonstrated that anthocyanins extracted from H. sabdariffa L., Hibiscus anthocyanins (HAs) (which are a group of natural pigments existing in the dried calyx of H. sabdariffa L.) exhibited antioxidant activity and liver protection. Therefore, in this study, we explored the effect of HAs on human cancer cells. The result showed that HAs could cause cancer cell apoptosis, especially in HL-60 cells. Using flow cytometry, we found that HAs treatment (0-4 mg/ml) markedly induced apoptosis in HL-60 cells in a dose- and time-dependent manner. The result also revealed increased phosphorylation in p38 and c-Jun, cytochrome c release, and expression of tBid, Fas, and FasL in the HAs-treated HL-60 cells. We further used SB203580 (p38 inhibitor), PD98059 (MEK inhibitor), SP600125 (JNK inhibitor), and wortmannin (phosphatidylinositol 3-kinase; PI-3K inhibitor) to evaluate their effect on the HAs-induced HL-60 death. The data showed that only SB203580 had strong potential in inhibiting HL-60 cell apoptosis and related protein expression and phosphorylation. Therefore, we suggested that HAs mediated HL-60 apoptosis via the p38-FasL and Bid pathway. According to these results, HAs could be developed as chemopreventive agents. However, further investigations into the specificity and mechanism(s) of HAs are needed.

  3. Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation

    PubMed Central

    Gerashchenko, Bogdan I.; Yamagata, Akira; Oofusa, Ken; Yoshizato, Katsutoshi; de Toledo, Sonia M.; Howell, Roger W.

    2010-01-01

    Recently (Cytometry 2003, 56A, 71–80), we reported that direct cell-to-cell contact is required for stimulating proliferation of bystander rat liver cells (WB-F344) cocultured with irradiated cells, and neither functional gap junction intercellular communication nor long-range extracellular factors appear to be involved in this proliferative bystander response (PBR). The molecular basis for this response is unknown. Confluent monolayers of WB-F344 cells were exposed to 5-Gray (Gy) of γ-rays. Irradiated cells were mixed with unirradiated cells and co-cultured for 24 h. Cells were harvested and protein expression was examined using 2-DE. Protein expression was also determined in cultures of unirradiated and 5-Gy irradiated cells. Proteins were identified by MS. Nucleophosmin (NPM)-1, a multifunctional nucleolar protein, was more highly expressed in bystander cells than in either unirradiated or 5-Gy irradiated cells. Enolase-α, a glycolytic enzyme, was present in acidic and basic variants in unirradiated cells. In bystander and 5-Gy irradiated cells, the basic variant was weakly expressed, whereas the acidic variant was overwhelmingly present. These data indicate that the presence of irradiated cells can affect NPM-1 and enolase-α in adjacent bystander cells. These proteins appear to participate in molecular events related to the PBR and suggest that this response may involve cellular defense, proliferation, and metabolism. PMID:17514680

  4. Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation.

    PubMed

    Gerashchenko, Bogdan I; Yamagata, Akira; Oofusa, Ken; Yoshizato, Katsutoshi; de Toledo, Sonia M; Howell, Roger W

    2007-06-01

    Recently (Cytometry 2003, 56A, 71-80), we reported that direct cell-to-cell contact is required for stimulating proliferation of bystander rat liver cells (WB-F344) cocultured with irradiated cells, and neither functional gap junction intercellular communication nor long-range extracellular factors appear to be involved in this proliferative bystander response (PBR). The molecular basis for this response is unknown. Confluent monolayers of WB-F344 cells were exposed to 5-Gray (Gy) of gamma-rays. Irradiated cells were mixed with unirradiated cells and co-cultured for 24 h. Cells were harvested and protein expression was examined using 2-DE. Protein expression was also determined in cultures of unirradiated and 5-Gy irradiated cells. Proteins were identified by MS. Nucleophosmin (NPM)-1, a multifunctional nucleolar protein, was more highly expressed in bystander cells than in either unirradiated or 5-Gy irradiated cells. Enolase-alpha, a glycolytic enzyme, was present in acidic and basic variants in unirradiated cells. In bystander and 5-Gy irradiated cells, the basic variant was weakly expressed, whereas the acidic variant was overwhelmingly present. These data indicate that the presence of irradiated cells can affect NPM-1 and enolase-alpha in adjacent bystander cells. These proteins appear to participate in molecular events related to the PBR and suggest that this response may involve cellular defense, proliferation, and metabolism.

  5. Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

    PubMed Central

    Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

    2016-01-01

    Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

  6. Cytoplasmic myosin-exposed apoptotic cells appear with caspase-3 activation and enhance CLL cell viability.

    PubMed

    Cui, X; Zhang, L; Magli, A R; Catera, R; Yan, X-J; Griffin, D O; Rothstein, T L; Barrientos, J; Kolitz, J E; Allen, S L; Rai, K R; Chiorazzi, N; Chu, C C

    2016-01-01

    The degree of chronic lymphocytic leukemia (CLL) B-cell antigen receptor (BCR) binding to myosin-exposed apoptotic cells (MEACs) correlates with worse patient outcomes, suggesting a link to disease activity. Therefore, we studied MEAC formation and the effects of MEAC binding on CLL cells. In cell line studies, both intrinsic (spontaneous or camptothecin-induced) and extrinsic (FasL- or anti-Fas-induced) apoptosis created a high percent of MEACs over time in a process associated with caspase-3 activation, leading to cytoplasmic myosin cleavage and trafficking to cell membranes. The involvement of common apoptosis pathways suggests that most cells can produce MEACs and indeed CLL cells themselves form MEACs. Consistent with the idea that MEAC formation may be a signal to remove dying cells, we found that natural IgM antibodies bind to MEACs. Functionally, co-culture of MEACs with CLL cells, regardless of immunoglobulin heavy-chain variable region gene mutation status, improved leukemic cell viability. Based on inhibitor studies, this improved viability involved BCR signaling molecules. These results support the hypothesis that stimulation of CLL cells with antigen, such as those on MEACs, promotes CLL cell viability, which in turn could lead to progression to worse disease. PMID:26220042

  7. Ultrastructural changes in tracheal epithelial cells exposed to oxygen

    NASA Technical Reports Server (NTRS)

    Philpott, D. E.; Harrison, G. A.; Turnbill, C.; Black, S.

    1977-01-01

    White albino rats were sacrificed after 24, 36, 48, 72, and 96 h of exposure to 100% O2 at 1 atm. Tissue was prepared for the scanning electron microscope (SEM) by Critical Point Drying and for the transmission electron microscope (TEM) by plastic embedding. Scanning microscopy showed a loss of microvilli after 48 h of exposure. Cilia appeared relatively normal with SEM, but TEM revealed changes in the outer membrane. In TEM, nonciliated cells appeared swollen and often encroached on the ciliated cells. A heavy mucous blanket remained even after processing. All the changes observed that are induced by oxygen exposure contribute to mucostasis, reducing and/or halting mucociliary clearance.

  8. Proliferation of human mammary cancer cells exposed to 27-hydroxycholesterol

    PubMed Central

    CRUZ, PAMELA; TORRES, CRISTIAN; RAMÍREZ, MARÍA EUGENIA; EPUÑÁN, MARÍA JOSÉ; VALLADARES, LUIS EMILIO; SIERRALTA, WALTER DANIEL

    2010-01-01

    The aim of the present study was to identify the possible mechanisms by which certain estradiol receptor (ER)-positive mammary tumor cells remain resistant to treatment with anti-estrogens or inhibitors of local estradiol (E2) production. To this end, we compared the proliferative effects on mammary cancer cells of the novel selective ER modulator 27-hydroxycholesterol (27OHC) to those of E2, and evaluated their inhibition by ICI 182,780 (ICI). Analysis of the effects on the cell cycle of 27OHC and E2 in the absence or presence of ICI was conducted. In ER-positive mammary tumor cells, we detected the blocking of 27OHC proliferation-stimulatory activity by simvastatin, as well as the inhibition of E2-stimulated proliferation by an α-fetoprotein-derived cyclic nonapeptide. The effects reported herein may be extrapolated to infiltrating mammary cancer, where the activity of local macrophages may stimulate tumor growth. We suggest that increased breast cancer growth in obese patients may be related to increased 27OHC circulatory levels. PMID:22993572

  9. Correlation between dielectric property by dielectrophoretic levitation and growth activity of cells exposed to electric field.

    PubMed

    Hakoda, Masaru; Hirota, Yusuke

    2013-09-01

    The purpose of this study is to develop a system analyzing cell activity by the dielectrophoresis method. Our previous studies revealed a correlation between the growth activity and dielectric property (Re[K(ω)]) of mouse hybridoma 3-2H3 cells using dielectrophoretic levitation. Furthermore, it was clarified that the differentiation activity of many stem cells could be evaluated by the Re[K(ω)] without differentiation induction. In this paper, 3-2H3 cells exposed to an alternating current (AC) electric field or a direct current (DC) electric field were cultivated, and the influence of damage by the electric field on the growth activity of the cells was examined. To evaluate the activity of the cells by measuring the Re[K(ω)], the correlation between the growth activity and the Re[K(ω)] of the cells exposed to the electric field was examined. The relations between the cell viability, growth activity, and Re[K(ω)] in the cells exposed to the AC electric field were obtained. The growth activity of the cells exposed to the AC electric field could be evaluated by the Re[K(ω)]. Furthermore, it was found that the adverse effects of the electric field on the cell viability and the growth activity were smaller in the AC electric field than the DC electric field.

  10. Autosomal mutants of proton-exposed kidney cells display frequent loss of heterozygosity on nonselected chromosomes.

    PubMed

    Grygoryev, Dmytro; Dan, Cristian; Gauny, Stacey; Eckelmann, Bradley; Ohlrich, Anna P; Connolly, Marissa; Lasarev, Michael; Grossi, Gianfranco; Kronenberg, Amy; Turker, Mitchell S

    2014-05-01

    High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton-exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome.

  11. Comparative Mitochondrial Proteomic Analysis of Raji Cells Exposed to Adriamycin

    PubMed Central

    Jiang, Yu-Jie; Sun, Qing; Fang, Xiao-Sheng; Wang, Xin

    2009-01-01

    The antitumor mechanisms of adriamycin (ADR) have been thought to contribute to induction of apoptosis and inefficiency of DNA repair, processes that are to a large extent mediated by mitochondria. This study aimed to investigate characteristics of ADR, including its antineoplastic activity, drug resistance, and unexpected toxicity in non-Hodgkin lymphoma (NHL) Raji cells at the mitochondrial proteomic level. The alterations of the mitochondrial proteome of Raji cells treated with ADR were analyzed by two-dimensional differential in-gel electrophoresis (2D-DIGE) coupled with linear ion trap quadrupole–electrospray ionization tandem mass spectrometry (LTQ-ESI-MS/MS).The altered patterns of three identified proteins were validated by Western blot and analyzed by pathway studio software. The results showed that 34 proteins were downregulated and 3 proteins upregulated in the study group compared with the control group. The differentially expressed proteins distributed their functions in reduction-oxidation reactions, DNA repair, cell cycle regulation, transporters and channels, and oxidative phosphorylation. Furthermore, heat shock protein 70 (HSP70), ATP-binding cassette transporter isoform B6 (ABCB6), and prohibitin (PHB) identified in this study may be closely related to chemoresistance and could serve as potential chemotherapeutic targets for NHL. Collectively, these results suggest that specific mitochondrial proteins are uniquely susceptible to alterations in abundance following exposure to ADR and carry implications for the investigation of therapeutic and prognostic markers. Further studies focusing on these identified proteins will be used to predict treatment response and reverse apoptosis resistance,and to explore drug-combination strategies associated with ADR for NHL therapy. PMID:19209238

  12. Modeling and analysis of retinoic acid induced differentiation of uncommitted precursor cells.

    PubMed

    Tasseff, Ryan; Nayak, Satyaprakash; Song, Sang Ok; Yen, Andrew; Varner, Jeffrey D

    2011-05-01

    Manipulation of differentiation programs has therapeutic potential in a spectrum of human cancers and neurodegenerative disorders. In this study, we integrated computational and experimental methods to unravel the response of a lineage uncommitted precursor cell-line, HL-60, to Retinoic Acid (RA). HL-60 is a human myeloblastic leukemia cell-line used extensively to study human differentiation programs. Initially, we focused on the role of the BLR1 receptor in RA-induced differentiation and G1/0-arrest in HL-60. BLR1, a putative G protein-coupled receptor expressed following RA exposure, is required for RA-induced cell-cycle arrest and differentiation and causes persistent MAPK signaling. A mathematical model of RA-induced cell-cycle arrest and differentiation was formulated and tested against BLR1 wild-type (wt) knock-out and knock-in HL-60 cell-lines with and without RA. The current model described the dynamics of 729 proteins and protein complexes interconnected by 1356 interactions. An ensemble strategy was used to compensate for uncertain model parameters. The ensemble of HL-60 models recapitulated the positive feedback between BLR1 and MAPK signaling. The ensemble of models also correctly predicted Rb and p47phox regulation and the correlation between p21-CDK4-cyclin D formation and G1/0-arrest following exposure to RA. Finally, we investigated the robustness of the HL-60 network architecture to structural perturbations and generated experimentally testable hypotheses for future study. Taken together, the model presented here was a first step toward a systematic framework for analysis of programmed differentiation. These studies also demonstrated that mechanistic network modeling can help prioritize experimental directions by generating falsifiable hypotheses despite uncertainty.

  13. Differential gene expression in pulmonary artery endothelial cells exposed to sickle cell plasma.

    PubMed

    Klings, Elizabeth S; Safaya, Surinder; Adewoye, Adeboye H; Odhiambo, Adam; Frampton, Garrett; Lenburg, Marc; Gerry, Norman; Sebastiani, Paola; Steinberg, Martin H; Farber, Harrison W

    2005-05-11

    Clinical variability in sickle cell disease (SCD) suggests a role for extra-erythrocytic factors in the pathogenesis of vasoocclusion. We hypothesized that endothelial cell (EC) dysfunction, one possible modifier of disease variability, results from induction of phenotypic changes by circulating factors. Accordingly, we analyzed gene expression in cultured human pulmonary artery ECs (HPAEC) exposed to plasma from 1) sickle acute chest syndrome (ACS) patients, 2) SCD patients at steady state, 3) normal volunteers, and 4) serum-free media, using whole genome microarrays (U133A-B GeneChip, Affymetrix). Data were analyzed by Bayesian analysis of differential gene expression (BADGE). Differential expression was defined by the probability of >1.5 fold change in signal intensity greater than 0.999 and a predicted score of 70-100, measured by cross-validation. Compared with normal plasma, plasma from SCD patients (steady state) resulted in differential expression of 50 genes in HPAEC. Of these genes, molecules involved in cholesterol biosynthesis and lipid transport, the cellular stress response, and extracellular matrix proteins were most prominent. Another 58 genes were differentially expressed in HPAEC exposed to plasma from ACS patients. The pattern of altered gene expression suggests that plasma from SCD patients induces an EC phenotype which is anti-apoptotic and favors cholesterol biosynthesis. An altered EC phenotype elicited by SCD plasma may contribute to the pathogenesis of sickle vasoocclusion.

  14. DNA Fragmentation in mammalian cells exposed to various light ions

    NASA Astrophysics Data System (ADS)

    Belli, M.; Cherubini, R.; Dalla Vecchia, M.; Dini, V.; Esposito, G.; Moschini, G.; Sapora, O.; Signoretti, C.; Simone, G.; Sorrentino, E.; Tabocchini, M. A.

    Elucidation of how effects of densely ionizing radiation at cellular level are linked to DNA damage is fundamental for a better understanding of the mechanisms leading to genomic damage (especially chromosome aberrations) and developing biophysical models to predict space radiation effects. We have investigated the DNA fragmentation patterns induced in Chinese hamster V79 cells by 31 keV/μm protons, 123 keV/μm helium-4 ions and γ-rays in the size range 0.023-5.7 Mbp, using calibrated Pulsed Field Gel Electrophoresis (PFGE). The frequency distributions of fragments induced by the charged particles were shifted towards smaller sizes with respct to that induced by comparable doses of γ-rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for γ-rays by a factor of about 1.9 and 1.2, respectively. However, these ratios do not adequately reflect the RBE observed on the same cells for inactivation and mutation induced by these beams. This is a further indication for the lack of correlation between the effects exerted at cellular level and the initial yield of DSB. The dependence on radiation quality of the fragmentation pattern suggests that it may have a role in damage reparability. We have analyzed these patterns with a "random breakage" model generalized in order to consider the initial non-random distribution of the DNA molecules. Our results suggest that a random breakage mechanism can describe with a reasonable approximation the DNA fragmentation induced by γ-rays, while the approximation is not so good for light ions, likely due to the interplay between ion tracks and chromatin organization at the loop level.

  15. Gene expression changes in human cells after exposure to mobile phone microwaves.

    PubMed

    Remondini, Daniel; Nylund, Reetta; Reivinen, Jukka; Poulletier de Gannes, Florence; Veyret, Bernard; Lagroye, Isabelle; Haro, Emmanuelle; Trillo, M Angeles; Capri, Miriam; Franceschi, Claudio; Schlatterer, Kathrin; Gminski, Richard; Fitzner, Rudolf; Tauber, Rudolf; Schuderer, Jurgen; Kuster, Niels; Leszczynski, Dariusz; Bersani, Ferdinando; Maercker, Christian

    2006-09-01

    Possible biological effects of mobile phone microwaves were investigated in vitro. In this study, which was part of the 5FP EU project REFLEX (Risk Evaluation of Potential Environmental Hazards From Low-Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods), six human cell types, immortalized cell lines and primary cells, were exposed to 900 and 1800 MHz. RNA was isolated from exposed and sham-exposed cells and labeled for transcriptome analysis on whole-genome cDNA arrays. The results were evaluated statistically using bioinformatics techniques and examined for biological relevance with the help of different databases. NB69 neuroblastoma cells, T lymphocytes, and CHME5 microglial cells did not show significant changes in gene expression. In EA.hy926 endothelial cells, U937 lymphoblastoma cells, and HL-60 leukemia cells we found between 12 and 34 up- or down-regulated genes. Analysis of the affected gene families does not point towards a stress response. However, following microwave exposure, some but not all human cells might react with an increase in expression of genes encoding ribosomal proteins and therefore up-regulating the cellular metabolism. PMID:16878293

  16. DETAIL OF ZINC CLEANER CELL INTERIOR (EXPOSED AT F/45 FOR ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    DETAIL OF ZINC CLEANER CELL INTERIOR (EXPOSED AT F/45 FOR DEPTH OF FIELD PURPOSES). NOTE GALIGHER STYLE BAFFLES AND TENDENCY OF ZINC TO BUILD UP ON CELL COMPONENTS. - Shenandoah-Dives Mill, 135 County Road 2, Silverton, San Juan County, CO

  17. Capsaicin Inhibits Preferentially the NADH Oxidase and Growth of Transformed Cells in Culture

    NASA Astrophysics Data System (ADS)

    Morre, D. James; Chueh, Pin-Ju; Morre, Dorothy M.

    1995-03-01

    A hormone- and growth factor-stimulated NADH oxidase of the mammalian plasma membrane, constitutively activated in transformed cells, was inhibited preferentially in HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells, all of human origin, by the naturally occurring quinone analog capsaicin (8-methyl-N-vanillyl-6-noneamide), compared with plasma membranes from human mammary epithelial, rat liver, normal rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. With cells in culture, capsaicin preferentially inhibited growth of HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells but was largely without effect on the mammary epithelial cells, rat kidney cells, or HL-60 cells induced to differentiate with dimethyl sulfoxide. Inhibited cells became smaller and cell death was accompanied by a condensed and fragmented appearance of the nuclear DNA, as revealed by fluorescence microscopy with 4',6-diamidino-2-phenylindole, suggestive of apoptosis. The findings correlate capsaicin inhibition of cell surface NADH oxidase activity and inhibition of growth that correlate with capsaicin-induced apoptosis.

  18. Extracellular vesicles released from cells exposed to reactive oxygen species increase annexin A2 expression and survival of target cells exposed to the same conditions.

    PubMed

    Grindheim, Ann Kari; Vedeler, Anni

    2016-01-01

    Annexin A2 (AnxA2) is present in multiple cellular compartments and interacts with numerous ligands including calcium, proteins, cholesterol, negatively charged phospholipids and RNA. These interactions are tightly regulated by its post-translational modifications. The levels of AnxA2 and its Tyr23 phosphorylated form (pTyr23AnxA2) are increased in many cancers and the protein is involved in malignant cell transformation, metastasis and angiogenesis. Our previous studies of rat pheochromocytoma (PC12) cells showed that reactive oxygen species (ROS) induce rapid, simultaneous and transient dephosphorylation of nuclear AnxA2, most likely associating with PML bodies, while AnxA2 associated with F-actin at the cell cortex undergoes Tyr23 phosphorylation. The pTyr23AnxA2 in the periphery of the cells is incorporated into intraluminal vesicles of multivesicular endosomes and subsequently released to the extracellular space. We show here that extracellular vesicles (EVs) from cells exposed to ROS prime untreated PC12 cells to better tolerate subsequent oxidative stress, thus enhancing their survival. There is an increase in the levels of pTyr23AnxA2 and AnxA2 in the primed cells, suggesting that AnxA2 is involved in their survival. This increase is due to an upregulation of AnxA2 expression both at the transcriptional and translational levels after relatively short term (2 h) exposure to primed EVs. PMID:27574537

  19. Activation of NK cells in subjects exposed to mild hyper- or hypothermic load.

    PubMed

    Lackovic, V; Borecký, L; Vigas, M; Rovenský, J

    1988-06-01

    The effect of mild hyper- and hypothermic stress on release of selected hormones (somatotropin, noradrenaline, etc.), interferon (IFN), and activity of NK cells in the blood was examined in groups of young males during a 30 min exposure to 39 degrees C and 4 degrees C. A quick release of somatotropin was registered in 44% of examinees in the hyperthermic group, while the persons exposed to 4 degrees C reacted with a release of noradrenaline only. Concurrently, an elevation of NK cell activity was observed both in the subgroup releasing somatotropin after hyperthermic stress and in the group exposed to cold. Since these forms of mild stress did not lead to an appearance of IFN in the serum, the possibility of an NK cell activating effect of somatotropin and/or the adrenal hormones was tested. While the adrenal hormones stimulated the NK cell activity in vitro, no support for a similar role for somatotropin was found. PMID:2457640

  20. Determining adaptive and adverse oxidative stress responses in human bronical epithelial cells exposed to zinc

    EPA Science Inventory

    Determining adaptive and adverse oxidative stress responses in human bronchial epithelial cells exposed to zincJenna M. Currier1,2, Wan-Yun Cheng1, Rory Conolly1, Brian N. Chorley1Zinc is a ubiquitous contaminant of ambient air that presents an oxidant challenge to the human lung...

  1. SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    SIGNALING MECHANISMS IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES
    Y.M. Kim, A.G. Lenz, R. Silbajoris, I. Jaspers and J.M. Samet. Department of Environmental Sciences and Engineering and Center for Environmental Medicine, University of North Carolina, ...

  2. Role of caspase-10 in the death of acute leukemia cells

    PubMed Central

    Guo, Wenjian; Dong, Aishu; Pan, Xiahui; Lin, Xiaoji; Lin, Ying; He, Muqing; Zhu, Baoling; Jin, Liming; Yao, Rongxing

    2016-01-01

    Autophagy can protect cells from stress, but can also induce cancer cell death. Caspase-10 is now considered to be a factor that is associated with autophagy in cancer. The present study therefore investigated whether caspase-10 affects autophagy in acute leukemia cells. The rates of survival vs. apoptosis in acute leukemia HL-60 and Jurkat cells treated with drugs were tested using cell viability assays and flow cytometry, and the levels of caspase-3 and −10 were tested by western blotting. In HL-60 cells that were treated with chemotherapy drugs combined with a caspase-10 inhibitor, the rate of survival decreased significantly compared with HL-60 cells treated with chemotherapy drugs alone. In contrast, the rate of survival of Jurkat cells treated with chemotherapy drugs combined with the caspase-10 inhibitor increased significantly compared with Jurkat cells treated with chemotherapy drugs alone. The results of the flow cytometry and western blotting showed that the changes in the survival rate may be caused by a change in the amount of apoptosis occurring in the Jurkat cells treated with chemotherapy drugs combined with the caspase-10 inhibitor. However, in HL-60 cells undergoing this combination treatment, the change in the survival rate was not caused by a change in the rate of apoptosis. When HL-60 cells were treated with the chemotherapy drugs combined with the caspase-10 inhibitor and the autophagy inhibitor 3-methyl adenine, the survival rate increased, whereas the rate of apoptosis did not change. These results show that caspase-10 may be associated with autophagy in acute myeloid leukemia cells, but not in acute lymphatic leukemia cells. PMID:27446483

  3. {sub p}53-Dependent Adaptive Responses in Human Cells Exposed to Space Radiations

    SciTech Connect

    Takahashi, Akihisa; Su Xiaoming; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki; Iwasaki, Toshiyasu; Ohnishi, Takeo

    2010-11-15

    Purpose: It has been reported that priming irradiation or conditioning irradiation with a low dose of X-rays in the range of 0.02-0.1 Gy induces a p53-dependent adaptive response in mammalian cells. The aim of the present study was to clarify the effect of space radiations on the adaptive response. Methods and Materials: Two human lymphoblastoid cell lines were used; one cell line bears a wild-type p53 (wtp53) gene, and another cell line bears a mutated p53 (mp53) gene. The cells were frozen during transportation on the space shuttle and while in orbit in the International Space Station freezer for 133 days between November 15, 2008 and March 29, 2009. After the frozen samples were returned to Earth, the cells were cultured for 6 h and then exposed to a challenging X-ray-irradiation (2 Gy). Cellular sensitivity, apoptosis, and chromosome aberrations were scored using dye-exclusion assays, Hoechst33342 staining assays, and chromosomal banding techniques, respectively. Results: In cells exposed to space radiations, adaptive responses such as the induction of radioresistance and the depression of radiation-induced apoptosis and chromosome aberrations were observed in wtp53 cells but not in mp53 cells. Conclusion: These results have confirmed the hypothesis that p53-dependent adaptive responses are apparently induced by space radiations within a specific range of low doses. The cells exhibited this effect owing to space radiations exposure, even though the doses in space were very low.

  4. Nuclear Envelope Composition Determines the Ability of Neutrophil-type Cells to Passage through Micron-scale Constrictions*

    PubMed Central

    Rowat, Amy C.; Jaalouk, Diana E.; Zwerger, Monika; Ung, W. Lloyd; Eydelnant, Irwin A.; Olins, Don E.; Olins, Ada L.; Herrmann, Harald; Weitz, David A.; Lammerding, Jan

    2013-01-01

    Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential. PMID:23355469

  5. T-cell activation in pulmonary lymph nodes of mice exposed to ozone

    SciTech Connect

    Dziedzic, D.; White, H.J.

    1985-12-01

    Groups of Cd-1 female mice were exposed to ozone at 0.3, 0.5, and 0.7 ppm, 20 hr per day, 7 days per week for 1-28 days. The effect of ozone exposure on lymphoid cells was determined by studying mediastinal lymph nodes at various times of exposure. It was found that lymphocyte numbers underwent a dose-dependent, four-phased change:cellular depletion (Days 1-2), followed by rapid hyperplasia (Days 3-4), incremental cell number reduction (Days 5-7), and a subsequent subacute phase of elevated lymphocyte numbers (Days 8-28). Using tritiated thymidine it was determined that cells underwent a rapid burst of division by Day 3 of exposure and that mitosis subsequently declined to near baseline values by 2 weeks of exposure. Autoradiographic analysis of histologic sections revealed that the paracortical T-cell areas of the nodes were particularly involved. In addition to the increase in thymidine uptake, several morphologic changes were evident in affected cells. By comparison, the B cells from ozone-exposed animals were virtually unaffected with respect to cell division or morphological alterations. Prior treatment of ozone-exposed animals with a monoclonal antibody that is cytotoxic for T cells eliminated the hyperplastic response. Immunologic aspects of T-cell reactivity were studied. T-cell responsiveness to mitogenic stimulation with concanavalin A showed little alteration during the first days of exposure; however, by Day 14 an increase in reactivity was observed. This change indicated that functional lymphocyte stimulation occurred during ozone exposure.

  6. Analysis of T-cell proliferation and cytokine secretion in the individuals exposed to arsenic.

    PubMed

    Biswas, R; Ghosh, P; Banerjee, N; Das, J K; Sau, T; Banerjee, A; Roy, S; Ganguly, S; Chatterjee, M; Mukherjee, A; Giri, A K

    2008-05-01

    Over six million people in nine districts of West Bengal, India are exposed to very high levels of arsenic primarily through their drinking water. More than 300,000 people showed arsenic-induced skin lesions in these districts. This is regarded as the greatest arsenic calamity in the world. Chronic arsenicosis causes varied dermatological signs ranging from pigmentation changes, hyperkeratosis to non-melanocytic cancer of skin, and also malignancies in different internal organs. Higher incidences of opportunistic infections are found in the arsenic-exposed individuals, indicating that their immune systems may be impaired somehow. We have thus investigated the effect of arsenic on T-cell proliferation and cytokine secretion in 20 individuals with arsenic-induced skin lesions and compared the results with 18 arsenic-unexposed individuals. A marked dose-dependent suppression of Concanavalin A (Con A) induced T-cell proliferation was observed in the arsenic-exposed individuals compared with the unexposed (P < 0.001) individuals. This correlated with a significant decrease in the levels of secreted cytokines by the T cells (TNF-alpha, IFN-gamma, IL2, IL10, IL5, and IL4) in the exposed individuals (P < 0.001). Thus it can be inferred that arsenic exposure can cause immunosuppression in humans.

  7. In vitro metabolism study of normal and tumor cells when exposed to red LED light

    NASA Astrophysics Data System (ADS)

    Stolbovskaya, Olga V.; Khairullin, Radik M.; Saenko, Yuri V.; Krasnikova, Ekaterina S.; Krasnikov, Aleksandr V.; Fomin, Aleksandr A.; Skaptsov, Aleksandr A.

    2016-04-01

    This work presents the results of studying the mitochondrial membrane potential, intracellular ROS, peculiarities of the cell cycle of cancer cells HCT-116 and the normal line of CHO cells when exposed to the red LED light with a wavelength range of 0.620-0.680 μm. A dose-dependent increase in mitochondrial membrane potential and intracellular ROS concentration in cancer cells HCT-116 was established. In normal CHO cell line a dose-dependent reduction of mitochondrial membrane potential and dose-dependent increase in intracellular ROS occur. It has been shown that the sensitivity of the studied cell lines to the red light depends on the stage of the cell cycle.

  8. Deerberry Fruit Extracts Inhibit Activator protein-1, Nuclear Factor-kappaB and Cell Proliferation, and Induce Apoptosis by Perturbing the Mitogenic Signaling Pathway in vitro in Culture Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fruit of deerberry [Vaccinium stamineum L.] were evaluated for their antioxidant capacity, antioxidant enzyme activity and anti-cancer properties in JB6 P+ mouse epidermal cells and human leukemia (HL-60) cells. Deerberries contain potent free radical scavenging activities and also had high activi...

  9. Mature natural killer cells reset their responsiveness when exposed to an altered MHC environment

    PubMed Central

    Joncker, Nathalie T.; Shifrin, Nataliya; Delebecque, Frédéric

    2010-01-01

    Some mature natural killer (NK) cells cannot be inhibited by major histocompatibility complex (MHC) I molecules, either because they lack corresponding inhibitory receptors or because the host lacks the corresponding MHC I ligands for the receptors. Such NK cells nevertheless remain self-tolerant and exhibit a generalized hyporesponsiveness to stimulation through activating receptors. To address whether NK cell responsiveness is set only during the NK cell differentiation process, we transferred mature NK cells from wild-type (WT) to MHC I–deficient hosts or vice versa. Remarkably, mature responsive NK cells from WT mice became hyporesponsive after transfer to MHC I–deficient mice, whereas mature hyporesponsive NK cells from MHC I–deficient mice became responsive after transfer to WT mice. Altered responsiveness was evident among mature NK cells that had not divided in the recipient animals, indicating that the cells were mature before transfer and that alterations in activity did not require cell division. Furthermore, the percentages of NK cells expressing KLRG1, CD11b, CD27, and Ly49 receptors specific for H-2b were not markedly altered after transfer. Thus, the functional activity of mature NK cells can be reset when the cells are exposed to a changed MHC environment. These findings have important implications for how NK cell functions may be curtailed or enhanced in the context of disease. PMID:20819928

  10. Ca2+ ion transport through patch-clamped cells exposed to magnetic fields.

    PubMed

    Höjevik, P; Sandblom, J; Galt, S; Hamnerius, Y

    1995-01-01

    The total current of Ca2+ ions through patch-clamped cell membranes was measured while exposing clonal insulin-producing beta-cells (RINm5F) to a combination of DC and AC magnetic fields at so-called cyclotron resonance conditions. Previous experimental evidence supports the theory that a resonant interaction between magnetic fields and organisms can exist. This experiment was designed to test one possible site of interaction: channels in the cell membrane. The transport of Ca2+ ions through the protein channels of the plasma membrane did not show any resonant behavior in the frequency range studied. PMID:7748201

  11. Accumulation of staphylococcal Panton-Valentine leukocidin in the detergent-resistant membrane microdomains on the target cells is essential for its cytotoxicity.

    PubMed

    Nishiyama, Akihito; Isobe, Hirokazu; Iwao, Yasuhisa; Takano, Tomomi; Hung, Wei-Chun; Taneike, Ikue; Nakagawa, Saori; Dohmae, Soshi; Iwakura, Nobuhiro; Yamamoto, Tatsuo

    2012-12-01

    The mechanisms for the cytotoxicity of staphylococcal Panton-Valentine leukocidin (PVL), a pore-forming toxin consisting of LukS-PV and LukF-PV, in human immune cells are still unclear. Because LukS-PV binds to ganglioside GM1, a constituent of detergent-resistant membrane microdomains (DRMs) of the plasma membrane, the role of DRMs in PVL cytotoxicity was examined in human polymorphonuclear neutrophils (PMNs), monocytes, HL-60 cells, and THP-1 cells. PVL binding capacities in HL-60 and THP-1 cells were higher than those in PMNs and monocytes; however, the PVL concentration to obtain more than 80% cell lysis in HL-60 cells was 10 times higher than that in PMNs and PVL even at such concentration induced < 10% cell lysis in THP-1 cells. After incubation of PMNs with LukS-PV, more than 90% of LukS-PV bound to the detergent-soluble membranes. Subsequent incubation with LukF-PV at 4 °C induced the accumulation of more than 70% of PVL components and 170- to 220-kDa complex formation in DRMs in an actin-independent manner. However, only 30% of PVL was found, and complex formation was under detectable level in DRMs in HL-60 cells. PVL did not accumulate in DRMs in THP-1 cells. Our observations strongly indicate that PVL accumulation in DRMs is essential for PVL cytotoxicity.

  12. Ajoene-induced cell death in human promyeloleukemic cells does not require JNK but is amplified by the inhibition of ERK.

    PubMed

    Antlsperger, Dorothee S M; Dirsch, Verena M; Ferreira, Dulce; Su, Jen-Liang; Kuo, Ming-Liang; Vollmar, Angelika M

    2003-01-30

    Treatment of human promyeloleukemic HL-60 cells with the experimental antileukemic drug ajoene induces the activation of the mitogen-activated protein kinases (MAPKs) c-Jun NH(2)-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK) 1/2 as well as the survival kinase Akt. JNK activation occurred in HL-60/neo, HL-60/bcl-x(L), and in HL-60 cells pretreated with the pan-caspase inhibitor zVAD-fmk, indicating that JNK activation is not dependent on ajoene-induced mitochondria perturbation and subsequent caspase activation. Cells overexpressing a dominant-negative JNK showed no altered sensitivity towards ajoene suggesting that the activation of JNK is not necessary for ajoene-induced cell death. Inhibition of p38 MAPK by SB 203580 had no influence on ajoene-mediated apoptosis. In contrast, inhibition of ERK1/2 vastly enhanced ajoene-induced cell death. The survival kinase Akt, in contrast, did not participate in ajoene-induced death signaling as shown by the use of the phosphatidylinositol-3-kinase inhibitor wortmannin. Thus in contrast to the previous findings regarding stress-induced cell death, ajoene-mediated activation of JNK and p38 has no impact on ajoene-induced apoptosis in HL-60 cells. Blockade of ERK1/2 but not Akt pathways leads to sensitization of cells against ajoene-mediated apoptosis supporting the view that inhibition of ERK1/2 is a valuable strategy to increase the sensitivity of promyeloleukemic cells towards ajoene.

  13. Arsenic-induced mitochondrial instability leading to programmed cell death in the exposed individuals.

    PubMed

    Banerjee, Nilanjana; Banerjee, Mayukh; Ganguly, Sudipto; Bandyopadhyay, Santu; Das, Jayanta K; Bandyopadhay, Apurba; Chatterjee, Mitali; Giri, Ashok K

    2008-04-18

    In West Bengal, India, more than 6 million people in nine districts are exposed to arsenic through drinking water. It is regarded as the greatest arsenic calamity in the world. Arsenic is a well-documented human carcinogen, which does not induce cancer in any other animal model. Interestingly, at lower concentrations, arsenic is known to induce apoptosis in various cancer cell lines in vitro. We have studied apoptosis in human peripheral blood mononuclear cells (PBMC) of 30 arsenic exposed skin lesion individuals by annexin V-FITC staining and compared with 28 unexposed individuals. The percentage of apoptotic cells in individuals with skin lesions was significantly higher (p<0.001) in comparison to unexposed individuals. In the exposed individuals with skin lesions, there were elevated levels of intracellular reactive oxygen species (ROS), mitochondrial membrane permeability and increased cytochrome c release, leading to increased downstream caspase activity. Arsenic-induced DNA damage was confirmed by DNA ladder formation and confocal microscopy. We also observed that chronic arsenic exposure reduced Bcl-2/Bax ratio and also resulted in cell cycle arrest of PBMC in G0/G1 phase. All these observations indicate that mitochondria-mediated pathway may be responsible for arsenic-induced apoptosis.

  14. α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke

    PubMed Central

    Berman, Reena; Jiang, Di; Wu, Qun; Chu, Hong Wei

    2016-01-01

    Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air–liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×104 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection. PMID:27354786

  15. Endometrial stem cell transplantation in MPTP- exposed primates: an alternative cell source for treatment of Parkinson's disease.

    PubMed

    Wolff, Erin F; Mutlu, Levent; Massasa, Efi E; Elsworth, John D; Eugene Redmond, D; Taylor, Hugh S

    2015-01-01

    Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. Cell-replacement therapies have emerged as a promising strategy to slow down or replace neuronal loss. Compared to other stem cell types, endometrium-derived stem cells (EDSCs) are an attractive source of stem cells for cellular therapies because of their ease of collection and vast differentiation potential. Here we demonstrate that endometrium-derived stem cells may be transplanted into an MPTP exposed monkey model of PD. After injection into the striatum, endometrium-derived stem cells engrafted, exhibited neuron-like morphology, expressed tyrosine hydroxylase (TH) and increased the numbers of TH positive cells on the transplanted side and dopamine metabolite concentrations in vivo. Our results suggest that endometrium-derived stem cells may provide a therapeutic benefit in the primate model of PD and may be used in stem cell based therapies.

  16. Effect of photobiomodulation on endothelial cell exposed to Bothrops jararaca venom.

    PubMed

    Franco, Ana Tereza Barufi; Silva, Luciana Miato Gonçalves; Costa, Marcília Silva; Zamuner, Silvia Fernanda; Vieira, Rodolfo Paula; de Fatima Pereira Teixeira, Catarina; Zamuner, Stella Regina

    2016-07-01

    Bleeding is a common feature in envenoming caused by Bothrops snake venom due to extensive damage to capillaries and venules, producing alterations in capillary endothelial cell morphology. It has been demonstrated, in vivo, that photobiomodulation (PBM) decreases hemorrhage after venom inoculation; however, the mechanism is unknown. Thus, the objective was to investigate the effects of PBM on a murine endothelial cell line (tEnd) exposed to Bothrops jararaca venom (BjV). Cells were exposed to BjV and irradiated once with either 660- or 780-nm wavelength laser light at energy densities of 4 and 5 J/cm(2), respectively, and irradiation time of 10 s. Cell integrity was analyzed by crystal violet and cell viability/mitochondrial metabolism by MTT assay. The release of lactic dehydrogenase (LDH) was quantified as a measure of cell damage. In addition, cytokine IL1-β levels were measured in the supernatant. PBM at 660 and 780 nm wavelength was able to increase cellular viability and decrease the release of LDH and the loss of cellular integrity. In addition, the concentration of pro-inflammatory cytokine IL1-β was reduced after PBM by both wavelengths. The data reported herein indicates that irradiation with red or near-infrared laser resulted in protection on endothelial cells after exposure to Bothrops venom and could be, at least in part, a reasonable explanation by the beneficial effects of PBM inhibiting the local effects induced by Bothrops venoms, in vivo. PMID:27147074

  17. BOSS on EXPOSE-R2-Comparative Investigations on Biofilm and Planktonic cells of Deinococcus geothermalis as Mission Preparation Tests

    NASA Astrophysics Data System (ADS)

    Panitz, C.; Rettberg, P.; Frösler, J.; Flemming, H.-C.; Rabbow, E.; Reitz, G.

    2013-09-01

    Biofilms are of interest for Astrobiological investigations since they are one of the oldest clear signs of life on Earth. In the experiment BOSS the hypothesis will be tested if the biofilm form of life with microorganisms embedded and aggregated in their EPS matrix is suited to support long-term survival of microorganisms under the harsh environmental conditions as they exist in space and on Mars and is superior to the same bacteria in the form of planktonic cultures. An additional protective role may be provided by particles associated in biofilms which may shield the organisms against radiation. The experiment will be flown on EXPOSE-R2 attached outside of the ISS on the Russian module. BOSS has participated the Experiment verification tests and will attend the upcoming Science verification test carried out in the Planetary and Space Simulation Facilities at DLR. The launch is scheduled for April 2014.

  18. DNA DAMAGE IN BUCCAL EPITHELIAL CELLS FROM INDIVIDUALS CHRONICALLY EXPOSED TO ARSENIC VIA DRINKING WATER IN INNER MONGOLIA, CHINA

    EPA Science Inventory

    The purpose of this pilot study was to assess DNA damage in buccal cells from individuals chronically exposed to arsenic via drinking water in Ba Men, Inner Mongolia. Buccal cells were collected from 19 Ba Men residents exposed to arsenic at 527.5 ? 23.7 g/L (mean ? SEM) and ...

  19. Phospholipidomic Profile Variation on THP-1 Cells Exposed to Skin or Respiratory Sensitizers and Respiratory Irritant.

    PubMed

    Martins, João D; Maciel, Elisabete A; Silva, Ana; Ferreira, Isabel; Ricardo, Fernando; Domingues, Pedro; Neves, Bruno M; Domingues, Maria Rosário M; Cruz, Maria Teresa

    2016-12-01

    Occupational exposure to low molecular weight reactive chemicals often leads to development of allergic reactions such as allergic contact dermatitis and respiratory allergies. Further insights into the interaction of these chemicals with physiopathological relevant cellular models might provide the foundations for novel non-animal approaches to safety assessment. In this work we used the human THP-1 cell line to determine phospholipidome changes induced by the skin sensitizer 1-fluoro-2,4-dinitrobenzene (DNFB), the respiratory allergen hexamethylene diisocyanate (HDI), and the irritant methyl salicylate (MESA). We detected that these chemicals differently induce lipid peroxidation and modulate THP-1 IL-1β, IL-12B, IL-8, CD86, and HMOX1 transcription. Decreased phosphatidylethanolamine content was detected in cells exposed to MESA, while profound alterations in the relative abundance of cardiolipin species were observed in cells exposed to DNFB. All chemicals tested induced a decrease in the relative abundance of plasmanyl phosphatidylcholine species PC (O-16:0e/18:1) and phosphatidylinositol species PI (34:1), while increasing PI (38:4). An increased abundance of oleic acid was observed in the phospholipids of cells exposed to DNFB while a decreased abundance of palmitic acid was detected in cells treated with MESA or DNFB. We conclude that both specific and common alterations at phospholipidome levels are triggered by the different chemicals, while not allowing a complete distinction between them using a Canonical Analysis of Principal Coordinates (CAP). The common effects observed at phospholipids level with all the chemicals tested might be related to unspecific cell cytotoxic mechanisms that nevertheless may contribute to the elicitation of specific immune responses. J. Cell. Physiol. 231: 2639-2651, 2016. © 2016 Wiley Periodicals, Inc. PMID:26946329

  20. Transcriptional modulation of a human monocytic cell line exposed to PM(10) from an urban area.

    PubMed

    Bastonini, Emanuela; Verdone, Loredana; Morrone, Stefania; Santoni, Angela; Settimo, Gaetano; Marsili, Giovanni; La Fortezza, Marco; Di Mauro, Ernesto; Caserta, Micaela

    2011-08-01

    Insight into the mechanisms by which ambient air particulate matter mediates adverse health effects is needed to provide biological plausibility to epidemiological studies demonstrating an association between PM(10) exposure and increased morbidity and mortality. In vitro studies of the effects of air pollution on human cells help to establish conditions for the analysis of cause-effect relationships. One of the major challenges is to test native atmosphere in its complexity, rather than the various components individually. We have developed an in vitro system in which human monocyte-macrophage U937 cells are directly exposed to filters containing different amounts of PM(10) collected in the city of Rome. Transcriptional profiling obtained after short exposure (1h) of cells to a filter containing 1666μg PM(10) (77.6μg/cm(2)) using a macroarray panel of 1176 genes reveals a significant change in the mRNA level (>2 fold) for 87 genes relative to cells exposed to a control filter. Overall, 9 out of 87 modulated genes were annotated as "lung cancer". qRT-PCR confirmed the induction of relevant genes involved in DNA repair and apoptosis, specifically: ERCC1, TDG, DAD1 and MCL1. In cells exposed for 10min, 1h and 3h to different amounts of PM(10), transcription of TNFα and TRAP1, which code for a key pro-inflammatory cytokine and a mitochondrial protein involved in cell protection from oxidative stress, respectively, was shown to be modulated in a time-dependent, but not a dose-dependent manner. Taken together, these data indicate that it is possible to analyze the effects of untreated particulate matter on human cells by the direct-exposure approach we have developed, possibly providing new clues to traffic-related health hazard.

  1. Clear cell renal carcinoma in a pregnant DES-exposed patient.

    PubMed

    Mansi, M L

    1989-07-01

    Several decades ago, diethylstilbestrol (DES) was prescribed to support the pregnancy of women who were diabetic, who had had consecutive abortions, or who were threatening to abort. The use of this estrogen substitute to support human gestation had ceased by the 1960s. In 1971, the first report was published in which DES exposure was linked with clear cell carcinoma of the vagina and cervix. Since then, many other documentations have been published on upper genital tract anomalies, poor reproductive performance, and the high incidence of fetal wastage in DES-exposed women. The author describes a case of clear cell carcinoma of the kidney in an 18-year-old pregnant woman with a prior history of vaginal adenosis who had been exposed to DES in utero.

  2. Mutagenicity and genotoxicity in gill erythrocyte cells of Poecilia reticulata exposed to a glyphosate formulation.

    PubMed

    De Souza Filho, José; Sousa, Caio César Neves; Da Silva, Cláudio Carlos; De Sabóia-Morais, Simone Maria Teixeira; Grisolia, Cesar Koppe

    2013-11-01

    Poecilia reticulata were exposed to herbicide Roundup Transorb(®) for micronucleus test, nuclear abnormalities and comet assay. The exposure-concentrations were based on CL50-96 h following 0, 1.41, 2.83, 4.24 and 5.65 μL L(-1) for 24 h. Micronucleus and comets were significantly increased in the gill erythrocyte cells after herbicide exposure compared with the non-exposed group. Results showed a gradual increase in the number of damaged cells, indicating a concentration-dependent effect and that this herbicide was mutagenic and genotoxic to P. reticulata and this effect could be attributed to a combination of compounds contained in the formulation with the active ingredient glyphosate. PMID:24042842

  3. Structural and function changes in organelles of liver cells in rats exposed to magnetic fields

    SciTech Connect

    Gorczynska, E. ); Wegrzynowicz, R. )

    1991-08-01

    Exposure of rats to magnetic fields of 10{sup {minus}3} and 10{sup {minus}2} T for 1 hr daily generated structural changes in hepatocytes mitochondria, endoplasmic reticulum, and ribosomes. Simultaneously there was an increase in the activities of the mitochondrial respiratory enzymes: NADH dehydrogenase, succinic dehydrogenase, and cytochrome oxidase. The extent of the changes in liver cell properties following exposure depend on the duration of exposure to and the strength of the applied magnetic fields. Ultrastructural studies did not reveal any changes in external membranes of hepatocytes or in the membranes of cell nuclei. An increase in the amount of glycogen in hepatocytes of rats exposed to both 10{sup {minus}3} and 10{sup {minus}2} T was noted. The high level of cortisol in serum of exposed rats suggests that magnetic field may be a stress generating factor.

  4. Natural killer cells in highly exposed hepatitis C-seronegative injecting drug users.

    PubMed

    Mina, M M; Cameron, B; Luciani, F; Vollmer-Conna, U; Lloyd, A R

    2016-06-01

    Injecting drug use remains the major risk factor for hepatitis C (HCV) transmission. A minority of long-term injecting drug users remain seronegative and aviraemic, despite prolonged exposure to HCV - termed highly exposed seronegative subjects. Natural killer (NK) cells have been implicated in this apparent protection. A longitudinal nested, three group case-control series of subjects was selected from a prospective cohort of seronegative injecting drug users who became incident cases (n = 11), remained seronegative (n = 11) or reported transient high-risk behaviour and remained uninfected (n = 11). The groups were matched by age, sex and initial risk behaviour characteristics. Stored peripheral blood mononuclear cells were assayed in multicolour flow cytometry to enumerate natural killer cell subpopulations and to assess functional activity using Toll-like receptor ligands before measurement of activation, cytokine production and natural cytotoxicity receptor expression. Principal components were derived to describe the detailed phenotypic characteristics of the major NK subpopulations (based on CD56 and CD16 co-expression), before logistic regression analysis to identify associations with exposed, seronegative individuals. The CD56(dim) CD16(+) (P = 0.05, OR 6.92) and CD56(dim) CD16(-) (P = 0.05, OR 6.07) principal components differed between exposed, seronegative individuals and pre-infection samples of the other two groups. These included CD56(dim) CD16(+) and CD56(dim) CD16(-) subsets with CD56(dim) CD16(+) IFN-γ and TNF-α on unstimulated cells, and CD56(dim) CD16(-) CD69(+) , CD107a(+) , IFN-γ and TNF-α following TLR stimulation. The cytotoxic CD56(dim) NK subset thus distinguished highly exposed, seronegative subjects, suggesting NK cytotoxicity may contribute to protection from HCV acquisition. Further investigation of the determinants of this association and prospective assessment of protection against HCV infection are warranted.

  5. 3-O-(E)-p-coumaroyl tormentic acid from Eriobotrya japonica leaves induces caspase-dependent apoptotic cell death in human leukemia cell line.

    PubMed

    Kikuchi, Takashi; Akazawa, Hiroyuki; Tabata, Keiichi; Manosroi, Aranya; Manosroi, Jiradej; Suzuki, Takashi; Akihisa, Toshihiro

    2011-01-01

    Eleven triterpene acids, 1-11, isolated from the leaves of Eriobotrya japonica, were evaluated for inhibition of DNA topoisomerase (Topo) I and cytotoxicity against human leukemia (HL60) and melanoma cell lines (CRL1579). Among the compounds tested, four compounds, δ-oleanolic acid (4), ursolic acid (5), 3-O-(E)-p-coumaroyl tormentic acid (8), and betulinic acid (10), exhibited potent Topo I inhibitory activity (IC(50) 20.3-36.5 µM) and cytotoxicity against HL60 (EC(50) 5.0-8.1 µM). Upon assessing the apoptosis-inducing activity in HL60 cells, compound 8 exhibited induction of apoptosis detected by the observation of DNA fragmentation and membrane phospholipid exposure in flow cytometry. Western blot analysis showed that compound 8 markedly reduced the levels of procaspases-3 and 9, while being increased the levels of cleaved caspases-3 and 9. On the other hand, compound 8 exerted almost no influence on the expression of caspase-8. In addition, compound 8 increased significantly Bax/Bcl-2 ratio and activated caspase-2. These results suggested that compound 8 induced apoptotic cell death in HL60 via mainly mitochondrial pathway by, at least in part, Topo I inhibition. Therefore, compound 8 may be promising lead compound for developing an effective drug for treatment of leukemia.

  6. Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

    NASA Technical Reports Server (NTRS)

    Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

  7. Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    PubMed Central

    2009-01-01

    Background Aspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response. Results The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to A. fumigatus was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1β antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during A. fumigatus defensin induction. Finally, induced defensin expression in

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

    PubMed

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

    2013-11-01

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

  9. Effects of selenocystine on lead-exposed Chinese hamster ovary (CHO) and PC-12 cells

    SciTech Connect

    Aykin-Burns, Nukhet; Ercal, Nuran . E-mail: nercal@umr.edu

    2006-07-15

    Lead is a pervasive environmental toxin that affects multiple organ systems, including the nervous, renal, reproductive, and hematological systems. Even though it is probably the most studied toxic metal, some of the symptoms of lead toxicity still cannot be explained by known molecular mechanisms. Therefore, lead-induced oxidative stress has recently started to gain attention. This in vitro study confirms the existence of oxidative stress due to lead exposure. Administration of lead acetate (PbA) to cultures of Chinese hamster ovary cells (CHO) had a concentration-dependent inhibitory effect on colony formation and cell proliferation. This inhibition was eliminated by 5 {mu}M selenocystine (SeCys). In order to evaluate the nature of SeCys's effect, we measured glutathione (GSH), its oxidized form glutathione disulfide (GSSG), malondialdehyde (MDA), catalase, and GSH peroxidase (GPx) activities in lead-exposed CHO cells both in the presence and absence of SeCys. Increases in MDA, catalase, and GPx activities were observed in cultures that received only PbA, but supplementation with SeCys returned these measures to pretreatment levels. The ratio of GSH to GSSG increased in lead-exposed cells incubated in SeCys-enhanced media but declined in cultures treated with PbA only. In order to determine whether SeCys also reverses lead-induced neurotoxicity, a neuronal cell line, PC-12 cells, was used. Lead's inhibition on neurite formation was significantly eliminated by SeCys in PC-12 cells. Our results suggest that SeCys can confer protection against lead-induced toxicity in CHO cells and neurotoxicity in PC-12 cells.

  10. Expression of AS3MT alters transcriptional profiles in human urothelial cells exposed to arsenite.

    PubMed

    Hester, Sd; Drobná, Z; Andrews, Dmk; Liu, J; Waalkes, Mp; Thomas, Dj; Styblo, M

    2009-01-01

    Inorganic arsenic (iAs) is an environmental toxicant and human carcinogen. The enzymatic methylation of iAs that is catalyzed by arsenic (+3 oxidation state)-methyltransferase (AS3MT) generates reactive methylated intermediates that contribute to the toxic and carcinogenic effects of iAs. We have shown that clonal human urothelial cells (UROtsa/F35) that express rat AS3MT and methylate iAs are more susceptible to acute toxicity of arsenite (iAs(III)) than parental UROtsa cells that do not express AS3MT and do not methylate iAs. The current work examines transcriptional changes associated with AS3MT expression and identifies specific categories of genes expressed in UROtsa and UROtsa/F35 cells in response to a 24-h exposure to 1 or 50 microM iAs(III). Here, the expression of 21,073 genes was assessed using Agilent Human 1A(V2) arrays. Venn analysis showed marked concentration-dependent differences between gene expression patterns in UROtsa and UROTsa/F35 cells exposed to iAs(III). Among 134 genes altered by exposure to subtoxic 1 microM iAs(III), only 14 were shared by both cell lines. Exposure to cytotoxic 50 microM iAs(III) uniquely altered 1389 genes in UROtsa/F35 and 649 genes in UROtsa cells; 5033 altered genes were associated with the chemical alone. In UROtsa, but not UROtsa/F35 cells exposure to 1 microM iAs(III) altered expression of genes associated with cell adhesion. In contrast, expression of genes involved in cell cycle regulation was significantly altered in UROtsa/F35 cells at this exposure level. At 50 microM iAs(III), pathways regulating cell cycle, cell death, transcription, and metabolism were affected in both cell lines. However, only Urotsa/F35 cells showed numerous G-protein and kinase pathway alterations as well as alterations in pathways involved in cell growth and differentiation. These data link the AS3MT-catalyzed methylation of iAs to specific genomic responses in human cells exposed to iAs(III). Further analysis of these responses will

  11. Identification of gene-based responses in human blood cells exposed to alpha particle radiation

    PubMed Central

    2014-01-01

    Background The threat of a terrorist-precipitated nuclear event places humans at danger for radiological exposures. Isotopes which emit alpha (α)-particle radiation pose the highest risk. Currently, gene expression signatures are being developed for radiation biodosimetry and triage with respect to ionizing photon radiation. This study was designed to determine if similar gene expression profiles are obtained after exposures involving α-particles. Methods Peripheral blood mononuclear cells (PBMCs) were used to identify sensitive and robust gene-based biomarkers of α-particle radiation exposure. Cells were isolated from healthy individuals and were irradiated at doses ranging from 0-1.5 Gy. Microarray technology was employed to identify transcripts that were differentially expressed relative to unirradiated cells 24 hours post-exposure. Statistical analysis identified modulated genes at each of the individual doses. Results Twenty-nine genes were common to all doses with expression levels ranging from 2-10 fold relative to control treatment group. This subset of genes was further assessed in independent complete white blood cell (WBC) populations exposed to either α-particles or X-rays using quantitative real-time PCR. This 29 gene panel was responsive in the α-particle exposed WBCs and was shown to exhibit differential fold-changes compared to X-irradiated cells, though no α-particle specific transcripts were identified. Conclusion Current gene panels for photon radiation may also be applicable for use in α-particle radiation biodosimetry. PMID:25017500

  12. In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells.

    PubMed

    Zar, Phyu Phyu Khine; Yano, Satoshi; Sakao, Kozue; Hashimoto, Fumio; Nakano, Takayuki; Fujii, Makoto; Hou, De-Xing

    2014-01-01

    Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.

  13. Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers.

    PubMed

    González-Robles, Arturo; Castañón, Guadalupe; Cristóbal-Ramos, Ana Ruth; Hernández-Ramírez, Verónica Ivonne; Omaña-Molina, Maritza; Martínez-Palomo, Adolfo

    2007-12-01

    Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.

  14. Phenotypic Modifications in Staphylococcus aureus Cells Exposed to High Concentrations of Vancomycin and Teicoplanin

    PubMed Central

    Gonçalves, Fábio D. A.; de Carvalho, Carla C. C. R.

    2016-01-01

    Bacterial cells are known to change the fatty acid (FA) composition of the phospholipids as a phenotypic response to environmental conditions and to the presence of toxic compounds such as antibiotics. In the present study, Staphylococcus aureus cells collected during the exponential growth phase were challenged with 50 and 100 mg/L of vancomycin and teicoplanin, which are concentrations high enough to kill the large majority of the cell population. Colony-forming unit counts showed biphasic killing kinetics, typical for persister cell enrichment, in both antibiotics and concentrations tested. However, fluorescence microscopy showed the existence of viable but non-culturable (VBNC) cells in a larger number than that of possible persister cells. The analysis of the FA composition of the cells showed that, following antibiotic exposure up to 6 h, the survivor cells have an increased percentage of saturated FAs, a significant reduced percentage of branched FAs and an increased iso/anteiso branched FA ratio when compared to cells exhibiting a regular phenotype. This should result in lower membrane fluidity. However, cells exposed for 8–24 h presented an increased branched/saturated and lower iso/anteiso branched FA ratios, and thus increased membrane fluidity. Furthermore, the phenotypic changes were transmitted to daughter cells grown in drug-free media. The fact that VBNC cells presented nearly the same FA composition as those obtained after cell growth in drug-free media, which could only be the result of growth of persister cells, suggest that VBNC and persister phenotypes share the same type of response to antibiotics at the lipid level. PMID:26834731

  15. Phenotypic Modifications in Staphylococcus aureus Cells Exposed to High Concentrations of Vancomycin and Teicoplanin.

    PubMed

    Gonçalves, Fábio D A; de Carvalho, Carla C C R

    2016-01-01

    Bacterial cells are known to change the fatty acid (FA) composition of the phospholipids as a phenotypic response to environmental conditions and to the presence of toxic compounds such as antibiotics. In the present study, Staphylococcus aureus cells collected during the exponential growth phase were challenged with 50 and 100 mg/L of vancomycin and teicoplanin, which are concentrations high enough to kill the large majority of the cell population. Colony-forming unit counts showed biphasic killing kinetics, typical for persister cell enrichment, in both antibiotics and concentrations tested. However, fluorescence microscopy showed the existence of viable but non-culturable (VBNC) cells in a larger number than that of possible persister cells. The analysis of the FA composition of the cells showed that, following antibiotic exposure up to 6 h, the survivor cells have an increased percentage of saturated FAs, a significant reduced percentage of branched FAs and an increased iso/anteiso branched FA ratio when compared to cells exhibiting a regular phenotype. This should result in lower membrane fluidity. However, cells exposed for 8-24 h presented an increased branched/saturated and lower iso/anteiso branched FA ratios, and thus increased membrane fluidity. Furthermore, the phenotypic changes were transmitted to daughter cells grown in drug-free media. The fact that VBNC cells presented nearly the same FA composition as those obtained after cell growth in drug-free media, which could only be the result of growth of persister cells, suggest that VBNC and persister phenotypes share the same type of response to antibiotics at the lipid level. PMID:26834731

  16. Adaptive responses and apoptosis in endothelial cells exposed to carbon monoxide

    PubMed Central

    Thom, Stephen R.; Fisher, Donald; Xu, Y. Anne; Notarfrancesco, Kathy; Ischiropoulos, Harry

    2000-01-01

    Prior studies have shown that exposure to carbon monoxide (CO) will elevate the steady-state concentration of nitric oxide (⋅NO) in several cell types and body organs and that some toxic effects of CO are directed toward endothelial cells. Studies reported in this paper were conducted with bovine pulmonary artery endothelial cells exposed to 10 to 100 ppm CO to achieve concentrations between 11 and 110 nM in air-saturated buffer. Exposure to 11 nM CO increased synthesis of manganous superoxide dismutase and conferred resistance against the lethal effects of 110 nM CO. At concentrations of 88 nM CO or more, exposures for 1 h or longer caused cell death that became apparent 18 h after the exposure ceased. Caspase-1 was activated in response to CO, and cell death was inhibited by a caspase-1 inhibitor. Alteration of proteolytic pathways by CO was indicated by the presence of ubiquitin-containing intracellular inclusion bodies. Morphological changes and caspase activation indicated that cell death was an apoptotic process. Cells exposed to 110 nM CO had higher concentrations of manganous superoxide dismutase and heme oxygenase-1 but no changes in glutathione peroxidase, glucose-6-phosphate dehydrogenase, thiols, or catalase. Elevated levels of antioxidant enzymes and apoptosis were inhibited by the nitric oxide synthase inhibitor, S-isopropylisothiourea, and the peroxynitrite scavenger, selenomethionine. These results show that biochemical effects of CO occur at environmentally relevant concentrations, that apoptotic cell death follows exposure to relatively high concentrations of CO, and that these actions of CO are mediated by nitric oxide. PMID:10655526

  17. Assessment of DNA integrity (COMET assay) in sperm cells of boron-exposed workers.

    PubMed

    Duydu, Yalçin; Başaran, Nurşen; Ustündağ, Aylin; Aydin, Sevtap; Undeğer, Ulkü; Ataman, Osman Yavuz; Aydos, Kaan; Düker, Yalçin; Ickstadt, Katja; Waltrup, Britta Schulze; Golka, Klaus; Bolt, Hermann M

    2012-01-01

    An extension of a male reproductive study conducted in a boric acid/borate production zone at Bandırma, Turkey, is presented. The relation between DNA-strand breaks (COMET assay, neutral and alkaline version) in sperm cells and previously described sperm quality parameters was investigated in boron-exposed males. A correlation between blood boron levels and mean DNA-strand breaks in sperm was weak, and DNA-strand breaks in sperm were statistically not different between control and exposed groups. Therefore, increasing boron exposures had no additional contribution in addition to already pre-existing DNA-strand breaks in the sperm cells. Weak but statistically significant correlations between DNA-strand breaks and motility/morphology parameters of sperm samples were observed in the neutral version of the COMET assay, while correlations between the same variables were statistically not significant in the alkaline version. A likely reason for these negative results, even in highly exposed humans, is that experimental exposures that had led to reproductive toxicity in animals were significantly higher than any boron exposures, which may be reached under realistic human conditions.

  18. Chromosomal Aberrations in Normal and AT Cells Exposed to High Dose of Low Dose Rate Irradiation

    NASA Technical Reports Server (NTRS)

    Kawata, T.; Shigematsu, N.; Kawaguchi, O.; Liu, C.; Furusawa, Y.; Hirayama, R.; George, K.; Cucinotta, F.

    2011-01-01

    Ataxia telangiectasia (A-T) is a human autosomally recessive syndrome characterized by cerebellar ataxia, telangiectases, immune dysfunction, and genomic instability, and high rate of cancer incidence. A-T cell lines are abnormally sensitive to agents that induce DNA double strand breaks, including ionizing radiation. The diverse clinical features in individuals affected by A-T and the complex cellular phenotypes are all linked to the functional inactivation of a single gene (AT mutated). It is well known that cells deficient in ATM show increased yields of both simple and complex chromosomal aberrations after high-dose-rate irradiation, but, less is known on how cells respond to low-dose-rate irradiation. It has been shown that AT cells contain a large number of unrejoined breaks after both low-dose-rate irradiation and high-dose-rate irradiation, however sensitivity for chromosomal aberrations at low-dose-rate are less often studied. To study how AT cells respond to low-dose-rate irradiation, we exposed confluent normal and AT fibroblast cells to up to 3 Gy of gamma-irradiation at a dose rate of 0.5 Gy/day and analyzed chromosomal aberrations in G0 using fusion PCC (Premature Chromosomal Condensation) technique. Giemsa staining showed that 1 Gy induces around 0.36 unrejoined fragments per cell in normal cells and around 1.35 fragments in AT cells, whereas 3Gy induces around 0.65 fragments in normal cells and around 3.3 fragments in AT cells. This result indicates that AT cells can rejoin breaks less effectively in G0 phase of the cell cycle? compared to normal cells. We also analyzed chromosomal exchanges in normal and AT cells after exposure to 3 Gy of low-dose-rate rays using a combination of G0 PCC and FISH techniques. Misrejoining was detected in the AT cells only? When cells irradiated with 3 Gy were subcultured and G2 chromosomal aberrations were analyzed using calyculin-A induced PCC technique, the yield of unrejoined breaks decreased in both normal and AT

  19. Induction of apoptosis in human leukemia cells by naturally fermented sugar cane vinegar (kibizu) of Amami Ohshima Island.

    PubMed

    Mimura, Akio; Suzuki, Yoshihiro; Toshima, Youhei; Yazaki, Shin-ichi; Ohtsuki, Takashi; Ui, Sadaharu; Hyodoh, Fuminori

    2004-01-01

    Naturally fermented vinegar such as Kibizu (sugar cane vinegar in Amami Ohshima, Japan), Kurozu (black rice vinegar in Kagoshima, Japan), Kouzu (black rice vinegar in China) and red wine vinegar in Italy had potent radical-scavenging activity analyzed by DPPH method. For the elucidation of food factor for cancer prevention contained in naturally fermented vinegar, the induction of apoptosis in human leukemia cell HL-60 was investigated with sugar cane vinegar Kibizu. Fraction eluted by 40% methanol from Amberlite XAD 2 chromatography of sugar cane vinegar showed potent radical scavenging activity. The fraction also showed the activity repressing growth of typical human leukemia cells such as HL-60, THP-1, Molt-4, U-937, Jurkat, Raji and K-562. On the other hand, the fraction did not have any growth inhibition activity against human fetal lung cell TIG-1. The most potent radical-scavenging activity and the growth repression activity of the leukemia cell were observed in the same chromatographic fraction of methanol 40%. From cell sorting FACS analyses, electron microscopic observations and cytochemical staining of chromatin and nuclear segments in human leukemia cell HL-60 treated with the active fraction, it was concluded that apoptosis was induced in the leukemia cell by the fraction of sugar cane vinegar and resulted in the repression of growth of the human leukemia cells. Chromatographic fraction of sugar cane juice eluted by 20% methanol showed potent activities of radical-scavenging and growth repression of HL-60. These results led us the consideration that active components in sugar cane juice could be converted to more lipophilic compounds with activity to induce apoptosis in HL-60 by microbial fermentation with yeast and acetic acid bacteria.

  20. Competitive binding of pentraxins and IgM to newly exposed epitopes on late apoptotic cells.

    PubMed

    Ciurana, Caroline L F; Hack, C Erik

    2006-01-01

    A random distribution of phospholipids among the inner and outer leaflet of the cell membrane occurs during apoptosis and is known as membrane flip-flop. Flip-flopped cells have binding sites for various plasma proteins, such as IgM and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP). In this study, we investigated whether pentraxins and IgM antibodies recognize the same binding sites on apoptotic cells, and whether phospholipids constitute these binding sites. Except for SAP which also bound to early apoptotic cells, pentraxins and IgM preferentially bound to late apoptotic cells. Competition experiments with different phosphatemonoesters revealed that CRP and SAP as well as part of the IgM bound to the phospholipids head groups, SAP mainly to phosphorylethanolamine, CRP to phosphorylcholine and phosphorylethanolamine and to a lesser extent to phosphorylserine, and IgM to phosphorylcholine and phosphorylserine. These results were confirmed in experiments in which proteins were adsorbed from plasma with artificial phospholipids particles. IgM and the pentraxins variably competed for the same binding sites on late apoptotic cells, SAP having the highest and CRP the lowest apparent affinity. We conclude that CRP, SAP, and part of the IgM bind to the phospholipid head groups exposed on apoptotic cells. This shared specificity as well as their shared capability to activate complement, suggest that IgM and the pentraxins CRP and SAP exert similar functions in the removal of apoptotic cells.

  1. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    PubMed Central

    2011-01-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis. PMID:27502666

  2. Cytoskeleton Modifications and Autophagy Induction in TCam-2 Seminoma Cells Exposed to Simulated Microgravity

    PubMed Central

    Ferranti, Francesca; Caruso, Maria; Cammarota, Marcella; Fabrizi, Cinzia; Fumagalli, Lorenzo; Schiraldi, Chiara; Catizone, Angela

    2014-01-01

    The study of how mechanical forces may influence cell behavior via cytoskeleton remodeling is a relevant challenge of nowadays that may allow us to define the relationship between mechanics and biochemistry and to address the larger problem of biological complexity. An increasing amount of literature data reported that microgravity condition alters cell architecture as a consequence of cytoskeleton structure modifications. Herein, we are reporting the morphological, cytoskeletal, and behavioral modifications due to the exposition of a seminoma cell line (TCam-2) to simulated microgravity. Even if no differences in cell proliferation and apoptosis were observed after 24 hours of exposure to simulated microgravity, scanning electron microscopy (SEM) analysis revealed that the change of gravity vector significantly affects TCam-2 cell surface morphological appearance. Consistent with this observation, we found that microtubule orientation is altered by microgravity. Moreover, the confocal analysis of actin microfilaments revealed an increase in the cell width induced by the low gravitational force. Microtubules and microfilaments have been related to autophagy modulation and, interestingly, we found a significant autophagic induction in TCam-2 cells exposed to simulated microgravity. This observation is of relevant interest because it shows, for the first time, TCam-2 cell autophagy as a biological response induced by a mechanical stimulus instead of a biochemical one. PMID:25140323

  3. Insights on cryoprotectant toxicity from gene expression profiling of endothelial cells exposed to ethylene glycol.

    PubMed

    Cordeiro, Rui Martins; Stirling, Soren; Fahy, Gregory M; de Magalhães, João Pedro

    2015-12-01

    Cryopreservation consists of preserving living cells or tissues generally at -80 °C or below and has many current applications in cell and tissue banking, and future potential for organ banking. Cryoprotective agents such as ethylene glycol (EG) are required for successful cryopreservation of most living systems, but have toxic side effects whose mechanisms remain largely unknown. In this work, we investigated the mechanisms of toxicity of ethylene glycol in human umbilical vein endothelial cells (HUVECs) as a model of the vascular endothelium in perfused organs. Exposing cells to 60% v/v EG for 2 h at 4 °C resulted in only a slight decrease in subsequent cell growth, suggesting only modest toxicity of EG for this cell type. Gene expression analysis with whole genome microarrays revealed signatures indicative of a generalized stress response at 24 h after EG exposure and a trend toward partial recovery at 72 h. The observed changes involved signalling pathways, glycoproteins, and genes involved in extracellular and transmembrane functions, the latter suggesting potential effects of ethylene glycol on membranes. These results continue to develop a new paradigm for understanding cryoprotectant toxicity and reveal molecular signatures helpful for future experiments in more completely elucidating the toxic effects of ethylene glycol in vascular endothelial cells and other cell types. PMID:26471925

  4. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    NASA Astrophysics Data System (ADS)

    Yuan, Hengguang; Hu, Shanglian; Huang, Peng; Song, Hua; Wang, Kan; Ruan, Jing; He, Rong; Cui, Daxiang

    2011-12-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  5. Cytoskeleton modifications and autophagy induction in TCam-2 seminoma cells exposed to simulated microgravity.

    PubMed

    Ferranti, Francesca; Caruso, Maria; Cammarota, Marcella; Masiello, Maria Grazia; Corano Scheri, Katia; Fabrizi, Cinzia; Fumagalli, Lorenzo; Schiraldi, Chiara; Cucina, Alessandra; Catizone, Angela; Ricci, Giulia

    2014-01-01

    The study of how mechanical forces may influence cell behavior via cytoskeleton remodeling is a relevant challenge of nowadays that may allow us to define the relationship between mechanics and biochemistry and to address the larger problem of biological complexity. An increasing amount of literature data reported that microgravity condition alters cell architecture as a consequence of cytoskeleton structure modifications. Herein, we are reporting the morphological, cytoskeletal, and behavioral modifications due to the exposition of a seminoma cell line (TCam-2) to simulated microgravity. Even if no differences in cell proliferation and apoptosis were observed after 24 hours of exposure to simulated microgravity, scanning electron microscopy (SEM) analysis revealed that the change of gravity vector significantly affects TCam-2 cell surface morphological appearance. Consistent with this observation, we found that microtubule orientation is altered by microgravity. Moreover, the confocal analysis of actin microfilaments revealed an increase in the cell width induced by the low gravitational force. Microtubules and microfilaments have been related to autophagy modulation and, interestingly, we found a significant autophagic induction in TCam-2 cells exposed to simulated microgravity. This observation is of relevant interest because it shows, for the first time, TCam-2 cell autophagy as a biological response induced by a mechanical stimulus instead of a biochemical one.

  6. Apoptotic effect of a novel kefir product, PFT, on multidrug-resistant myeloid leukemia cells via a hole-piercing mechanism

    PubMed Central

    GHONEUM, MAMDOOH; GIMZEWSKI, JAMES

    2014-01-01

    We examined the apoptotic effect of a novel Probiotics Fermentation Technology (PFT) kefir grain product; PFT is a natural mixture composed primarily of Lactobacillus kefiri P-IF, a specific strain of L. kefiri with unique growth characteristics. The aim of this study was to examine the apoptotic effect of PFT on human multidrug-resistant (MDR) myeloid leukemia (HL60/AR) cells in vitro and explore the mechanistic approach underlying its effect. HL60/AR cells were cultured with PFT (0.6–5.0 mg/ml) for 3 days. The apoptotic effect of PFT was assessed through examination of percent apoptosis, caspase 3 activation, Bcl-2 expression levels and changes in mitochondrial membrane potential (MMP). PFT induced apoptosis in HL60/AR cells in a dose-dependent manner which was maximal at 67.5% for 5 mg/ml. Induction of apoptosis was associated with activation of caspase 3, decreased expression of Bcl-2 and decreased polarization of MMP. In addition, PFT showed a unique characteristic of piercing holes in HL60/AR cells, as indicated by AFM studies. This hole induction may be responsible for the apoptotic effect on cancer cells. These results suggest that PFT may act as a potential therapy for the treatment of MDR leukemia. PMID:24430613

  7. Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays.

    PubMed

    Noguchi, M; Kanari, Y; Yokoya, A; Narita, A; Fujii, K

    2015-09-01

    Morphological changes in mitochondria induced by X-irradiation in normal murine mammary gland cells were studied with a live-cell microscopic imaging technique. Mitochondria were visualised by staining with a specific fluorescent probe in the cells, which express fluorescent ubiquitination-based cell-cycle indicator 2 (Fucci2) probes to visualise cell cycle. In unirradiated cells, the number of cells with fragmented mitochondria was about 20 % of the total cells through observation period (96 h). In irradiated cells, the population with fragmented mitochondria significantly increased depending on the absorbed dose. Particularly, for 8 Gy irradiation, the accumulation of fragmentation persists even in the cells whose cell cycle came to a stand (80 % in G1 (G0-like) phase). The fraction reached to a maximum at 96 h after irradiation. The kinetics of the fraction with fragmented mitochondria was similar to that for cells in S/G2/M phase (20 %) through the observation period (120 h). The evidences show that, in irradiated cells, some signals are continually released from a nucleus or cytoplasm even in the G0-like cells to operate some sort of protein machineries involved in mitochondrial fission. It is inferred that this delayed mitochondrial fragmentation is strongly related to their dysfunction, and hence might modulate radiobiological effects such as mutation or cell death.

  8. The experiment on the protein exposed to acoustic wave in model of hair cells.

    PubMed

    Takeda, H

    1976-01-01

    Why do animals have intense troubles mainly in the basal turns of their cochleae by exposure to sound and the ototoxity? I thought that the protein in cells had an important relation to this subject. So I performed this experiment. I made model of hair cells of silicon and poured three kinds of solutions of protein into them. And I checked the ionization of the protein and its denaturation by giving strong energy of oscillation to them like the same idea of photoelectric effect. As the result, as for the protein liquid, I noticed the increase of volts just after it was exposed to sound and about 3 hours later, I observed the figure decreased gradually. From this phenomenon, I presumed that the protein in sensory cells became a factor of the stimulation by receiving strong energy conversely. Low-percent protein liquid had less effect caused by sound. PMID:7097

  9. QSAR model for predicting cell viability of human embryonic kidney cells exposed to SiO₂ nanoparticles.

    PubMed

    Manganelli, Serena; Leone, Caterina; Toropov, Andrey A; Toropova, Alla P; Benfenati, Emilio

    2016-02-01

    A predictive model for the viability (%) of cultured human embryonic kidney cells (HEK293) exposed to 20 and 50 nm silica nanoparticles was built using 'optimal descriptors' as mathematical functions of size, concentration and exposure time. The calculation was carried out with CORAL software (http://www.insilico.eu/coral/) on five random splits of combined systems (particle size-particle concentration-cell exposure time) into training, calibration, and validation sets. The R(2) values of the best models were above 0.68. The average statistical quality of the model for the viability (%) of HEK293 exposed to different concentrations of silica nanoparticles measured by MTT assay is satisfactory. PMID:26439516

  10. Chromosome Aberrations in Normal and Ataxia-Telangiectasia Cells Exposed to Heavy Ions

    NASA Technical Reports Server (NTRS)

    Kawata, T.; Ito, H.; Liu, C.; Shigematsu, N.; George, K.; Cucinotta, F. A.

    2007-01-01

    Although cells derived from Ataxia Telangiectasia (AT) patients are known to exhibit abnormal responses to ionizing radiations, its underlying mechanism still remains unclear. Previously, the authors reported that at the same gamma-irradiation dose AT cells show higher frequencies of misrepair and deletions compared to normal human fibroblast cells. In this study, we investigated the effects of heavy ions beams on chromosomal aberrations in normal and AT cells. Normal and AT fibroblast cells arrested at G0/G1 phase were irradiated with 2 Gy of X-rays, 490 MeV/u Silicon (LET 55 keV/m), 500 MeV/u Iron (LET 185 keV/m) and 200 MeV/u Iron (LET 440 keV/m) particles, and then cells were allowed to repair for 24 hours at 37 degrees before subculture. Calyculin-A induced PCC method was employed to collect G2/M chromosomes and whole DNA probes 1 and 3 were used to analyze chromosomal aberrations such as color-junctions, deletions, simple exchanges (incomplete and reciprocal exanges) and complex-type exchanges. The percentages of aberrant cells were higher when normal and AT cells were exposed to heavy ions compared to X-rays, and had a tendency to increase with increasing LET up to 185 keV/m and then decreased at 440 keV/m. When the frequency of color-junctions per cell was compared after X-ray exposure, AT cells had around three times higher frequency of color-junctions (mis-rejoining) than normal cells. However, at 185 keV/m there was no difference in the frequency of color-junctions between two cell lines. It was also found that the frequency of simple exchanges per cell was almost constant in AT cells regardless LET levels, but it was LET dependent for normal cells. Interestingly, the frequency of simple exchanges was higher for AT cells when it was compared at 185 keV/m but AT cells had more complex-type exchanges at the same LET levels. Heavy ions are more efficient in inducing chromosome aberrations in normal and AT cells compared to X-rays, and the aberration types

  11. Fate of D3 mouse embryonic stem cells exposed to X-rays or carbon ions.

    PubMed

    Luft, S; Pignalosa, D; Nasonova, E; Arrizabalaga, O; Helm, A; Durante, M; Ritter, S

    2014-01-15

    The risk of radiation exposure during embryonic development is still a major problem in radiotoxicology. In this study we investigated the response of the murine embryonic stem cell (mESC) line D3 to two radiation qualities: sparsely ionizing X-rays and densely ionizing carbon ions. We analyzed clonogenic cell survival, proliferation, induction of chromosome aberrations as well as the capability of cells to differentiate to beating cardiomyocytes up to 3 days after exposure. Our results show that, for all endpoints investigated, carbon ions are more effective than X-rays at the same radiation dose. Additionally, in long term studies (≥8 days post-irradiation) chromosomal damage and the pluripotency state were investigated. These studies reveal that pluripotency markers are present in the progeny of cells surviving the exposure to both radiation types. However, only in the progeny of X-ray exposed cells the aberration frequency was comparable to that of the control population, while the progeny of carbon ion irradiated cells harbored significantly more aberrations than the control, generally translocations. We conclude that cells surviving the radiation exposure maintain pluripotency but may carry stable chromosomal rearrangements after densely ionizing radiation.

  12. Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

    NASA Astrophysics Data System (ADS)

    Liu, Jianguo; Zhang, Xiaoli; Sun, Yanhong; Lin, Wei

    2010-01-01

    The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O{2/-}). The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H. pluvialis during exposure to reactive oxygen species (ROS) such as O{2/-}. Astaxanthin reacted with ROS much faster than did the protective enzymes, and had the strongest antioxidative capacity to protect against lipid peroxidation. The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells. Astaxanthin-enriched red cells had the strongest antioxidative capacity, followed by brown cells, and astaxanthin-deficient green cells. Although there was no significant increase in expression of protective enzymes, the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin, which quenched O{2/-} before the protective enzymes could act. In green cells, astaxanthin is very low or absent; therefore, scavenging of ROS is inevitably reliant on antioxidative enzymes. Accordingly, in green cells, these enzymes play the leading role in scavenging ROS, and the expression of these enzymes is rapidly increased to reduce excessive ROS. However, because ROS were constantly increased in this study, the enhance enzyme activity in the green cells was not able to repair the ROS damage, leading to elevated MDA content. Of the four defensive enzymes measured in astaxanthin-deficient green cells, SOD eliminates O{2/-}, POD eliminates H2O2, which is a by-product of SOD activity, and APX and CAT are then initiated to scavenge excessive ROS.

  13. Radiation-Induced Bystander Effects in A549 Cells Exposed to 6 MV X-rays.

    PubMed

    Yang, Shuning; Xu, Jing; Shao, Weixian; Geng, Chong; Li, Jia; Guo, Feng; Miao, Hui; Shen, Wenbin; Ye, Tao; Liu, Yazhou; Xu, Haiting; Zhang, Xuguang

    2015-07-01

    The aim of the study is to explore the bystander effects in A549 cells that have been exposed to 6MV X-ray. Control group, irradiated group, irradiated conditioned medium (ICM)-received group, and fresh medium group were designed in this study. A549 cells in the logarithmic growth phase were irradiated with 6MV X-ray at 0, 0.5, 1, 1.5, and 2. In ICM-received group, post-irradiation A549 cells were cultured for 3 h and were transferred into non-irradiated A549 cells for further cultivation. Clone forming test was applied to detect the survival fraction of cells. Annexin V-FITC/PI double-staining assay was used to detect the apoptosis of A549 cells 24, 48, 72, and 96 h after 2-Gy 6MV X-ray irradiation, and the curves of apoptosis were drawn. The changes in the cell cycles 4, 48, 72, and 96 h after 2-Gy 6MV X-ray irradiation were detected using PI staining flow cytometry. With the increase of irradiation dose, the survival fraction of A549 cells after the application of 0.5 Gy irradiation was decreasing continuously. In comparison to the control group, the apoptosis rate of the ICM-received group was increased in a time-dependent pattern, with the highest apoptosis rate observed at 72 h (p < 0.05). Cell count in G2/M stages was obviously increased compared with that of the control group (p < 0.05), with the highest count observed at 72 h, after which G2/M stage arrest was diminished. ICM can cause apparent A549 cell damage, indicating that 6MV X-ray irradiation can induce bystander effect on A549 cells, which reaches a peak at 72 h.

  14. Detection of 8-oxodG in Dreissena polymorpha gill cells exposed to model contaminants.

    PubMed

    Michel, Cécile; Vincent-Hubert, Françoise

    2012-01-24

    Genotoxic end-points are routinely measured in various sentinel organisms in aquatic environments in order to monitor the impact of water pollution on organisms. As a first step towards the evaluation of oxidative DNA damage (8-oxodG) in organisms exposed to chemical water pollution, we have optimized the association between the comet assay and the hOGG1 enzyme for use on zebra mussel (Dreissena polymorpha) gill cells by in vitro exposure to H₂O₂. Firstly, we observed that in vitro exposure of D. polymorpha gill cells to benzo[a]pyrene (B[a]P, 98.4nM) induced an increase of the Olive Tail Moment (OTM) in both the comet-hOGG1 and comet-Fpg assays, indicating that B[a]P causes oxidative DNA damage. By contrast, methylmethane sulfonate (MMS, 33μM) only induced an increase of the Fpg-sensitive sites, indicating that MMS caused alkylating DNA damage and confirming that hOGG1 does not detect alkylating damage. Thus, the hOGG1 enzyme seems to be more specific towards oxidative DNA damage, such as 8-oxodG than Fpg. Secondly, as was observed in vitro, the in vivo exposure of D. polymorpha to B[a]P (24.6 and 98.4nM) increased oxidative DNA damage in gill cells, whereas only Fpg-sensitive sites were detected in mussels exposed to MMS (240μM). These results show that the comet-hOGG1 assay detects oxidative DNA lesions induced in vitro by H₂O₂ and in vivo with BaP. The comet-hOGG1 assay will be used to detect oxidative DNA lesions (8-oxodG) in mussels exposed in situ. PMID:22009068

  15. Diesel Exhaust Particle-Exposed Human Bronchial Epithelial Cells Induce Dendritic Cell Maturation and Polarization via Thymic Stromal Lymphopoietin

    PubMed Central

    Bleck, Bertram; Tse, Doris B.; Curotto de Lafaille, Maria A.; Zhang, Feijie

    2009-01-01

    Human exposure to air pollutants, including ambient particulate matter, has been proposed as a mechanism for the rise in allergic disorders. Diesel exhaust particles, a major component of ambient particulate matter, induce sensitization to neoallergens, but the mechanisms by which sensitization occur remain unclear. We show that diesel exhaust particles upregulate thymic stromal lymphopoietin in human bronchial epithelial cells in an oxidant-dependent manner. Thymic stromal lymphopoietin induced by diesel exhaust particles was associated with maturation of myeloid dendritic cells, which was blocked by anti-thymic stromal lymphopoietin antibodies or silencing epithelial cell-derived thymic stromal lymphopoietin. Dendritic cells exposed to diesel exhaust particle-treated human bronchial epithelial cells induced Th2 polarization in a thymic stromal lymphopoietin-dependent manner. These findings provide new insight into the mechanisms by which diesel exhaust particles modify human lung mucosal immunity. PMID:18049884

  16. Proteomic signature of Arabidopsis cell cultures exposed to magnetically induced hyper- and microgravity environments.

    PubMed

    Herranz, Raul; Manzano, Ana I; van Loon, Jack J W A; Christianen, Peter C M; Medina, F Javier

    2013-03-01

    Earth-based microgravity simulation techniques are required due to space research constraints. Using diamagnetic levitation, we exposed Arabidopsis thaliana in vitro callus cultures to environments with different levels of effective gravity and magnetic field strengths (B) simultaneously. The environments included simulated 0 g* at B=10.1 T, an internal 1 g* control (B=16.5 T), and hypergravity (2 g* at B=10.1 T). Furthermore, samples were also exposed to altered gravity environments that were created with mechanical devices, such as the Random Positioning Machine (simulated μg) and the Large Diameter Centrifuge (2 g). We have determined the proteomic signature of cell cultures exposed to these altered-gravity environments by means of the difference gel electrophoresis (DiGE) technique, and we have compared the results with microarray-based transcriptomes from the same samples. The magnetic field itself produced a low number of proteomic alterations, but the combination of gravitational alteration and magnetic field exposure produced synergistic effects on the proteome of plants (the number of significant changes is 3-7 times greater). Tandem mass spectrometry identification of 19 overlapping spots in the different conditions corroborates a major role of abiotic stress and secondary metabolism proteins in the molecular adaptation of plants to unusual environments, including microgravity.

  17. Proteomic signature of Arabidopsis cell cultures exposed to magnetically induced hyper- and microgravity environments.

    PubMed

    Herranz, Raul; Manzano, Ana I; van Loon, Jack J W A; Christianen, Peter C M; Medina, F Javier

    2013-03-01

    Earth-based microgravity simulation techniques are required due to space research constraints. Using diamagnetic levitation, we exposed Arabidopsis thaliana in vitro callus cultures to environments with different levels of effective gravity and magnetic field strengths (B) simultaneously. The environments included simulated 0 g* at B=10.1 T, an internal 1 g* control (B=16.5 T), and hypergravity (2 g* at B=10.1 T). Furthermore, samples were also exposed to altered gravity environments that were created with mechanical devices, such as the Random Positioning Machine (simulated μg) and the Large Diameter Centrifuge (2 g). We have determined the proteomic signature of cell cultures exposed to these altered-gravity environments by means of the difference gel electrophoresis (DiGE) technique, and we have compared the results with microarray-based transcriptomes from the same samples. The magnetic field itself produced a low number of proteomic alterations, but the combination of gravitational alteration and magnetic field exposure produced synergistic effects on the proteome of plants (the number of significant changes is 3-7 times greater). Tandem mass spectrometry identification of 19 overlapping spots in the different conditions corroborates a major role of abiotic stress and secondary metabolism proteins in the molecular adaptation of plants to unusual environments, including microgravity. PMID:23510084

  18. Antioxidant Activities and Anti-Cancer Cell Proliferation Properties of Natsuhaze (Vaccinium oldhamii Miq.), Shashanbo (V. bracteatum Thunb.) and Blueberry Cultivars

    PubMed Central

    Tsuda, Hirotoshi; Kunitake, Hisato; Kawasaki-Takaki, Ryoko; Nishiyama, Kazuo; Yamasaki, Masao; Komatsu, Haruki; Yukizaki, Chizuko

    2013-01-01

    Antioxidants are abundant in blueberries, and while there are many studies concerning the bioactive compound of fruit, it is only recently that the wild Vaccinium species has attracted attention for their diverse and abundant chemical components. The aim of this study was to investigate the bioactive compounds of blueberry cultivars and wild species found in Japan. Among the five extracts of the Vaccinium species, Natsuhaze (Vaccinium oldhamii Miq.) was found to be the most effective at inhibiting the growth of HL-60 human leukemia cells in vitro. Although all ethanol extracts showed a growth inhibitory effect on HL-60 cells, the degree of the effects differed among the species. The extract of Natsuhaze induced apoptotic bodies and nucleosomal DNA fragmentation in the HL-60 cells. Of the extracts tested, that of Natsuhaze contained the largest amount of total polyphenols and showed the greatest antioxidant activity, but the anthocyanin content of Natsuhaze was similar to that of rabbiteye blueberry (V. virgatum Ait.). The results showed that total polyphenols contributed to the high antioxidant activity and growth inhibitory effect on HL-60 human leukemia cells of Natsuhaze extract. PMID:27137366

  19. Structural development of benzhydrol-type 1'-acetoxychavicol acetate (ACA) analogs as human leukemia cell-growth inhibitors based on quantitative structure-activity relationship (QSAR) analysis.

    PubMed

    Misawa, Takashi; Aoyama, Hiroshi; Furuyama, Taniyuki; Dodo, Kosuke; Sagawa, Morihiko; Miyachi, Hiroyuki; Kizaki, Masahiro; Hashimoto, Yuichi

    2008-10-01

    Benzhydrol-type analogs of 1'-acetoxychavicol acetate (ACA) were developed as inhibitors of human leukemia HL-60 cell growth based on quantitative structure-activity relationship (QSAR) analysis. An analog containing an anthracenyl moiety (8) was a potent inhibitor with the IC(50) value of 0.12 microM.

  20. Nanosecond pulsed electric fields (nsPEF) induce direct electric field effects and biological effects on human colon carcinoma cells.

    PubMed

    Hall, Emily H; Schoenbach, Karl H; Beebe, Stephen J

    2005-05-01

    Nanosecond pulsed electric fields (nsPEFs) are ultrashort pulses with high electric field intensity (kV/cm) and high power (megawatts), but low energy density (mJ/cc). To determine roles for p53 in response to nsPEFs, HCT116 cells (p53+/+ and p53-/-) were exposed to nsPEF and analyzed for membrane integrity, phosphatidylserine externalization, caspase activation, and cell survival. Decreasing plasma membrane effects were observed in both HCT116p53+/+ and p53-/- cells with decreasing pulse durations and/or decreasing electric fields. However, addition of ethidium homodimer-1 and Annexin-V-FITC post-pulse demonstrated greater fluorescence in p53-/- versus p53+/+ cells, suggesting a postpulse p53-dependent biological effect at the plasma membrane. Caspase activity was significantly higher than nonpulsed cells only in the p53-/- cells. HCT116 cells exhibited greater survival in response to nsPEFs than HL-60 and Jurkat cells, but survival was more evident for HCT116p53+/+ cells than for HCT116p53-/- cells. These results indicate that nsPEF effects on HCT116 cells include (1) apparent direct electric field effects, (2) biological effects that are p53-dependent and p53-independent, (3) actions on mechanisms that originate at the plasma membranes and at intracellular structures, and (4) an apparent p53 protective effect. NsPEF applications provide a means to explore intracellular structures and functions that can reveal mechanisms in health and disease.

  1. Evaluation of cell types for assessment of cytogenetic damage in arsenic exposed population

    PubMed Central

    Ghosh, Pritha; Basu, Arindam; Singh, Keshav K; Giri, Ashok K

    2008-01-01

    Background Cytogenetic biomarkers are essential for assessing environmental exposure, and reflect adverse human health effects such as cellular damage. Arsenic is a potential clastogen and aneugen. In general, the majority of the studies on clastogenic effects of arsenic are based on frequency of micronuclei (MN) study in peripheral lymphocytes, urothelial and oral epithelial cells. To find out the most suitable cell type, here, we compared cytogenetic damage through MN assay in (a) various populations exposed to arsenic through drinking water retrieved from literature review, as also (b) arsenic-induced Bowen's patients from our own survey. Results For literature review, we have searched the Pubmed database for English language journal articles using the following keywords: "arsenic", "micronuclei", "drinking water", and "human" in various combinations. We have selected 13 studies consistent with our inclusion criteria that measured micronuclei in either one or more of the above-mentioned three cell types, in human samples. Compared to urothelial and buccal mucosa cells, the median effect sizes measured by the difference between people with exposed and unexposed, lymphocyte based MN counts were found to be stronger. This general pattern pooled from 10 studies was consistent with our own set of three earlier studies. MN counts were also found to be stronger for lymphocytes even in arsenic-induced Bowen's patients (cases) compared to control individuals having arsenic-induced non-cancerous skin lesions. Conclusion Overall, it can be concluded that MN in lymphocytes may be superior to other epithelial cells for studying arsenic-induced cytogenetic damage. PMID:18505595

  2. Low Doses of Cisplatin Induce Gene Alterations, Cell Cycle Arrest, and Apoptosis in Human Promyelocytic Leukemia Cells

    PubMed Central

    Velma, Venkatramreddy; Dasari, Shaloam R.; Tchounwou, Paul B.

    2016-01-01

    Cisplatin is a known antitumor drug, but its mechanisms of action are not fully elucidated. In this research, we studied the anticancer potential of cisplatin at doses of 1, 2, or 3 µM using HL-60 cells as a test model. We investigated cisplatin effects at the molecular level using RNA sequencing, cell cycle analysis, and apoptotic assay after 24, 48, 72, and 96 hours of treatment. The results show that many genes responsible for molecular and cellular functions were significantly altered. Cisplatin treatment also caused the cells to be arrested at the DNA synthesis phase, and as the time increases, the cells gradually accumulated at the sub-G1 phase. Also, as the dose increases, a significant number of cells entered into the apoptotic and necrotic stages. Altogether, the data show that low doses of cisplatin significantly impact the viability of HL-60 cells, through modulation of gene expression, cell cycle, and apoptosis. PMID:27594783

  3. Low Doses of Cisplatin Induce Gene Alterations, Cell Cycle Arrest, and Apoptosis in Human Promyelocytic Leukemia Cells

    PubMed Central

    Velma, Venkatramreddy; Dasari, Shaloam R.; Tchounwou, Paul B.

    2016-01-01

    Cisplatin is a known antitumor drug, but its mechanisms of action are not fully elucidated. In this research, we studied the anticancer potential of cisplatin at doses of 1, 2, or 3 µM using HL-60 cells as a test model. We investigated cisplatin effects at the molecular level using RNA sequencing, cell cycle analysis, and apoptotic assay after 24, 48, 72, and 96 hours of treatment. The results show that many genes responsible for molecular and cellular functions were significantly altered. Cisplatin treatment also caused the cells to be arrested at the DNA synthesis phase, and as the time increases, the cells gradually accumulated at the sub-G1 phase. Also, as the dose increases, a significant number of cells entered into the apoptotic and necrotic stages. Altogether, the data show that low doses of cisplatin significantly impact the viability of HL-60 cells, through modulation of gene expression, cell cycle, and apoptosis.

  4. Low Doses of Cisplatin Induce Gene Alterations, Cell Cycle Arrest, and Apoptosis in Human Promyelocytic Leukemia Cells.

    PubMed

    Velma, Venkatramreddy; Dasari, Shaloam R; Tchounwou, Paul B

    2016-01-01

    Cisplatin is a known antitumor drug, but its mechanisms of action are not fully elucidated. In this research, we studied the anticancer potential of cisplatin at doses of 1, 2, or 3 µM using HL-60 cells as a test model. We investigated cisplatin effects at the molecular level using RNA sequencing, cell cycle analysis, and apoptotic assay after 24, 48, 72, and 96 hours of treatment. The results show that many genes responsible for molecular and cellular functions were significantly altered. Cisplatin treatment also caused the cells to be arrested at the DNA synthesis phase, and as the time increases, the cells gradually accumulated at the sub-G1 phase. Also, as the dose increases, a significant number of cells entered into the apoptotic and necrotic stages. Altogether, the data show that low doses of cisplatin significantly impact the viability of HL-60 cells, through modulation of gene expression, cell cycle, and apoptosis. PMID:27594783

  5. Cell wall chitosaccharides are essential components and exposed patterns of the phytopathogenic oomycete Aphanomyces euteiches.

    PubMed

    Badreddine, Ilham; Lafitte, Claude; Heux, Laurent; Skandalis, Nicholas; Spanou, Zacharoula; Martinez, Yves; Esquerré-Tugayé, Marie-Thérèse; Bulone, Vincent; Dumas, Bernard; Bottin, Arnaud

    2008-11-01

    Chitin is an essential component of fungal cell walls, where it forms a crystalline scaffold, and chitooligosaccharides derived from it are signaling molecules recognized by the hosts of pathogenic fungi. Oomycetes are cellulosic fungus-like microorganisms which most often lack chitin in their cell walls. Here we present the first study of the cell wall of the oomycete Aphanomyces euteiches, a major parasite of legume plants. Biochemical analyses demonstrated the presence of ca. 10% N-acetyl-D-glucosamine (GlcNAc) in the cell wall. Further characterization of the GlcNAc-containing material revealed that it corresponds to noncrystalline chitosaccharides associated with glucans, rather than to chitin per se. Two putative chitin synthase (CHS) genes were identified by data mining of an A. euteiches expressed sequence tag collection and Southern blot analysis, and full-length cDNA sequences of both genes were obtained. Phylogeny analysis indicated that oomycete CHS diversification occurred before the divergence of the major oomycete lineages. Remarkably, lectin labeling showed that the Aphanomyces euteiches chitosaccharides are exposed at the cell wall surface, and study of the effect of the CHS inhibitor nikkomycin Z demonstrated that they are involved in cell wall function. These data open new perspectives for the development of antioomycete drugs and further studies of the molecular mechanisms involved in the recognition of pathogenic oomycetes by the host plants. PMID:18806214

  6. Overexpression of GSTA2 protects against cell cycle arrest and apoptosis induced by the DNA inter-strand crosslinking nitrogen mustard, mechlorethamine.

    PubMed

    Xie, Jingping; Shults, Keith; Flye, Leanne; Jiang, Fen; Head, David R; Briggs, Robert C

    2005-05-15

    The effectiveness of bifunctional alkylating nitrogen mustard compounds in chemotherapy is related to their ability to form DNA inter-strand crosslinks. Patients exposed to DNA inter-strand crosslinking (ICL) agents subsequently experience an elevated incidence of myelodysplastic syndromes (MDS) and MDS related acute myeloid leukemia. Fanconi's anemia (FA) patients are deficient in the repair of crosslink DNA damage and they experience a high incidence of MDS. These observations indicate that hematopoietic cells are specific target for the transforming effects of DNA crosslinking damage. Changes in transcript levels were characterized in human hematopoietic cells occurring in response to the nitrogen mustard, mechlorethamine (HN2), but not in response to monofunctional analogs. Only modest changes in a few gene transcripts were detected in HL60 cells exposed to levels of HN2 tittered to maximal dose that caused growth suppression with minimal cell death and allowed eventual resumption of normal cell growth. Under conditions of transient growth suppression, a subset of glutathione-S-transferase (GST) isoenzyme genes was consistently upregulated three to fourfold by HN2, but not by monofunctional analogs. Subsequent efforts to confirm the changes detected by microarray analyses revealed an unexpected dependence on treatment conditions. The GST alpha class A2 subfamily member transcripts were upregulated 24 h after a 1 h exposure to HN2 that caused an extensive, but transient block in late S/G2 cell cycle phase, but were minimally altered with continuous exposure. The 1-h exposure to HN2 caused a transient late S/G2 cell cycle arrest in both the HL-60 cell line and the Colo 320HSR human colon cancer cell line. Overexpression of GSTA2 by transient transfection protected Colo 320HSR cells against both cycle arrest and apoptosis following exposure to HN2. Overexpression of GSTA2 in Colo 320HSR cells induced after exposure to HN2 did not alter cycle arrest or apoptosis

  7. Genome exposure and regulation in mammalian cells.

    PubMed

    Puck, T T; Webb, P; Johnson, R

    1998-09-01

    A method of measurement of exposed DNA (i.e. hypersensitive to DNase I hydrolysis) as opposed to sequestered (hydrolysis resistant) DNA in isolated nuclei of mammalian cells is described. While cell cultures exhibit some differences in behavior from day to day, the general pattern of exposed and sequestered DNA is satisfactorily reproducible and agrees with results previously obtained by other methods. The general pattern of DNA hydrolysis exhibited by all cells tested consists of a curve which at first rises sharply with increasing DNase I, and then becomes almost horizontal, indicating that roughly about half of the nuclear DNA is highly sequestered. In 4 cases where transformed cells (Raszip6, CHO, HL60 and PC12) were compared, each with its more normal homolog (3T3, and the reverse transformed versions of CHO, HL60 and PC12, achieved by dibutyryl cyclic AMP [DBcAMP], retinoic acid, and nerve growth factor [NGF] respectively), the transformed form displayed less genome exposure than the nontransformed form at every DNase I dose tested. When Ca++ was excluded from the hydrolysis medium in both the Raszip6-3T3 and the CHO-DBcAMP systems, the normal cell forms lost their increased exposure reverting to that of the transformed forms. Therefore Ca++ appears necessary for maintenance of the DNA in the more highly exposed state characteristic of the nontransformed phenotype. LiCl increases the DNA exposure of all transformed cells tested. Dextran sulfate and heparin each can increase the DNA exposure of several different cancers. Colcemid prevents the increase of exposure of CHO by DBcAMP but it must be administered before or simultaneously with the latter compound. Measurements on mouse biopsies reveal large differences in exposure in different normal tissues. Thus, the exposure from adult liver cells was greater than that of adult brain, but both fetal liver and fetal brain had significantly greater exposure than their adult counterparts. Exposure in normal human

  8. Disrupted Endothelial Cell Layer and Exposed Extracellular Matrix Proteins Promote Capture of Late Outgrowth Endothelial Progenitor Cells.

    PubMed

    Zhao, Jing; Mitrofan, Claudia-Gabriela; Appleby, Sarah L; Morrell, Nicholas W; Lever, Andrew M L

    2016-01-01

    Late outgrowth endothelial progenitor cells (LO-EPC) possess a high proliferative potential, differentiate into vascular endothelial cells (EC), and form networks, suggesting they play a role in vascular repair. However, due to their scarcity in the circulation there is a requirement for ex vivo expansion before they could provide a practical cell therapy and it is currently unclear if they would home and engraft to an injury site. Using an in vitro flow system we studied LO-EPC under simulated injury conditions including EC activation, ischaemia, disrupted EC integrity, and exposed basement membrane. Perfused LO-EPC adhered to discontinuous EC paracellularly at junctional regions between adjacent cells under shear stress 0.7 dyn/cm(2). The interaction was not adhesion molecule-dependent and not enhanced by EC activation. LO-EPC expressed high levels of the VE-Cadherin which may explain these findings. Ischaemia reperfusion injury decreased the interaction with LO-EPC due to cell retraction. LO-EPC interacted with exposed extracellular matrix (ECM) proteins, fibronectin and vitronectin. The interaction was mediated by integrins α5β3, αvβ1, and αvβ3. This study has demonstrated that an injured local environment presents sufficient adhesive signals to capture flow perfused LO-EPC in vitro and that LO-EPC have properties consistent with their potential role in vascular repair.

  9. Disrupted Endothelial Cell Layer and Exposed Extracellular Matrix Proteins Promote Capture of Late Outgrowth Endothelial Progenitor Cells.

    PubMed

    Zhao, Jing; Mitrofan, Claudia-Gabriela; Appleby, Sarah L; Morrell, Nicholas W; Lever, Andrew M L

    2016-01-01

    Late outgrowth endothelial progenitor cells (LO-EPC) possess a high proliferative potential, differentiate into vascular endothelial cells (EC), and form networks, suggesting they play a role in vascular repair. However, due to their scarcity in the circulation there is a requirement for ex vivo expansion before they could provide a practical cell therapy and it is currently unclear if they would home and engraft to an injury site. Using an in vitro flow system we studied LO-EPC under simulated injury conditions including EC activation, ischaemia, disrupted EC integrity, and exposed basement membrane. Perfused LO-EPC adhered to discontinuous EC paracellularly at junctional regions between adjacent cells under shear stress 0.7 dyn/cm(2). The interaction was not adhesion molecule-dependent and not enhanced by EC activation. LO-EPC expressed high levels of the VE-Cadherin which may explain these findings. Ischaemia reperfusion injury decreased the interaction with LO-EPC due to cell retraction. LO-EPC interacted with exposed extracellular matrix (ECM) proteins, fibronectin and vitronectin. The interaction was mediated by integrins α5β3, αvβ1, and αvβ3. This study has demonstrated that an injured local environment presents sufficient adhesive signals to capture flow perfused LO-EPC in vitro and that LO-EPC have properties consistent with their potential role in vascular repair. PMID:27413378

  10. Disrupted Endothelial Cell Layer and Exposed Extracellular Matrix Proteins Promote Capture of Late Outgrowth Endothelial Progenitor Cells

    PubMed Central

    Mitrofan, Claudia-Gabriela; Appleby, Sarah L.; Morrell, Nicholas W.; Lever, Andrew M. L.

    2016-01-01

    Late outgrowth endothelial progenitor cells (LO-EPC) possess a high proliferative potential, differentiate into vascular endothelial cells (EC), and form networks, suggesting they play a role in vascular repair. However, due to their scarcity in the circulation there is a requirement for ex vivo expansion before they could provide a practical cell therapy and it is currently unclear if they would home and engraft to an injury site. Using an in vitro flow system we studied LO-EPC under simulated injury conditions including EC activation, ischaemia, disrupted EC integrity, and exposed basement membrane. Perfused LO-EPC adhered to discontinuous EC paracellularly at junctional regions between adjacent cells under shear stress 0.7 dyn/cm2. The interaction was not adhesion molecule-dependent and not enhanced by EC activation. LO-EPC expressed high levels of the VE-Cadherin which may explain these findings. Ischaemia reperfusion injury decreased the interaction with LO-EPC due to cell retraction. LO-EPC interacted with exposed extracellular matrix (ECM) proteins, fibronectin and vitronectin. The interaction was mediated by integrins α5β3, αvβ1, and αvβ3. This study has demonstrated that an injured local environment presents sufficient adhesive signals to capture flow perfused LO-EPC in vitro and that LO-EPC have properties consistent with their potential role in vascular repair. PMID:27413378

  11. Transcriptional and Secretomic Profiling of Epidermal Cells Exposed to Alpha Particle Radiation

    PubMed Central

    Chauhan, Vinita; Howland, Matthew; Greene, Hillary Boulay; Wilkins, Ruth C

    2012-01-01

    Alpha (α)-particle emitters are probable isotopes to be used in a terrorist attack. The development of biological assessment tools to identify those who have handled these difficult to detect materials would be an asset to our current forensic capacity. In this study, for the purposes of biomarker discovery, human keratinocytes were exposed to α-particle and X-radiation (0.98 Gy/h at 0, 0.5, 1.0, 1.5 Gy) and assessed for differential gene and protein expression using microarray and Bio-Plex technology, respectively. Secretomic analysis of supernatants showed expression of two pro-inflammatory cytokines (IL-13 and PDGF-bb) to be exclusively affected in α-particle exposed cells. The highest dose of α-particle radiation modulated a total of 67 transcripts (fold change>|1.5|, (False discovery rate) FDR<0.05) in exposed cells. Several genes which responded with high expression levels (>2 fold) included KIF20A, NEFM, C7orf10, HIST1H2BD, BMP6, and HIST1H2AC. Among the high expressing genes, five (CCNB2, BUB1, NEK2, CDC20, AURKA) were also differentially expressed at the medium (1.0 Gy) dose however, these genes were unmodulated following exposure to X-irradiation. Networks of these genes clustered around tumor protein-53 and transforming growth factor-beta signaling. This study has identified some potential gene /protein responses and networks that may be validated further to confirm their specificity and potential to be signature biomarkers of α-particle exposure. PMID:23002402

  12. Tumor necrosis factor alpha gene expression in human monocytic THP-1 cells exposed to beryllium.

    PubMed

    Galbraith, G M; Pandey, J P; Schmidt, M G; Arnaud, P; Goust, J M

    1996-01-01

    Chronic beryllium disease, which results from occupational exposure to particulate beryllium, is characterized by the development of lung granulomas and progressive pulmonary fibrosis. Increased production of proinflammatory cytokines (e.g., tumor necrosis factor alpha and interleukin-1 beta) by pulmonary alveolar macrophages occurs in many chronic fibrotic lung diseases and is thought to contribute to the disease process. The purpose of the present study was to investigate cytokine production by human monocytic cells exposed to beryllium in vitro. The results indicated that such cells respond to beryllium ions in the presence of fluoride by accumulation of messenger ribonucleic acid for both tumor necrosis factor alpha and interleukin-1 beta. These findings suggest that inhaled beryllium may directly stimulate the production of these cytokines by alveolar macrophages in vitro. PMID:8629860

  13. From the Cover: Exposing Imidacloprid Interferes With Neurogenesis Through Impacting on Chick Neural Tube Cell Survival.

    PubMed

    Liu, Meng; Wang, Guang; Zhang, Shi-Yao; Zhong, Shan; Qi, Guo-Long; Wang, Chao-Jie; Chuai, Manli; Lee, Kenneth Ka Ho; Lu, Da-Xiang; Yang, Xuesong

    2016-09-01

    As a neonicotinoid pesticide, imidacloprid is widely used to control insects in agriculture and fleas on domestic animals. However, it is not known whether imidacloprid exposure negatively affects neurogenesis during embryonic development. In this study, using a chick embryo model, we investigated the effects of imidacloprid exposure on neurogenesis at the earliest stage and during late-stage embryo development. Exposing HH0 chick embryos to imidacloprid in EC culture caused neural tube defects (NTDs) and neuronal differentiation dysplasia as determined by NF/Tuj1 labeling. Furthermore, we found that F-actin accumulation on the apical side of the neural tube was suppressed by exposure to imidacloprid, and the expression of BMP4 and Shh on the dorsal and ventral sides of the neural tubes, respectively, were also reduced, which in turn affects the dorsolateral hinge points during bending of the neural plate. In addition, exposure to imidacloprid reduced cell proliferation and increased cell apoptosis, as determined by pHIS3 labeling and TUNEL staining, respectively, also contributing to the malformation. We obtained similar results in late-stage embryos exposed to imidacloprid. Finally, a bioinformatics analysis was employed to determine which genes identified in this study were involved in NTDs. The experimental evidence and bioinformatics analysis suggested that imidacloprid exposure during chick embryo development could increase the risk of NTDs and neural dysplasia.

  14. From the Cover: Exposing Imidacloprid Interferes With Neurogenesis Through Impacting on Chick Neural Tube Cell Survival.

    PubMed

    Liu, Meng; Wang, Guang; Zhang, Shi-Yao; Zhong, Shan; Qi, Guo-Long; Wang, Chao-Jie; Chuai, Manli; Lee, Kenneth Ka Ho; Lu, Da-Xiang; Yang, Xuesong

    2016-09-01

    As a neonicotinoid pesticide, imidacloprid is widely used to control insects in agriculture and fleas on domestic animals. However, it is not known whether imidacloprid exposure negatively affects neurogenesis during embryonic development. In this study, using a chick embryo model, we investigated the effects of imidacloprid exposure on neurogenesis at the earliest stage and during late-stage embryo development. Exposing HH0 chick embryos to imidacloprid in EC culture caused neural tube defects (NTDs) and neuronal differentiation dysplasia as determined by NF/Tuj1 labeling. Furthermore, we found that F-actin accumulation on the apical side of the neural tube was suppressed by exposure to imidacloprid, and the expression of BMP4 and Shh on the dorsal and ventral sides of the neural tubes, respectively, were also reduced, which in turn affects the dorsolateral hinge points during bending of the neural plate. In addition, exposure to imidacloprid reduced cell proliferation and increased cell apoptosis, as determined by pHIS3 labeling and TUNEL staining, respectively, also contributing to the malformation. We obtained similar results in late-stage embryos exposed to imidacloprid. Finally, a bioinformatics analysis was employed to determine which genes identified in this study were involved in NTDs. The experimental evidence and bioinformatics analysis suggested that imidacloprid exposure during chick embryo development could increase the risk of NTDs and neural dysplasia. PMID:27444676

  15. Altered gene expression in HepG2 cells exposed to a methanolic coal dust extract.

    PubMed

    Guerrero-Castilla, Angelica; Olivero-Verbel, Jesus

    2014-11-01

    Exposure to coal dust has been associated with different chronic diseases and mortality risk. This airborne pollutant is produced during coal mining and transport activities, generating environmental and human toxicity. The aim of this study was to determine the effects of a coal dust methanolic extract on HepG2, a human liver hepatocellular carcinoma cell line. Cells were exposed to 5-100ppm methanolic coal extract for 12h, using DMSO as control. MTT and comet assays were used for the evaluation of cytotoxicity and genotoxicity, respectively. Real time PCR was utilized to quantify relative expression of genes related to oxidative stress, xenobiotic metabolism and DNA damage. Coal extract concentrations did not induce significant changes in HepG2 cell viability after 12h exposure; however, 50 and 100ppm of the coal extract produced a significant increase in genetic damage index with respect to negative control. Compared to vehicle control, mRNA CYP1A1 (up to 163-fold), NQO1 (up to 4.7-fold), and GADD45B (up to 4.7-fold) were up regulated, whereas PRDX1, SOD, CAT, GPX1, XPA, ERCC1 and APEX1 remained unaltered. This expression profile suggests that cells exposed to coal dust extract shows aryl hydrocarbon receptor-mediated alterations, changes in cellular oxidative status, and genotoxic effects. These findings share some similarities with those observed in liver of mice captured near coal mining areas, and add evidence that living around these industrial operations may be negatively impacting the biota and human health. PMID:25305735

  16. Altered gene expression in HepG2 cells exposed to a methanolic coal dust extract.

    PubMed

    Guerrero-Castilla, Angelica; Olivero-Verbel, Jesus

    2014-11-01

    Exposure to coal dust has been associated with different chronic diseases and mortality risk. This airborne pollutant is produced during coal mining and transport activities, generating environmental and human toxicity. The aim of this study was to determine the effects of a coal dust methanolic extract on HepG2, a human liver hepatocellular carcinoma cell line. Cells were exposed to 5-100ppm methanolic coal extract for 12h, using DMSO as control. MTT and comet assays were used for the evaluation of cytotoxicity and genotoxicity, respectively. Real time PCR was utilized to quantify relative expression of genes related to oxidative stress, xenobiotic metabolism and DNA damage. Coal extract concentrations did not induce significant changes in HepG2 cell viability after 12h exposure; however, 50 and 100ppm of the coal extract produced a significant increase in genetic damage index with respect to negative control. Compared to vehicle control, mRNA CYP1A1 (up to 163-fold), NQO1 (up to 4.7-fold), and GADD45B (up to 4.7-fold) were up regulated, whereas PRDX1, SOD, CAT, GPX1, XPA, ERCC1 and APEX1 remained unaltered. This expression profile suggests that cells exposed to coal dust extract shows aryl hydrocarbon receptor-mediated alterations, changes in cellular oxidative status, and genotoxic effects. These findings share some similarities with those observed in liver of mice captured near coal mining areas, and add evidence that living around these industrial operations may be negatively impacting the biota and human health.

  17. Gene expression profile of Jurkat cells exposed to high power terahertz radiation

    NASA Astrophysics Data System (ADS)

    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.

  18. Genotoxic changes to rodent cells exposed in vitro to tungsten, nickel, cobalt and iron.

    PubMed

    Bardack, Stephanie; Dalgard, Clifton L; Kalinich, John F; Kasper, Christine E

    2014-03-10

    Tungsten-based materials have been proposed as replacements for depleted uranium in armor-penetrating munitions and for lead in small-arms ammunition. A recent report demonstrated that a military-grade composition of tungsten, nickel, and cobalt induced a highly-aggressive, metastatic rhabdomyosarcoma when implanted into the leg muscle of laboratory rats to simulate a shrapnel wound. The early genetic changes occurring in response to embedded metal fragments are not known. In this study, we utilized two cultured rodent myoblast cell lines, exposed to soluble tungsten alloys and the individual metals comprising the alloys, to study the genotoxic effects. By profiling cell transcriptomes using microarray, we found slight, yet distinct and unique, gene expression changes in rat myoblast cells after 24 h metal exposure, and several genes were identified that correlate with impending adverse consequences of ongoing exposure to weapons-grade tungsten alloy. These changes were not as apparent in the mouse myoblast cell line. This indicates a potential species difference in the cellular response to tungsten alloy, a hypothesis supported by current findings with in vivo model systems. Studies examining genotoxic-associated gene expression changes in cells from longer exposure times are warranted.

  19. Ultrastructure and calcium balance in meristem cells of pea roots exposed to extremely low magnetic fields

    NASA Astrophysics Data System (ADS)

    Belyavskaya, N. A.

    2001-01-01

    Investigations of low magnetic field (LMF) effects on biological systems have attracted attention of biologists due to planned space flights to other planets where the field intensity does not exceed 10 -5 Oe. Pea ( Pisum sativum L.) seeds were grown in an environment of LMF 3 days. In meristem cells of roots exposed to LMF, one could observe such ultrastructural peculiarities as a noticeable accumulation of lipid bodies, development of a lytic compartment (vacuoles, cytosegresomes and paramural bodies), and reduction of phytoferritin in plastids. Mitochondria were the most sensitive organelle to LMF application. Their size and relative volume in cells increased, matrix was electron-transparent, and cristae reduced. Because of the significant role of calcium signalling in plant responses to different environmental factors, calcium participation in LMF effects was investigated using a pyroantimonate method to identify the localization of free calcium ions. The intensity of cytochemical reaction in root cells after LMF application was strong. The Ca 2+ pyroantimonate deposits were observed both in all organelles and in a hyaloplasm of the cells. Data obtained suggest that the observed LMF effects on ultrastructure of root cells were due to disruptions in different metabolic systems including effects on Ca 2+ homeostasis.

  20. Effects of nicotine on cellular proliferation, macromolecular synthesis and cell cycle phase distribution in human and murine cells

    SciTech Connect

    Konno, S.; Chiao, J.; Rossi, J.; Wang, C.H.; Wu, J.M.

    1986-05-01

    Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in established human and murine cells. In the human promyelocytic HL-60 leukemic cells, 3 mM nicotine results in a 50% inhibition of cellular proliferation after 80 h. Nicotine was also found to affect the cell cycle distribution of HL-60 cells. Treatment with 4 mM nicotine for 20 h causes an increase in proportion of Gl-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes cell arrest in the Gl-phase which may in part account for its effects on cell growth. To determine whether nicotine has a primary effect on the uptake/transport of macromolecular precursors into cells, HL-60 cells were treated with 2-6 mM nicotine for 30 h/sub 3/ at the end of which time cells were labeled with (/sup 3/H)thymidine, (/sup 3/H)uridine, (/sup 14/C)lysine and (/sup 35/S)methionine, the trichloroacetic acid (TCA) soluble and insoluble radioactivities from each of the labeling conditions were determined. These studies show that nicotine primarily affect the synthesis of proteins.

  1. PD-L2 induction on dendritic cells exposed to Mycobacterium avium downregulates BCG-specific T cell response.

    PubMed

    Mendoza-Coronel, Elizabeth; Camacho-Sandoval, Rosa; Bonifaz, Laura C; López-Vidal, Yolanda

    2011-01-01

    The exposure to certain species of Nontuberculous Mycobacteria (NTM) can modulate the immune response induced by Mycobacterium bovis BCG. Mycobacterium avium has been postulated as a weak inducer of dendritic cell (DC) maturation. However, how the DC exposure to M. avium could contribute to the modulation of a BCG-specific CD4+ T cell response and the molecules involved remain unknown. Here, we exposed bone marrow-derived DCs (BMDCs) to M. avium either prior to exposure to BCG or as a unique stimulus. We found that M. avium induces high expression of PD-L2 (B7-DC) in BMDCs. This was dependent on IL-10 production through the TLR2-p38 MAPK signaling pathway. Exposure to M. avium prior to BCG results in BMDCs that do not express co-stimulatory molecules and pro-inflammatory cytokines, while the expression of PD-L2 and IL-10 was maintained. BMDCs exposed to M. avium impaired the activation of BCG-specific T cells through the PD-1: PD-L interaction. This suggests that a M. avium-induced phenotype in DCs might be implicated in the induction of mechanisms of tolerance that could impact the T cell response induced by BCG vaccination.

  2. Human epithelial cells exposed to functionalized multiwalled carbon nanotubes: interactions and cell surface modifications.

    PubMed

    Fanizza, C; Casciardi, S; Incoronato, F; Cavallo, D; Ursini, C L; Ciervo, A; Maiello, R; Fresegna, A M; Marcelloni, A M; Lega, D; Alvino, A; Baiguera, S

    2015-09-01

    With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 μg/mL for 1, 2, 4 h; 5, 10, 20 μg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 μg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to

  3. Analysis of target cell susceptibility as a basis for the development of a chemoprotective strategy against benzene-induced hematotoxicities.

    PubMed Central

    Trush, M A; Twerdok, L E; Rembish, S J; Zhu, H; Li, Y

    1996-01-01

    A goal of our research is to identify biochemical factors that underlie the susceptibility of bone marrow cell populations to benzene metabolites so as to develop a mechanistically based chemoprotective strategy that may be used in susceptible humans exposed to benzene. By doing biochemical risk analysis of bone marrow stromal cells from mice and rats and the human myeloid cell lines, HL-60 and ML-1; and by using buthionine sulfoximine and dicumarol we have observed that the susceptibility of these cell populations to hydroquinone (HQ) correlates with their concentration of glutathione (GSH) and activity of quinone reductase (QR). Accordingly, the induction of QR and GSH by 1,2-dithiole-3-thione (D3T) in these cell populations has resulted in a significant protection against the following hydroquinone-mediated toxicities: inhibition of cell proliferation and viability; reduced ability of stromal cells to support myelopoiesis; and altered differentiated of ML-1 cells to monocytes/macrophages. Preliminary in vivo experiments indicate that feeding mice D3T results in an induction of QR in the bone marrow compartment such that stromal cells are more resistant to hydroquinone-induced cytotoxicity in vitro. Overall, these studies suggest that in addition to hepatic cytochrome P4502E1, bone marrow QR and GSH are factors that could determine an individual's relative susceptibility to the toxic effects of benzene. PMID:9118897

  4. Global gene expression profiling in human lung cells exposed to cobalt

    PubMed Central

    Malard, Veronique; Berenguer, Frederic; Prat, Odette; Ruat, Sylvie; Steinmetz, Gerard; Quemeneur, Eric

    2007-01-01

    Background It has been estimated that more than 1 million workers in the United States are exposed to cobalt. Occupational exposure to 59 Co occurs mainly via inhalation and leads to various lung diseases. Cobalt is classified by the IARC as a possible human carcinogen (group 2B). Although there is evidence for in vivo and in vitro toxicity, the mechanisms of cobalt-induced lung toxicity are not fully known. The purpose of this work was to identify potential signatures of acute cobalt exposure using a toxicogenomic approach. Data analysis focused on some cellular processes and protein targets that are thought to be relevant for carcinogenesis, transport and biomarker research. Results A time course transcriptome analysis was performed on A549 human pulmonary cells, leading to the identification of 85 genes which are repressed or induced in response to soluble 59 Co. A group of 29 of these genes, representing the main biological functions, was assessed by quantitative RT-PCR. The expression profiles of six of them were then tested by quantitative RT-PCR in a time-dependent manner and three modulations were confirmed by Western blotting. The 85 modulated genes include potential cobalt carriers (FBXL2, ZNT1, SLC12A5), tumor suppressors or transcription factors (MAZ, DLG1, MYC, AXL) and genes linked to the stress response (UBC, HSPCB, BNIP3L). We also identified nine genes coding for secreted proteins as candidates for biomarker research. Of those, TIMP2 was found to be down-regulated and this modulation was confirmed, in a dose-dependent manner, at protein level in the supernatant of exposed cells. Conclusion Most of these genes have never been described as related to cobalt stress and provide original hypotheses for further study of the effects of this metal ion on human lung epithelial cells. A putative biomarker of cobalt toxicity was identified. PMID:17553155

  5. Induction of micronuclei in haemocytes and gill cells of zebra mussels, Dreissena polymorpha, exposed to clastogens.

    PubMed

    Mersch, J; Beauvais, M N; Nagel, P

    1996-11-01

    Zebra mussels, Dreissena polymorpha, were exposed to four directly acting reference clastogens (mitomycin C, bleomycin, dimethylarsinic acid and potassium chromate) under laboratory conditions. The aim was to examine the inducibility of micronuclei (MN) in haemocytes and gill cells. Positive responses were observed in both tissues for all four substances used under the given test conditions. The mean MN frequencies in treated mussels ranged between 3.2 and 6.9/1000 in haemocytes and between 5.4 and 6.7/1000 in gill cells. The spontaneous MN levels averaged 1.2 and 2.8/1000 in haemocytes and gill cells, respectively. The MN induction capacity of the different chemicals was equivalent in both tissues, except for the treatment with dimethylarsinic acid which generated a significantly higher MN rate in gill cells than in haemocytes. Several characteristics suggest that haemolymph is the more appropriate test tissue for environmental genotoxicity assessment: (1) a shorter preparation time of slides, (2) a more accurate identification of unambiguous MN, (3) a lower baseline MN frequency and a higher induction factor.

  6. The Bioeffects Resulting from Prokaryotic Cells and Yeast Being Exposed to an 18 GHz Electromagnetic Field.

    PubMed

    Nguyen, The Hong Phong; Pham, Vy T H; Nguyen, Song Ha; Baulin, Vladimir; Croft, Rodney J; Phillips, Brian; Crawford, Russell J; Ivanova, Elena P

    2016-01-01

    The mechanisms by which various biological effects are triggered by exposure to an electromagnetic field are not fully understood and have been the subject of debate. Here, the effects of exposing typical representatives of the major microbial taxa to an 18 GHz microwave electromagnetic field (EMF)were studied. It appeared that the EMF exposure induced cell permeabilisation in all of the bacteria and yeast studied, while the cells remained viable (94% throughout the exposure), independent of the differences in cell membrane fatty acid and phospholipid composition. The resulting cell permeabilisation was confirmed by detection of the uptake of propidium iodine and 23 nm fluorescent silica nanospheres using transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Upon EMF exposure, the bacterial cell membranes are believed to become permeable through quasi-endocytosis processes. The dosimetry analysis revealed that the EMF threshold level required to induce the uptake of the large (46 nm) nanopsheres was between three and six EMF doses, with a specific absorption rate (SAR) of 3 kW/kg and 5 kW/kg per exposure, respectively, depending on the bacterial taxa being studied. It is suggested that the taxonomic affiliation and lipid composition (e.g. the presence of phosphatidyl-glycerol and/or pentadecanoic fatty acid) may affect the extent of uptake of the large nanospheres (46 nm). Multiple 18 GHz EMF exposures over a one-hour period induced periodic anomalous increases in the cell growth behavior of two Staphylococcus aureus strains, namely ATCC 25923 and CIP 65.8T.

  7. The Bioeffects Resulting from Prokaryotic Cells and Yeast Being Exposed to an 18 GHz Electromagnetic Field

    PubMed Central

    Pham, Vy T. H.; Nguyen, Song Ha; Baulin, Vladimir; Croft, Rodney J.; Phillips, Brian; Crawford, Russell J.

    2016-01-01

    The mechanisms by which various biological effects are triggered by exposure to an electromagnetic field are not fully understood and have been the subject of debate. Here, the effects of exposing typical representatives of the major microbial taxa to an 18 GHz microwave electromagnetic field (EMF)were studied. It appeared that the EMF exposure induced cell permeabilisation in all of the bacteria and yeast studied, while the cells remained viable (94% throughout the exposure), independent of the differences in cell membrane fatty acid and phospholipid composition. The resulting cell permeabilisation was confirmed by detection of the uptake of propidium iodine and 23 nm fluorescent silica nanospheres using transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Upon EMF exposure, the bacterial cell membranes are believed to become permeable through quasi-endocytosis processes. The dosimetry analysis revealed that the EMF threshold level required to induce the uptake of the large (46 nm) nanopsheres was between three and six EMF doses, with a specific absorption rate (SAR) of 3 kW/kg and 5 kW/kg per exposure, respectively, depending on the bacterial taxa being studied. It is suggested that the taxonomic affiliation and lipid composition (e.g. the presence of phosphatidyl-glycerol and/or pentadecanoic fatty acid) may affect the extent of uptake of the large nanospheres (46 nm). Multiple 18 GHz EMF exposures over a one-hour period induced periodic anomalous increases in the cell growth behavior of two Staphylococcus aureus strains, namely ATCC 25923 and CIP 65.8T. PMID:27391488

  8. The Bioeffects Resulting from Prokaryotic Cells and Yeast Being Exposed to an 18 GHz Electromagnetic Field.

    PubMed

    Nguyen, The Hong Phong; Pham, Vy T H; Nguyen, Song Ha; Baulin, Vladimir; Croft, Rodney J; Phillips, Brian; Crawford, Russell J; Ivanova, Elena P

    2016-01-01

    The mechanisms by which various biological effects are triggered by exposure to an electromagnetic field are not fully understood and have been the subject of debate. Here, the effects of exposing typical representatives of the major microbial taxa to an 18 GHz microwave electromagnetic field (EMF)were studied. It appeared that the EMF exposure induced cell permeabilisation in all of the bacteria and yeast studied, while the cells remained viable (94% throughout the exposure), independent of the differences in cell membrane fatty acid and phospholipid composition. The resulting cell permeabilisation was confirmed by detection of the uptake of propidium iodine and 23 nm fluorescent silica nanospheres using transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Upon EMF exposure, the bacterial cell membranes are believed to become permeable through quasi-endocytosis processes. The dosimetry analysis revealed that the EMF threshold level required to induce the uptake of the large (46 nm) nanopsheres was between three and six EMF doses, with a specific absorption rate (SAR) of 3 kW/kg and 5 kW/kg per exposure, respectively, depending on the bacterial taxa being studied. It is suggested that the taxonomic affiliation and lipid composition (e.g. the presence of phosphatidyl-glycerol and/or pentadecanoic fatty acid) may affect the extent of uptake of the large nanospheres (46 nm). Multiple 18 GHz EMF exposures over a one-hour period induced periodic anomalous increases in the cell growth behavior of two Staphylococcus aureus strains, namely ATCC 25923 and CIP 65.8T. PMID:27391488

  9. Enhanced histamine release from lung mast cells of guinea pigs exposed to sulfuric acid aerosols

    SciTech Connect

    Fujimaki, Hidekazu ); Katayama, Noboru; Wakamori, Kazuo )

    1992-06-01

    To clarify the relationship between air pollution and mast cell response, the effects of sulfuric acid aerosols on histamine release from lung mast cells of guinea pigs were investigated. Guinea pigs were exposed to 0.3, 1.0 and 3.2 mg/m{sup 3} sulfuric acid (H{sub 2}SO{sub 4}) aerosols or 4 ppm nitrogen dioxide (NO{sub 2}) for 2 and 4 weeks. After the exposure, lung mast cell suspensions were isolated by collagenase treatment and antigen- or A23187-induced histamine release was measured. Antigen-induced histamine release from mast cells was significantly enhanced by the exposure to 1.0 and 3.2 mg/m{sup 3} H{sub 2}SO{sub 4} for 2 weeks, but exposure to H{sub 2}SO{sub 4} for 4 weeks did not show the enhancement of antigen-induced histamine release. A23187-induced histamine release was significantly enhanced by the exposure to 1.0 mg/m{sup 3} H{sub 2}SO{sub 4} or 4 ppm NO{sub 2} for 2 weeks, but suppression of histamine release from lung mast cells stimulated with A23187 was observed by the exposure to 3.2 mg/m{sup 3} H{sub 2}So{sub 4} for 4 weeks. The exposure to 0.3 mg/m{sup 3} H{sub 2}So{sub 4} showed no changes in antigen- and A23187-induced histamine release. The combination of 1.0 mg/m{sup 3} H{sub 2}So{sub 4} with 4 ppm NO{sub 2} for 2 weeks resulted in no changes in antigen- and A23187-induced histamine release. These results suggested that functional properties of lung mast cells may be altered by a low concentration of H{sub 2}So{sub 4} aerosol exposure.

  10. Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging.

    PubMed

    Chatre, Laurent; Ricchetti, Miria

    2013-02-15

    Mitochondrial DNA (mtDNA) replication and transcription are crucial for cell function, but these processes are poorly understood at the single-cell level. We describe a novel fluorescence in situ hybridization protocol, called mTRIP (mitochondrial transcription and replication imaging protocol), that reveals simultaneously mtDNA and RNA, and that can also be coupled to immunofluorescence for in situ protein examination. mTRIP reveals mitochondrial structures engaged in initiation of DNA replication by identification of a specific sequence in the regulatory D-loop, as well as unique transcription profiles in single human cells. We observe and quantify at least three classes of mitochondrial structures: (i) replication initiation active and transcript-positive (Ia-Tp); (ii) replication initiation silent and transcript-positive (Is-Tp); and (iii) replication initiation silent and transcript-negative (Is-Tn). Thus, individual mitochondria are dramatically heterogeneous within the same cell. Moreover, mTRIP exposes a mosaic of distinct nucleic acid patterns in the D-loop, including H-strand versus L-strand transcripts, and uncoupled rRNA transcription and mtDNA initiation of replication, which might have functional consequences in the regulation of the mtDNA. Finally, mTRIP identifies altered mtDNA processing in cells with unbalanced mtDNA content and function, including in human mitochondrial disorders. Thus, mTRIP reveals qualitative and quantitative alterations that provide additional tools for elucidating the dynamics of mtDNA processing in single cells and mitochondrial dysfunction in diseases.

  11. Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement.

    PubMed

    Gac, Séverine Le; Zwaan, Ed; van den Berg, Albert; Ohl, Claus-Dieter

    2007-12-01

    We report here the sonoporation of HL60 (human promyelocytic leukemia) suspension cells in a microfluidic confinement using a single laser-induced cavitation bubble. Cavitation bubbles can induce membrane poration of cells located in their close vicinity. Membrane integrity of suspension cells placed in a microfluidic chamber is probed through either the calcein release out of calcein-loaded cells or the uptake of trypan blue. Cells that are located farther away than four times Rmax (maximum bubble radius) from the cavitation bubble center remain fully unaffected, while cells closer than 0.75 Rmax become porated with a probability of >75%. These results enable us to define a distance of 0.75 Rmax as a critical interaction distance of the cavitation bubble with HL60 suspension cells. These experiments suggest that flow-induced poration of suspension cells is applicable in lab-on-a-chip systems, and this might be an interesting alternative to electroporation.

  12. Natural Products Mediated Regulation of Oxidative Stress and DNA Damage in Ultraviolet Exposed Skin Cells.

    PubMed

    Farooqi, Ammad A; Li, Ruei-Nian; Huang, Hurng-Wern; Ismail, Muhammad; Yuan, Shyng-Shiou F; Wang, Hui-Min D; Liu, Jing-Ru; Tang, Jen-Yang; Chang, Hsueh-Wei

    2015-01-01

    Data obtained through high-throughput technologies have gradually revealed that a unique stratified epithelial architecture of human skin along with the antioxidant-response pathways provided vital defensive mechanisms against UV radiation. However, it is noteworthy that skin is a major target for toxic insult by UV radiations that can alter its structure and function. Substantial fraction of information has been added into the existing pool of knowledge related to natural products mediated biological effects in UV exposed skin cells. Accumulating evidence has started to shed light on the potential of these bioactive ingredients as protective natural products in cosmetics against UV photodamage by exerting biological effects mainly through wide ranging intracellular signalling cascades of oxidative stress and modulation of miRNAs. In this review, we have summarized recently emerging scientific evidences addressing underlying mechanisms of UV induced oxidative stress and deregulation of signalling cascades and how natural products can be used tactfully to protect against UV induced harmful effects.

  13. Formation of lipofuscin in cultured retinal pigment epithelial cells exposed to pre-oxidized photoreceptor outer segments.

    PubMed

    Wihlmark, U; Wrigstad, A; Roberg, K; Brunk, U T; Nilsson, S E

    1996-04-01

    Accumulation of lipofuscin in the retinal pigment epithelium (RPE) with increasing age may affect essential supportive functions for the photoreceptors. Earlier, we described a model system for the study of lipofuscinogenesis in RPE cell cultures and showed that mild oxidative stress enhances lipofuscin formation from phagocytized photoreceptor outer segments (POS). In the present study, bovine POS were photo-oxidized, and turned into a lipofuscin-like material, by irradiation with UV light. Transmission electron microscopy of irradiated POS showed loss of the normal stacks of the disk membranes with conversion into an amorphous osmiophilic electron-dense mass. The formation of thiobarbituric acid reactive substances (TBARS), estimated during the irradiation process, indicated lipid peroxidation. Irradiated POS also showed a strong granular yellow autofluorescence. RPE cell cultures, kept at 21% ambient oxygen, were fed daily for 3, 5 or 7 days with either (i) UV-peroxidized POS, (ii) native POS or (iii) culture medium only. RPE cells fed irradiated POS showed significantly higher levels of lipofuscin-specific autofluorescence compared to cells exposed to native POS after 3 days (p = 0.0056), 5 days (p = 0.0037) and 7 days (p = 0.0020), and to the non-exposed control cells (3 days: p = 0.005, 5 days: p = 0.0037, 7 days: p = 0.0094). The lipofuscin content of cells exposed to irradiated POS increased significantly between days 3 and 7 (p = 0.0335). Ultrastructural studies showed much more numerous and larger lipofuscin-like inclusions in RPE cells fed irradiated POS compared to cells exposed to native POS. In the control cells, lipofuscin-like granules were small and sparse. It appears that exposing RPE cells to previously peroxidized POS, thus artificially converted to lipofuscin and obviously not digestible by the lysosomal enzymes, accelerates the formation of severely lipofuscin-loaded cells. The results will be useful for further studies of possible harmful

  14. Genome-wide gene expression analysis of mouse embryonic stem cells exposed to p-dichlorobenzene.

    PubMed

    Tani, Hidenori; Takeshita, Jun-Ichi; Aoki, Hiroshi; Abe, Ryosuke; Toyoda, Akinobu; Endo, Yasunori; Miyamoto, Sadaaki; Gamo, Masashi; Torimura, Masaki

    2016-09-01

    Because of the limitations of whole animal testing approaches for toxicological assessment, new cell-based assay systems have been widely studied. In this study, we focused on two biological products for toxicological assessment: mouse embryonic stem cells (mESCs) and long noncoding RNAs (lncRNAs). mESCs possess the abilities of self-renewal and differentiation into multiple cell types. LlncRNAs are an important class of pervasive non-protein-coding transcripts involved in the molecular mechanisms associated with responses to chemicals. We exposed mESCs to p-dichlorobenzene (p-DCB) for 1 or 28 days (daily dose), extracted total RNA, and performed deep sequencing analyses. The genome-wide gene expression analysis indicated that mechanisms modulating proteins occurred following acute and chronic exposures, and mechanisms modulating genomic DNA occurred following chronic exposure. Moreover, our results indicate that three novel lncRNAs (Snora41, Gm19947, and Scarna3a) in mESCs respond to p-DCB exposure. We propose that these lncRNAs have the potential to be surrogate indicators of p-DCB responses in mESCs. PMID:26975756

  15. Proteomic analysis of cell wall in four pathogenic species of Candida exposed to oxidative stress.

    PubMed

    Ramírez-Quijas, Mayra Denisse; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2015-10-01

    In order for Candida species to adhere and colonize human host cells they must express cell wall proteins (CWP) and adapt to reactive oxygen species (ROS) generated by phagocytic cells of the human host during the respiratory burst. However, how these pathogens change the expression of CWP in response to oxidative stress (OSR) is not known. Here, fifteen moonlight-like CWP were identified that expressed differentially in four species of Candida after they were exposed to H2O2 or menadione (O2(-)). These proteins included: (i) glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (Gapdh), fructose-bisphosphate aldolase (Fba1), phosphoglycerate mutase (Gpm1), phosphoglycerate kinase (Pgk), pyruvate kinase (Pk) and enolase (Eno1); (ii) the heat shock proteins Ssb1 and Ssa2; (iii) OSR proteins such as peroxyredoxin (Tsa1), the stress protein Ddr48 (Ddr48) and glutathione reductase (Glr1); (iv) other metabolic enzymes such as ketol-acid reductoisomerase (Ilv5) and pyruvate decarboxylase (Pdc1); and (v) other proteins such as elongation factor 1-beta (Efb1) and the 14-3-3 protein homolog. RT-PCR revealed that transcription of the genes coding for some of the identified CWP are differentially regulated. To our knowledge this is the first report showing that moonlight-like CWP are the first line of defense of Candida against ROS, and that they are differentially regulated in each of these pathogens.

  16. Investigation of micronucleus induction in MTH1 knockdown cells exposed to UVA, UVB or UVC.

    PubMed

    Fotouhi, Asal; Cornella, Nicola; Ramezani, Mehrafarin; Wojcik, Andrzej; Haghdoost, Siamak

    2015-11-01

    The longer wave parts of UVR can increase the production of reactive oxygen species (ROS) which can oxidize nucleotides in the DNA or in the nucleotide pool leading to mutations. Oxidized bases in the DNA are repaired mainly by the DNA base excision repair system and incorporation of oxidized nucleotides into newly synthesized DNA can be prevented by the enzyme MTH1. Here we hypothesize that the formation of several oxidized base damages (from pool and DNA) in close proximity, would cause a high number of base excision repair events, leading to DNA double strand breaks (DSB) and therefore giving rise to cytogenetic damage. If this hypothesis is true, cells with low levels of MTH1 will show higher cytogenetic damage after the longer wave parts of UVR. We analyzed micronuclei induction (MN) as an endpoint for cytogenetic damage in the human lymphoblastoid cell line, TK6, with a normal and a reduced level of MTH1 exposed to UVR. The results indicate a higher level of micronuclei at all incubation times after exposure to the longer wave parts of UVR. There is no significant difference between wildtype and MTH1-knockdown TK6 cells, indicating that MTH1 has no protective role in UVR-induced cytogenetic damage. This indicates that DSBs induced by UV arise from damage forms by direct interaction of UV or ROS with the DNA rather than through oxidation of dNTP. PMID:26520386

  17. Oxidative stress response in neural stem cells exposed to different superparamagnetic iron oxide nanoparticles.

    PubMed

    Pongrac, Igor M; Pavičić, Ivan; Milić, Mirta; Brkić Ahmed, Lada; Babič, Michal; Horák, Daniel; Vinković Vrček, Ivana; Gajović, Srećko

    2016-01-01

    Biocompatibility, safety, and risk assessments of superparamagnetic iron oxide nanoparticles (SPIONs) are of the highest priority in researching their application in biomedicine. One improvement in the biological properties of SPIONs may be achieved by different functionalization and surface modifications. This study aims to investigate how a different surface functionalization of SPIONs - uncoated, coated with d-mannose, or coated with poly-l-lysine - affects biocompatibility. We sought to investigate murine neural stem cells (NSCs) as important model system for regenerative medicine. To reveal the possible mechanism of toxicity of SPIONs on NSCs, levels of reactive oxygen species, intracellular glutathione, mitochondrial membrane potential, cell-membrane potential, DNA damage, and activities of SOD and GPx were examined. Even in cases where reactive oxygen species levels were significantly lowered in NSCs exposed to SPIONs, we found depleted intracellular glutathione levels, altered activities of SOD and GPx, hyperpolarization of the mitochondrial membrane, dissipated cell-membrane potential, and increased DNA damage, irrespective of the surface coating applied for SPION stabilization. Although surface coating should prevent the toxic effects of SPIONs, our results showed that all of the tested SPION types affected the NSCs similarly, indicating that mitochondrial homeostasis is their major cellular target. Despite the claimed biomedical benefits of SPIONs, the refined determination of their effects on various cellular functions presented in this work highlights the need for further safety evaluations. This investigation helps to fill the knowledge gaps on the criteria that should be considered in evaluating the biocompatibility and safety of novel nanoparticles. PMID:27217748

  18. In situ real-time monitoring of apoptosis on leukemia cells by surface infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Ryo-taro; Hirano-Iwata, Ayumi; Kimura, Yasuo; Niwano, Michio; Miyamoto, Ko-ichiro; Isoda, Hiroko; Miyazaki, Hitoshi

    2009-01-01

    We have investigated in situ real-time monitoring of apoptosis on human promyelocytic leukemia (HL-60) cells using infrared absorption spectroscopy with the multiple internal reflection (MIR-IRAS) geometry. Actinomycin D (Act D)-induced apoptosis on HL-60 cells was monitored for 24 h. Apoptotic cells showed two strong peaks around the protein amide I and amide II bands probably due to the leakage of cytoplasmic proteins, while growing viable cells showed a peak corresponding to the secretion of metabolites and two downward peaks corresponding to uptake of nutrients from culture media. In addition, IR absorption peak intensity of the amide I and amide II bands was proportional to the extracellular concentration of lactate dehydrogenase, a marker protein for cell damage. These results demonstrate that our MIR-IRAS method is useful for discrimination of apoptotic cells from viable ones and cell apoptotic processes can be monitored in situ by analyzing the amide I and amide II peak intensity.

  19. DNA damage in buccal epithelial cells from individuals chronically exposed to arsenic via drinking water in Inner Mongolia, China.

    PubMed

    Feng, Z; Xia, Y; Tian, D; Wu, K; Schmitt, M; Kwok, R K; Mumford, J L

    2001-01-01

    The purpose of this pilot study was to assess DNA damage in buccal cells from individuals chronically exposed to arsenic via drinking water in Ba Men, Inner Mongolia. Buccal cells were collected from 19 Ba Men residents exposed to arsenic at 527.5 +/- 23.7 micrograms/L (mean +/- SEM) and 13 controls exposed to arsenic at 4.4 +/- 1.0 micrograms/L. DNA fragmentation by the DNA ladder and TUNEL assay were used to detect DNA damage in buccal cells. In the DNA ladder assay, 89% (17/19) of the arsenic-exposed group showed < 100 bp DNA fragments, in contrast to 15% (2/13) of the controls (p < 0.0001). For the TUNEL assay, the mean frequencies of positive cells were higher in the exposed group (15.1%) than in the controls (2.0%) (p < 0.0001). This study showed that high arsenic exposure via drinking water resulted in DNA damage and DNA fragmentation in buccal cells thus may be an appropriate biomarker for assessing chronic effects of arsenic in humans. A study investigating DNA fragmentation from the individuals with low levels of arsenic exposure in this population is in progress.

  20. Study of damage to red blood cells exposed to different doses of γ-ray irradiation

    PubMed Central

    Xu, Deyi; Peng, Mingxi; Zhang, Zhe; Dong, Guofei; Zhang, Yiqin; Yu, Hongwei

    2012-01-01

    Background. The aims of this research were to study alterations in the ultrastructure of red blood cells, the changes in concentrations of plasma electrolytes and the killing effect of lymphocytes in samples of blood exposed to different doses of γ-ray irradiation. Materials and methods. Blood samples were treated with different doses of γ-ray irradiation and then preserved for different periods. Specimens were prepared for standard electron microscopy and transmission electron microscopy. At the same time, changes in the concentrations of Na+, K+ and Cl− and pH values in the plasma as well as Fas and FasL expression of lymphocytes before and after irradiation were determined. Results. The proportions of reversibly and irreversibly transformed cells, for example, echinocytes, sphero-echinocytes, and degenerated forms, increased with increasing doses of irradiation and storage period, while the number of discocyte shaped red blood cells decreased. The change in K+ concentration was greater than that of Na+ or Cl− after irradiation and was dosage-dependent. Plasma pH was influenced by different doses of radiation and storage time. After exposure to 137Cs γ-irradiation, the expression of both Fas and FasL in lymphocytes differed significantly from that in the control group: the expression was positively correlated with irradiation dose (r=0.95, 0.96), but no significant difference in the Fas/FasL ratio was observed (P>0.05). Discussion. We conclude that the ultrastructure of red blood cells is not changed obviously by irradiation with some doses of γ-rays and various periods of storage. However, irradiation does have some dose-dependent and time-dependent adverse effects on the erythrocytes. PMID:22682338

  1. Early and delayed reproductive death in human cells exposed to high energy iron-ion beams

    NASA Astrophysics Data System (ADS)

    Bettega, D.; Calzolari, P.; Doneda, L.; Durante, M.; Tallone, L.

    For radiation protection of the astronauts it is important to know both the acute and the late effects of charged particles. Iron is the most abundant high charge and energy (HZE) specie in galactic cosmic radiation. (HZE) ions are considered to be the major contributors to equivalent dose in space, but the Relative Biological Effectiveness of HZE particles has large uncertainties, expecially for late effects. We have determined early and delayed reproductive death in human fibroblast cells (AG1522) exposed to iron ion beams of energies between 0.2 and 1 GeV/n. The cells were irradiated at the HIMAC accelerator in Chiba (0.2 and 0.5 GeV/n) and at the AGS accelerator at the NASA Space Radiation Laboratory in Brookhaven (1 GeV/n). For each beam the dose--effect curves were measured at least twice in the dose range between 0.5 and 2 Gy. 60 Co gamma rays were used as reference radiation. The following results were obtained: 1) the 1 GeV/n beam effectiveness for inactivation of the AG1522 cells is higher than that of any other beam. 2) the progeny of the irradiated cells show the presence of delayed damage in the form of reproductive death for all the beams with the 1 GeV/n being the most effective. 3) the relative biological effectiveness of the iron beams is higher for delayed compared to early reproductive death. A comparison with preliminary results obtained with 970 MeV/n Ti and 490 MeV/n Si ions will be also reported .

  2. Antioxidant status and selected biochemical parameters of porcine ovarian granulosa cells exposed to lead in vitro.

    PubMed

    Capcarová, Marcela; Kolesárová, Adriana; Lukác, Norbert; Sirotkin, Alexander; Roychoudhury, Shubhadeep

    2009-12-01

    The objective of this study was to determine the activity of superoxide dismutase (SOD), total antioxidant status (TAS) and release of calcium, phosphorus, magnesium, sodium, potassium, total lipids, totals proteins, glucose, cholesterol and triglycerides by porcine ovarian granulosa cells cultured in vitro after lead acetate administration. The parameters were analyzed using semi-automated clinical chemistry analyzer Microlab 300, microprocessor-controlled analyzer EasyLite and spectrophotometer Genesys 10. Cells were cultured with lead acetate trihydrate [Pb(CH(3)COO)(2).3H(2)O] as follows: group Max (5 mg Pb(CH(3)COO)(2).3H(2)O/10 mL), group A (2.5 mg/10 mL), group B (0.83 mg/10 mL), group C (0.625 mg/10 mL), group D (0.455 mg/10 mL) and the control group without lead exposure for 18 hrs. The highest TAS was estimated in the control group without lead treatment in comparison with other groups (MAX, A, B, C, D). Statistical analyses showed significantly lower value (P < 0.05) in group B. The activity of SOD was the lowest in the control group in comparison to those exposed to in vitro lead culture. A significant decrease (P < 0.05) of calcium content in group MAX in comparison with control group was determined. Release of phosphorus by ovarian granulosa cells was significantly lower (P < 0.05; 0.01; 0.001) in all the treated groups in comparison with control group. Lead was found to stimulate the release of magnesium and potassium by granulosa cells, but the increase remained statistically insignificant. The highest concentration of glucose was noted in control group, but the differences were not significant either. No significant differences (P > 0.05) were detected in concentration of other studied parameters among observed groups, too.

  3. Rapid identification of the multidrug resistance in the human leukemic cells by near-infrared Fourier transform Raman microspectroscopy

    NASA Astrophysics Data System (ADS)

    Beljebbar, Abdelilah; Morjani, Hamid; Sockalingum, Ganesh D.; Manfait, Michel

    1998-04-01

    In this work, we have studied the cancer cell lines sharing a common feature: the multi-drug resistance where P- glycoprotein is responsible for the active efflux of the drug out of the cell. For this, we have used two types of cells, MDR-human leukemic K562 cells and non-MDR acute promyelocytic leukemic HL60 cells. The comparison between normalized micro FT-Raman spectra of resistant and sensitive K652 cells shows a decrease in the intensity of the amide I and III bands and a down shift of the amide I band. On the other hand, control experiments with HL60 cells do not show any remarkable changes. Analysis of micro-FT-Raman spectra by resolution enhancement methods and by chemometrics tools reveal further information concerning the conformational changes of the cell constituents involved in the expression of the MDR-phenotype.

  4. Chitosan-based nanocoatings for hypothermic storage of living cells.

    PubMed

    Bulwan, Maria; Antosiak-Iwańska, Magdalena; Godlewska, Ewa; Granicka, Ludomira; Zapotoczny, Szczepan; Nowakowska, Maria

    2013-11-01

    The formation of ultrathin chitosan-based nanocoating on HL-60 model cells and their protective function in hypothermic storage are presented. HL-60 cells are encapsulated in ultrathin shells by adsorbing cationic and anionic chitosan derivatives in a stepwise, layer-by-layer, procedure carried out in an aqueous medium under mild conditions. The chitosan-based films are also deposited on model lipid bilayer and the interactions are studied using ellipsometry and atomic force microscopy. The cells covered with the chitosan-based films and stored at 4 °C for 24 h express viability comparable to that of the control sample incubated at 37 °C, while the unprotected cells stored under the same conditions do not show viability. It is shown that the chitosan-based shell protects HL-60 cells against damaging effect of hypothermic storage. Such nanocoatings provide protection, mechanical stability, and support the cell membrane, while ensuring penetration of small molecules such as nutrients/gases what is essential for cell viability.

  5. Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin.

    PubMed Central

    Colotta, F.; Sciacca, F. L.; Sironi, M.; Luini, W.; Rabiet, M. J.; Mantovani, A.

    1994-01-01

    Thrombin, in addition to being a key enzyme in hemostasis, affects a series of endothelial and leukocyte functions and thus may be involved in the regulation of inflammatory reactions. Because leukocyte recruitment and activation are important events in inflammatory and thrombotic processes, in this study we have examined the possibility that thrombin induces the production of a cytokine chemotactic for mononuclear phagocytes. Human peripheral blood mononuclear cells (PBMC) exposed in vitro to thrombin expressed transcripts of monocyte chemotactic protein-1 (MCP-1; alternative acronyms: JE, monocyte chemotactic and activating factor, tumor-derived chemotactic factor). Thrombin was two- to threefold less effective than endotoxin in inducing MCP-1 transcripts in PBMC. Among circulating mononuclear cells, monocytes were identified as the cells expressing MCP-1 in response to thrombin. Monocytes expressed thrombin receptor transcripts. Boiling, hirudin, antithrombin III, and mutation of the catalytic site serine 205 into alanine) blocked the capacity of thrombin to induce MCP-1 expression. The thrombin receptor-activating peptide mimicked the effect of thrombin in inducing MCP-1 expression. Induction of MCP-1 transcript by thrombin was not reduced by blocking interleukin-1 and tumor necrosis factor, suggesting that these mediators are not involved in thrombin-induced expression of MCP-1. In addition to monocytes, endothelial cells (EC) also expressed MCP-1 in response to thrombin, although at lower levels compared with monocytes. Actinomycin D experiments indicated that induction of MCP-1 by thrombin in PBMC and EC was gene transcription dependent. The inhibition of protein synthesis blocked thrombin-induced MCP-1 expression in PBMC, whereas it superinduced both constitutive and thrombin-inducible expression of MCP-1 in EC, indicating different mechanisms of regulation of this gene in mononuclear phagocytes versus endothelial cells. Thrombin stimulated mononuclear

  6. Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

    NASA Astrophysics Data System (ADS)

    Miura, Taichi; Ando, Ayumi; Hirano, Kazumi; Ogura, Chika; Kanazawa, Tatsuya; Ikeguchi, Masamichi; Seki, Atsushi; Nishihara, Shoko; Hamaguchi, Satoshi

    2014-11-01

    Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H2O2), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells.

  7. Distribution of Chromosome Breakpoints in Human Epithelial Cells Exposed to Low- and High-LET Radiations

    NASA Technical Reports Server (NTRS)

    Hada, Megumi; Cucinotta, Francis; Wu, Honglu

    2009-01-01

    The advantage of the multicolor banding in situ hybridization (mBAND) technique is not only its ability to identify simultaneously both inter- and intrachromosome exchanges, but also the ability to measure the breakpoint location along the length of the chromosome in a precision that is unmatched with other traditional banding techniques. Breakpoints on specific regions of a chromosome have been known to associate with specific cancers. The breakpoint distribution in cells after low- and high-LET radiation exposures will also provide the data for biophysical modeling of the chromatin structure, as well as the data for the modeling the formation of radiation-induced chromosome aberrations. In a series of experiments, we studied low- and high-LET radiation-induced chromosome aberrations using the mBAND technique with chromosome 3 painted in 23 different colored bands. Human epithelial cells (CH1 84B5F5/M10) were exposed in vitro to Cs- 137 rays at both low and high dose rates, secondary neutrons with a broad energy spectrum at a low dose rate and 600 MeV/u Fe ions at a high dose rate. The data of both inter- and intrachromosome aberrations involving the painted chromosome have been reported previously. Here we present data of the location of the chromosome breaks along the length of chromosome 3 in the cells after exposures to each of the four radiation scenarios. In comparison to the expected breakpoint distribution based on the length of the bands, the observed distribution appeared to be non-random for both the low- and high-LET radiations. In particular, hot spots towards both ends of the chromosome were found after low-LET irradiations of either low or high dose rates. For both high-LET radiation types (Fe ions and neutrons), the breakpoint distributions were similar, and were much smoother than that for low-LET radiation. The dependence of the breakpoint distribution on the radiation quality requires further investigations.

  8. DNA methylation analysis using CpG microarrays is impaired in benzopyrene exposed cells

    SciTech Connect

    Sadikovic, Bekim; Andrews, Joseph; Rodenhiser, David I.

    2007-12-15

    Epigenetic alterations have emerged as a key mechanism involved in tumorigenesis. These disruptions are partly due to environmental factors that change normal DNA methylation patterns necessary for transcriptional regulation and chromatin compaction. Microarray technologies are allowing environmentally susceptible epigenetic patterns to be mapped and the precise targets of environmentally induced alterations to be identified. Previously, we observed BaP-induced epigenetic events and cell cycle disruptions in breast cancer cell lines that included time- and concentration-dependent loss of proliferation as well as sequence-specific hypo- and hypermethylation events. In this present report, we further characterized epigenetic changes in BaP-exposed MCF-7 cells. We analyzed DNA methylation on a CpG island microarray platform with over 5400 unique genomic regions. Depleted and enriched microarray targets, representative of putative DNA methylation changes, were identified across the genome; however, subsequent sodium bisulfite analyses revealed no changes in DNA methylation at a number of these loci. Instead, we found that the identification of DNA methylation changes using this restriction enzyme-based microarray approach corresponded with the regions of DNA bound by the BaP derived DNA adducts. This DNA adduct formation occurs at both methylated and unmethylated CpG dinucleotides and affects PCR amplification during sample preparation. Our data suggest that caution should be exercised when interpreting data from comparative microarray experiments that rely on enzymatic reactions. These results are relevant to genome screening approaches involving environmental exposures in which DNA adduct formation at specific nucleotide sites may bias target acquisition and compromise the correct identification of epigenetically responsive genes.

  9. Proteomic responses of human intestinal Caco-2 cells exposed to silver nanoparticles and ionic silver.

    PubMed

    Oberemm, Axel; Hansen, Ulf; Böhmert, Linda; Meckert, Christine; Braeuning, Albert; Thünemann, Andreas F; Lampen, Alfonso

    2016-03-01

    Even although quite a number of studies have been performed so far to demonstrate nanoparticle-specific effects of substances in living systems, clear evidence of these effects is still under debate. The present study was designed as a comparative proteomic analysis of human intestinal cells exposed to a commercial silver nanoparticle reference material and ions from AgNO3. A two-dimensional gel electrophoresis/MALDI mass spectrometry (MS)-based proteomic analysis was conducted after 24-h incubation of differentiated Caco-2 cells with non-cytotoxic and low cytotoxic silver concentrations (2.5 and 25 µg ml(-1) nanosilver, 0.5 and 5 µg ml(-1) AgNO3). Out of an overall number of 316 protein spots differentially expressed at a fold change of ≥ 1.4 or ≤ -1.4 in all treatments, 169 proteins could be identified. In total, 231 spots were specifically deregulated in particle-treated groups compared with 41 spots, which were limited to AgNO3-treatments. Forty-four spots (14 %) were commonly deregulated by both types of treatment. A considerable fraction of the proteins differentially expressed after treatment with nanoparticles is related to protein folding, synthesis or modification of proteins as well as cellular assembly and organization. Overlays of networks obtained for particulate and ionic treatments showed matches, indicating common mechanisms of combined particle and ionic silver exposure and exclusive ionic silver treatment. However, proteomic responses of Caco-2 cells treated with higher concentrations of silver species also showed some differences, for example regarding proteins related to fatty acid and energy metabolism, suggesting an induction of also some different molecular mechanisms for particle exposure and ionic treatment.

  10. Comet assay in gill cells of Prochilodus lineatus exposed in vivo to cypermethrin.

    PubMed

    Poletta, G L; Gigena, F; Loteste, A; Parma, M J; Kleinsorge, E C; Simoniello, M F

    2013-11-01

    Agricultural chemicals can induce genetic alterations on aquatic organisms that have been associated with effects on growth, reproduction and population dynamics. The evaluation of DNA damage in fish using the comet assay (CA) frequently involves the utilization of erythrocytes. However, epithelial gill cells (EGC) can be more sensitive, as they are constantly dividing and in direct contact with potentially stressing compounds from the aquatic environment. The aim of the present study was to evaluate (1) the sensitivity and suitability of epithelial gill cells of Prochilodus lineatus in response to different genotoxic agents through the application of the CA, (2) the induction of DNA damage in this cell population after in vivo exposure to cypermethrin. Baseline value of the CA damage index (DI) for EGC of juvenile P. lineatus was 144.68±5.69. Damage increased in a dose-dependent manner after in vitro exposure of EGC to methyl methanesulfonate (MMS) and H2O2, two known genotoxic agents. In vivo exposure of fish to cypermethrin induced a significant increase in DNA DI of EGC at 0.150μg/l (DI: 239.62±6.21) and 0.300μg/l (270.63±2.09) compared to control (150.25±4.38) but no effect was observed at 0.075μg/l (168.50±10.77). This study shows that EGC of this species are sensitive for the application of the CA, demonstrating DNA damage in response to alkylation (MMS), oxidative damage (H2O2), and to the insecticide cypermethryn. These data, together with our previous study on DNA damage induction on erythrocytes of this species, provides useful information for future work involving biomonitoring in regions where P. lineatus is naturally exposed to pesticides and other genotoxic agents.

  11. Oxidative stress response in neural stem cells exposed to different superparamagnetic iron oxide nanoparticles

    PubMed Central

    Pongrac, Igor M; Pavičić, Ivan; Milić, Mirta; Brkić Ahmed, Lada; Babič, Michal; Horák, Daniel; Vinković Vrček, Ivana; Gajović, Srećko

    2016-01-01

    Biocompatibility, safety, and risk assessments of superparamagnetic iron oxide nanoparticles (SPIONs) are of the highest priority in researching their application in biomedicine. One improvement in the biological properties of SPIONs may be achieved by different functionalization and surface modifications. This study aims to investigate how a different surface functionalization of SPIONs – uncoated, coated with d-mannose, or coated with poly-l-lysine – affects biocompatibility. We sought to investigate murine neural stem cells (NSCs) as important model system for regenerative medicine. To reveal the possible mechanism of toxicity of SPIONs on NSCs, levels of reactive oxygen species, intracellular glutathione, mitochondrial membrane potential, cell-membrane potential, DNA damage, and activities of SOD and GPx were examined. Even in cases where reactive oxygen species levels were significantly lowered in NSCs exposed to SPIONs, we found depleted intracellular glutathione levels, altered activities of SOD and GPx, hyperpolarization of the mitochondrial membrane, dissipated cell-membrane potential, and increased DNA damage, irrespective of the surface coating applied for SPION stabilization. Although surface coating should prevent the toxic effects of SPIONs, our results showed that all of the tested SPION types affected the NSCs similarly, indicating that mitochondrial homeostasis is their major cellular target. Despite the claimed biomedical benefits of SPIONs, the refined determination of their effects on various cellular functions presented in this work highlights the need for further safety evaluations. This investigation helps to fill the knowledge gaps on the criteria that should be considered in evaluating the biocompatibility and safety of novel nanoparticles. PMID:27217748

  12. Curcumin enhances the cytogenotoxic effect of etoposide in leukemia cells through induction of reactive oxygen species.

    PubMed

    Papież, Monika A; Krzyściak, Wirginia; Szade, Krzysztof; Bukowska-Straková, Karolina; Kozakowska, Magdalena; Hajduk, Karolina; Bystrowska, Beata; Dulak, Jozef; Jozkowicz, Alicja

    2016-01-01

    Curcumin may exert a more selective cytotoxic effect in tumor cells with elevated levels of free radicals. Here, we investigated whether curcumin can modulate etoposide action in myeloid leukemia cells and in normal cells of hematopoietic origin. HL-60 cell line, normal myeloid progenitor cluster of differentiation (CD)-34(+) cells, and granulocytes were incubated for 4 or 24 hours at different concentrations of curcumin and/or etoposide. Brown Norway rats with acute myeloid leukemia (BNML) were used to prove the influence of curcumin on etoposide action in vivo. Rats were treated with curcumin for 23 days and etoposide was administered for the final 3 days of the experiment. Curcumin synergistically potentiated the cytotoxic effect of etoposide, and it intensified apoptosis and phosphorylation of the histone H2AX induced by this cytostatic drug in leukemic HL-60 cells. In contrast, curcumin did not significantly modify etoposide-induced cytotoxicity and H2AX phosphorylation in normal CD34(+) cells and granulocytes. Curcumin modified the cytotoxic action of etoposide in HL-60 cells through intensification of free radical production because preincubation with N-acetyl-l-cysteine (NAC) significantly reduced the cytotoxic effect of curcumin itself and a combination of two compounds. In contrast, NAC did not decrease the cytotoxic effect of etoposide. Thus, oxidative stress plays a greater role in the cytotoxic effect of curcumin than that of etoposide in HL-60 cells. In vitro results were confirmed in a BNML model. Pretreatment with curcumin enhanced the antileukemic activity of etoposide in BNML rats (1.57-fold tumor reduction versus etoposide alone; P<0.05) and induced apoptosis of BNML cells more efficiently than etoposide alone (1.54-fold change versus etoposide alone; P<0.05), but this treatment protected nonleukemic B-cells from apoptosis. Thus, curcumin can increase the antileukemic effect of etoposide through reactive oxygen species in sensitive myeloid

  13. Curcumin enhances the cytogenotoxic effect of etoposide in leukemia cells through induction of reactive oxygen species

    PubMed Central

    Papież, Monika A; Krzyściak, Wirginia; Szade, Krzysztof; Bukowska-Straková, Karolina; Kozakowska, Magdalena; Hajduk, Karolina; Bystrowska, Beata; Dulak, Jozef; Jozkowicz, Alicja

    2016-01-01

    Curcumin may exert a more selective cytotoxic effect in tumor cells with elevated levels of free radicals. Here, we investigated whether curcumin can modulate etoposide action in myeloid leukemia cells and in normal cells of hematopoietic origin. HL-60 cell line, normal myeloid progenitor cluster of differentiation (CD)-34+ cells, and granulocytes were incubated for 4 or 24 hours at different concentrations of curcumin and/or etoposide. Brown Norway rats with acute myeloid leukemia (BNML) were used to prove the influence of curcumin on etoposide action in vivo. Rats were treated with curcumin for 23 days and etoposide was administered for the final 3 days of the experiment. Curcumin synergistically potentiated the cytotoxic effect of etoposide, and it intensified apoptosis and phosphorylation of the histone H2AX induced by this cytostatic drug in leukemic HL-60 cells. In contrast, curcumin did not significantly modify etoposide-induced cytotoxicity and H2AX phosphorylation in normal CD34+ cells and granulocytes. Curcumin modified the cytotoxic action of etoposide in HL-60 cells through intensification of free radical production because preincubation with N-acetyl-l-cysteine (NAC) significantly reduced the cytotoxic effect of curcumin itself and a combination of two compounds. In contrast, NAC did not decrease the cytotoxic effect of etoposide. Thus, oxidative stress plays a greater role in the cytotoxic effect of curcumin than that of etoposide in HL-60 cells. In vitro results were confirmed in a BNML model. Pretreatment with curcumin enhanced the antileukemic activity of etoposide in BNML rats (1.57-fold tumor reduction versus etoposide alone; P<0.05) and induced apoptosis of BNML cells more efficiently than etoposide alone (1.54-fold change versus etoposide alone; P<0.05), but this treatment protected nonleukemic B-cells from apoptosis. Thus, curcumin can increase the antileukemic effect of etoposide through reactive oxygen species in sensitive myeloid leukemia

  14. Semisynthetic homoharringtonine induces apoptosis via inhibition of protein synthesis and triggers rapid myeloid cell leukemia-1 down-regulation in myeloid leukemia cells.

    PubMed

    Tang, Ruoping; Faussat, Anne-Marie; Majdak, Patricia; Marzac, Christophe; Dubrulle, Sabine; Marjanovic, Zora; Legrand, Ollivier; Marie, Jean-Pierre

    2006-03-01

    Semisynthetic homoharringtonine (ssHHT) is now being evaluated in phase II clinical trials for the treatment of chronic myelogenous leukemia and acute myelogenous leukemia patients. Here, we examined the mechanism of the apoptosis induced by ssHHT in myeloid leukemia cells. First, we have shown that ssHHT induces apoptosis in HL60 and HL60/MRP cell lines in a time- and dose-dependent manner, and independently of the expression of Bax. The decrease of mitochondrial membrane potential and the release of cytochrome c were observed in the apoptotic cells induced by ssHHT. To unveil the relationship between ssHHT and the mitochondrial disruption, we have shown that ssHHT decreased myeloid cell leukemia-1 (Mcl-1) expression and induced Bcl-2 cleavage in HL60 and HL60/MRP cell lines. The Bcl-2 cleavage could be inhibited by the Z-VAD.fmk caspase inhibitor. However, Mcl-1 turnover was very rapid and occurred before caspase activation. The Mcl-1 turnover was only induced by ssHHT and cycloheximide, but not by daunorubicin and cytosine arabinoside, and could be restored by proteasome inhibitors. Second, we confirmed that ssHHT rapidly induced massive apoptosis in acute myelogenous leukemia patient cells. We have also confirmed the release of cytochrome c and a rapid turnover of Mcl-1 in these patient cells, taking place only in apoptotic cells induced by ssHHT but not in cells undergoing spontaneous apoptosis. Finally, we have shown that ssHHT inhibits protein synthesis in both cell line and patient cells. We suggest that the inhibition of protein synthesis and resulting Mcl-1 turnover play a key role in the apoptosis induced by ssHHT. Our results encourage further clinical trials for the use of ssHHT in acute myelogenous leukemia.

  15. Microfluidic device capable of medium recirculation for non-adherent cell culture

    PubMed Central

    Dixon, Angela R.; Rajan, Shrinidhi; Kuo, Chuan-Hsien; Bersano, Tom; Wold, Rachel; Futai, Nobuyuki; Takayama, Shuichi; Mehta, Geeta

    2014-01-01

    We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells. PMID:24753733

  16. Transcriptomic analysis of cultured whale skin cells exposed to hexavalent chromium [Cr(VI)].

    PubMed

    Pabuwal, Vagmita; Boswell, Mikki; Pasquali, Amanda; Wise, Sandra S; Kumar, Suresh; Shen, Yingjia; Garcia, Tzintzuni; Lacerte, Carolyne; Wise, John Pierce; Wise, John Pierce; Warren, Wesley; Walter, Ronald B

    2013-06-15

    Hexavalent chromium Cr(VI) is known to produce cytotoxic effects in humans and is a highly toxic environmental contaminant. Interestingly, it has been shown that free ranging sperm whales (Phyester macrocephalus) may have exceedingly high levels of Cr in their skin. Also, it has been demonstrated that skin cells from whales appear more resistant to both cytotoxicity and clastogenicity upon Cr exposure compared to human cells. However, the molecular genetic mechanisms employed in whale skin cells that might lead to Cr tolerance are unknown. In an effort to understand the underlying mechanisms of Cr(VI) tolerance and to illuminate global gene expression patterns modulated by Cr, we exposed whale skin cells in culture to varying levels of Cr(VI) (i.e., 0.0, 0.5, 1.0 and 5.0 μg/cm²) followed by short read (100 bp) next generation RNA sequencing (RNA-seq). RNA-seq reads from all exposures (≈280 million reads) were pooled to generate a de novo reference transcriptome assembly. The resulting whale reference assembly had 11K contigs and an N50 of 2954 bp. Using the reads from each dose (0.0, 0.5, 1.0 and 5.0 μg/cm²) we performed RNA-seq based gene expression analysis that identified 35 up-regulated genes and 19 down-regulated genes. The experimental results suggest that low dose exposure to Cr (1.0 μg/cm²) serves to induce up-regulation of oxidative stress response genes, DNA repair genes and cell cycle regulator genes. However, at higher doses (5.0 μg/cm²) the DNA repair genes appeared down-regulated while other genes that were induced suggest the initiation of cytotoxicity. The set of genes identified that show regulatory modulation at different Cr doses provide specific candidates for further studies aimed at determination of how whales exhibit resistance to Cr toxicity and what role(s) reactive oxygen species (ROS) may play in this process.

  17. Apoptosis of peripheral blood mononuclear cells in children exposed to arsenic and fluoride.

    PubMed

    Rocha-Amador, Diana O; Calderón, Jaqueline; Carrizales, Leticia; Costilla-Salazar, Rogelio; Pérez-Maldonado, Iván Nelinho

    2011-11-01

    In this study, we evaluated apoptosis induction in human immune cells in children exposed to arsenic (As) and fluoride (F). Children living in two areas in Mexico (Soledad de Graciano Sanchez (SGS) in San Luis Potosí and Colonia 5 de Febrero in Durango) were studied. Water, urine and blood samples were collected. Approximately 90% of the water samples in 5 de Febrero had As and F levels above the World Health Organization intervention guideline (10 μg/L and 1.5mg/L, respectively). In SGS, 0% of the water samples exceeded Mexican guidelines. Urinary As and F levels in children living in 5 de Febrero were significantly higher than the levels found in children living in SGS. In addition, the level of apoptosis was higher in children from the 5 de Febrero community when compared with the level of apoptosis in children living in SGS. Thus, in a worldwide context, our study demonstrates the health risks to children living in these regions. PMID:22004959

  18. Ion transport into cells exposed to monopolar and bipolar nanosecond pulses

    PubMed Central

    Schoenbach, Karl H.; Pakhomov, Andrei G.; Semenov, Iurii; Xiao, Shu; Pakhomova, Olga N.; Ibey, Bennet L.

    2014-01-01

    Experiments with CHO cells exposed to 60 and 300 ns pulsed electric fields with amplitudes in the range from several kV/cm to tens of kV/cm, showed a decrease of the uptake of calcium ions by more than an order of magnitude when, immediately after a first pulse, a second one of opposite polarity was applied. This effect is assumed to be due to the reversal of the electrophoretic transport of ions through the electroporated membrane during the second phase of the bipolar pulse. This assumption, however, is only valid if electrophoresis is the dominant transport mechanism, rather than diffusion. Comparison of calculated calcium ion currents with experimental results showed that for nanosecond pulses, electrophoresis is at least as important as diffusion. By delaying the second pulse with respect to the first one, the effect of reverse electrophoresis is reduced. Consequently, separating nanosecond pulses of opposite polarity by up to approximately hundred microseconds allows us to vary the uptake of ions from very small values to that obtained with two pulses of the same polarity. The measured calcium ion uptake obtained with bipolar pulses also allowed us to determine the membrane pore recovery time. The calculated recovery time constants are on the order of ten microseconds. PMID:25212701

  19. A biosensor of SRC family kinase conformation by exposable tetracysteine useful for cell-based screening.

    PubMed

    Irtegun, Sevgi; Wood, Rebecca; Lackovic, Kurt; Schweiggert, Jörg; Ramdzan, Yasmin M; Huang, David C S; Mulhern, Terrence D; Hatters, Danny M

    2014-07-18

    We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology.

  20. Disrupted NOS signaling in lymphatic endothelial cells exposed to chronically increased pulmonary lymph flow.

    PubMed

    Datar, Sanjeev A; Gong, Wenhui; He, Youping; Johengen, Michael; Kameny, Rebecca J; Raff, Gary W; Maltepe, Emin; Oishi, Peter E; Fineman, Jeffrey R

    2016-07-01

    Associated abnormalities of the lymphatic circulation are well described in congenital heart disease. However, their mechanisms remain poorly elucidated. Using a clinically relevant ovine model of a congenital cardiac defect with chronically increased pulmonary blood flow (shunt), we previously demonstrated that exposure to chronically elevated pulmonary lymph flow is associated with: 1) decreased bioavailable nitric oxide (NO) in pulmonary lymph; and 2) attenuated endothelium-dependent relaxation of thoracic duct rings, suggesting disrupted lymphatic endothelial NO signaling in shunt lambs. To further elucidate the mechanisms responsible for this altered NO signaling, primary lymphatic endothelial cells (LECs) were isolated from the efferent lymphatic of the caudal mediastinal node in 4-wk-old control and shunt lambs. We found that shunt LECs (n = 3) had decreased bioavailable NO and decreased endothelial nitric oxide synthase (eNOS) mRNA and protein expression compared with control LECs (n = 3). eNOS activity was also low in shunt LECs, but, interestingly, inducible nitric oxide synthase (iNOS) expression and activity were increased in shunt LECs, as were total cellular nitration, including eNOS-specific nitration, and accumulation of reactive oxygen species (ROS). Pharmacological inhibition of iNOS reduced ROS in shunt LECs to levels measured in control LECs. These data support the conclusion that NOS signaling is disrupted in the lymphatic endothelium of lambs exposed to chronically increased pulmonary blood and lymph flow and may contribute to decreased pulmonary lymphatic bioavailable NO.

  1. Ion transport into cells exposed to monopolar and bipolar nanosecond pulses.

    PubMed

    Schoenbach, Karl H; Pakhomov, Andrei G; Semenov, Iurii; Xiao, Shu; Pakhomova, Olga N; Ibey, Bennett L

    2015-06-01

    Experiments with CHO cells exposed to 60 and 300 ns pulsed electric fields with amplitudes in the range from several kV/cm to tens of kV/cm showed a decrease of the uptake of calcium ions by more than an order of magnitude when, immediately after a first pulse, a second one of opposite polarity was applied. This effect is assumed to be due to the reversal of the electrophoretic transport of ions through the electroporated membrane during the second phase of the bipolar pulse. This assumption, however, is only valid if electrophoresis is the dominant transport mechanism, rather than diffusion. Comparison of calculated calcium ion currents with experimental results showed that for nanosecond pulses, electrophoresis is at least as important as diffusion. By delaying the second pulse with respect to the first one, the effect of reverse electrophoresis is reduced. Consequently, separating nanosecond pulses of opposite polarity by up to approximately hundred microseconds allows us to vary the uptake of ions from very small values to those obtained with two pulses of the same polarity. The measured calcium ion uptake obtained with bipolar pulses also allowed us to determine the membrane pore recovery time. The calculated recovery time constants are on the order of 10 μs.

  2. Apoptosis of peripheral blood mononuclear cells in children exposed to arsenic and fluoride.

    PubMed

    Rocha-Amador, Diana O; Calderón, Jaqueline; Carrizales, Leticia; Costilla-Salazar, Rogelio; Pérez-Maldonado, Iván Nelinho

    2011-11-01

    In this study, we evaluated apoptosis induction in human immune cells in children exposed to arsenic (As) and fluoride (F). Children living in two areas in Mexico (Soledad de Graciano Sanchez (SGS) in San Luis Potosí and Colonia 5 de Febrero in Durango) were studied. Water, urine and blood samples were collected. Approximately 90% of the water samples in 5 de Febrero had As and F levels above the World Health Organization intervention guideline (10 μg/L and 1.5mg/L, respectively). In SGS, 0% of the water samples exceeded Mexican guidelines. Urinary As and F levels in children living in 5 de Febrero were significantly higher than the levels found in children living in SGS. In addition, the level of apoptosis was higher in children from the 5 de Febrero community when compared with the level of apoptosis in children living in SGS. Thus, in a worldwide context, our study demonstrates the health risks to children living in these regions.

  3. Persistent oxidative stress in human neural stem cells exposed to low fluences of charged particles.

    PubMed

    Baulch, Janet E; Craver, Brianna M; Tran, Katherine K; Yu, Liping; Chmielewski, Nicole; Allen, Barrett D; Limoli, Charles L

    2015-08-01

    Exposure to the space radiation environment poses risks for a range of deleterious health effects due to the unique types of radiation encountered. Galactic cosmic rays are comprised of a spectrum of highly energetic nuclei that deposit densely ionizing tracks of damage along the particle trajectory. These tracks are distinct from those generated by the more sparsely ionizing terrestrial radiations, and define the geometric distribution of the complex cellular damage that results when charged particles traverse the tissues of the body. The exquisite radiosensitivity of multipotent neural stem and progenitor cells found within the neurogenic regions of the brain predispose the central nervous system to elevated risks for radiation induced sequelae. Here we show that human neural stem cells (hNSC) exposed to different charged particles at space relevant fluences exhibit significant and persistent oxidative stress. Radiation induced oxidative stress was found to be most dependent on total dose rather than on the linear energy transfer of the incident particle. The use of redox sensitive fluorogenic dyes possessing relative specificity for hydroxyl radicals, peroxynitrite, nitric oxide (NO) and mitochondrial superoxide confirmed that most irradiation paradigms elevated reactive oxygen and nitrogen species (ROS and RNS, respectively) in hNSC over a 1 week interval following exposure. Nitric oxide synthase (NOS) was not the major source of elevated nitric oxides, as the use of NOS inhibitors had little effect on NO dependent fluorescence. Our data provide extensive evidence for the capability of low doses of charged particles to elicit marked changes in the metabolic profile of irradiated hNSC. Radiation induced changes in redox state may render the brain more susceptible to the development of neurocognitive deficits that could affect an astronaut's ability to perform complex tasks during extended missions in deep space. PMID:25800120

  4. Fibrogenic response of hepatic stellate cells in ovariectomised rats exposed to ketogenic diet.

    PubMed

    Bobowiec, R; Wojcik, M; Jaworska-Adamu, J; Tusinska, E

    2013-02-01

    The discrepancy about the role of estrogens in hepatic fibrogenesis and lack of studies addressed of ketogenic diet (KD) on hepatic stellate cells (HSC), prompted us to investigate the activity of HSC in control, KD- and thioacetamide (TAA)-administrated rats with different plasma concentration of estradiol (E2). HSC were isolated by the collagenase perfusion methods and separated by the Percoll gradient centrifugation. After the 4(th) and 8(th) day of incubation, lysates of HSC and the media were collected for further analysis. The HSC derived from KD-rats released remarkably more transforming growth factor (TGF)-β1 than cells obtained from animals fed with a standard diet. The ovariectomy of KD-rats markedly intensified the secretion of this fibrogenic cytokine on the 8(th) day of incubation (201.33 ±1 7.15 pg/ml). In HSC of rats exposed to E2, the TGF-β1 concentration did not exceed 157 ± 34.39 pg/ml. In respect to the collagen type I, the HSC obtained from ovariectomised KD-rats released an augmented amount of this ECM protein after the 8(th) day of culture (1.83 ± 0.14 U/ml). In the same time, higher quantities of ASMA appeared in the KD rats (1.41 ± 0.3 pg/mg protein). Exposition of rats to E2 did not markedly decrease the amount of ASMA. In summary, KD was able to induce morphological and functional changes in HSC, especially derived from rats deprived of ovarian estrogens. However, the preservation of E2 in ovariectomised rats didn't substantially alter the activation of HSC.

  5. Persistent oxidative stress in human neural stem cells exposed to low fluences of charged particles

    PubMed Central

    Baulch, Janet E.; Craver, Brianna M.; Tran, Katherine K.; Yu, Liping; Chmielewski, Nicole; Allen, Barrett D.; Limoli, Charles L.

    2015-01-01

    Exposure to the space radiation environment poses risks for a range of deleterious health effects due to the unique types of radiation encountered. Galactic cosmic rays are comprised of a spectrum of highly energetic nuclei that deposit densely ionizing tracks of damage along the particle trajectory. These tracks are distinct from those generated by the more sparsely ionizing terrestrial radiations, and define the geometric distribution of the complex cellular damage that results when charged particles traverse the tissues of the body. The exquisite radiosensitivity of multipotent neural stem and progenitor cells found within the neurogenic regions of the brain predispose the central nervous system to elevated risks for radiation induced sequelae. Here we show that human neural stem cells (hNSC) exposed to different charged particles at space relevant fluences exhibit significant and persistent oxidative stress. Radiation induced oxidative stress was found to be most dependent on total dose rather than on the linear energy transfer of the incident particle. The use of redox sensitive fluorogenic dyes possessing relative specificity for hydroxyl radicals, peroxynitrite, nitric oxide (NO) and mitochondrial superoxide confirmed that most irradiation paradigms elevated reactive oxygen and nitrogen species (ROS and RNS, respectively) in hNSC over a 1 week interval following exposure. Nitric oxide synthase (NOS) was not the major source of elevated nitric oxides, as the use of NOS inhibitors had little effect on NO dependent fluorescence. Our data provide extensive evidence for the capability of low doses of charged particles to elicit marked changes in the metabolic profile of irradiated hNSC. Radiation induced changes in redox state may render the brain more susceptible to the development of neurocognitive deficits that could affect an astronaut’s ability to perform complex tasks during extended missions in deep space. PMID:25800120

  6. Host cell reactivation of sunlamp-exposed adenovirus in fibroblasts from patients with Bloom's syndrome, ataxia telangiectasia, and Huntington's disease

    SciTech Connect

    Rainbow, A.J. )

    1991-01-01

    In this study, a sensitive host cell reactivation (HCR) technique was used to examine the repair capacity for DNA damaged by sunlamp exposure in fibroblast strains derived from 5 normal individuals and 8 patients representing three different diseases associated with DNA repair deficiencies. Adenovirus type 2 (Ad 2) was exposed to radiation from a GE 275 W sunlamp and subsequently used to infect fibroblast monolayers. At 48 hr after infection, cells were scored for the presence of viral structural antigens (Vag) using indirect immunofluorescent staining. Previous reports using this technique showed a substantial reduction in the HCR of sunlamp-exposed Ad 2 for infection of excision repair deficient fibroblasts from patients with xeroderma pigmentosum. In contrast, the HCR of Vag synthesis for sunlamp-exposed Ad 2 was in the normal range for the three ataxia telangiectasia, three Bloom's syndrome, and two Huntington's disease fibroblasts strains.

  7. Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells.

    PubMed

    Lam, R K K; Han, Wei; Yu, K N

    2015-12-01

    We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased significantly. PMID:26524645

  8. Unirradiated cells rescue cells exposed to ionizing radiation: Activation of NF-κB pathway in irradiated cells.

    PubMed

    Lam, R K K; Han, Wei; Yu, K N

    2015-12-01

    We studied the involvement of NF-κB pathway activation in the rescue effect in HeLa and NIH/3T3 cells irradiated by α particles. Firstly, upon irradiation by 5 cGy of α particles, for both cell lines, the numbers of 53BP1 foci/cell at 12 h post-irradiation were significantly smaller when only 2.5% of the cell population was irradiated as compared to 100% irradiation, which demonstrated the rescue effect. Secondly, we studied the effect of NF-κB on the rescue effect through the use of the NF-κB activation inhibitor BAY-11-7082. Novel experimental setup and procedures were designed to prepare the medium (CM) which had conditioned the bystander cells previously partnered with irradiated cells, to ensure physical separation between rescue and bystander signals. BAY-11-7082 itself did not inflict DNA damages in the cells or have effects on activation of the NF-κB response pathway in the irradiated cells through direct irradiation. The rescue effect was induced in both cell lines by the CM, which was abrogated if BAY-11-7082 was added to the CM. Thirdly, we studied the effect of NF-κB on the rescue effect through staining for phosphorylated NF-κB (p-NF-κB) expression using the anti-NF-κB p65 (phospho S536) antibody. When the fraction of irradiated cells dropped from 100% to 2.5%, the p-NF-κB expression in the cell nuclei of irradiated NIH/3T3 cells increased significantly, while that in the cell nuclei of irradiated HeLa cells also increased although not significantly. Moreover, the p-NF-κB expression in the cell nuclei of irradiated HeLa cells and NIH/3T3 cells treated with CM also increased significantly.

  9. Phosphatidylserine index as a marker of the procoagulant phenotype of acute myelogenous leukemia cells

    NASA Astrophysics Data System (ADS)

    Tormoen, Garth W.; Recht, Olivia; Gruber, András; Levine, Ross L.; McCarty, Owen J. T.

    2013-10-01

    Patients with acute myelogenous leukemia (AML) are at risk for thrombotic complications. Risk to develop thrombosis is closely tied to leukemia subtype, and studies have shown an association between leukocytosis and thrombosis in AML M3. We evaluated the relative roles of cell count and the surface expression of tissue factor (TF) and phosphatidylserine (PS) in the procoagulant phenotype of AML cell lines. The TF-positive AML M3 cell lines, NB4 and HL60, and AML M2 cell line, AML14, exhibited both extrinsic tenase and prothrombinase activity in a purified system and promoted experimental thrombus formation. In contrast, the TF-negative AML cell line, HEL, exhibited only prothrombinase activity and did not affect the rate of occlusive thrombus formation. In plasma, NB4, HL60 and AML14 shortened clotting times in a cell-count, PS- and TF-dependent manner. Exposure of cultured NB4, HL60, and AML14 cells to the chemotherapeutic agent daunorubicin increased their extrinsic tenase activity and PS expression. Clot initiation time inversely correlated with logarithm of PS index, defined as the product of multiplying leukocyte count with cell surface PS exposure. We propose that leukemia cell PS index may serve as a biomarker for procoagulant activity.

  10. INCREASED IL-8 AND IL-6 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    EPA Science Inventory

    INCREASED IL-6 AND IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES.
    R Silbajoris1, A G Lenz2, I Jaspers3, J M Samet1. 1NHEERL, USEPA, RTP, NC, USA; 2GSF-Institute for Inhalation Biology, Neuherberg, Germany; 3 CEMLB, UNC-CH, Chapel Hill, ...

  11. GENE EXPRESSION PROFILES IN HUMAN AND RAT VASCULAR ENDOTHELIAL CELLS EXPOSED TO RESIDUAL OIL FLY ASH (ROFA) AND VANADIUM (V)

    EPA Science Inventory

    Gene expression profiles in human and rat vascular endothelial cells exposed to residual oil fly ash (ROFA) or vanadium (V).
    Srikanth S. Nadadur, Darrell W. Winsett and Daniel L. Costa, US EPA, ORD, NHEERL (ETD, Pulmonary Toxicology Branch), Research Triangle Park, NC 27711.

  12. IN VITRO EFFECTS OF PARTICULATE MATTER ON AIRWAY EPITHELIAL CELLS ISOLATED FROM CONCENTRATED AIR PARTICLES-EXPOSED SPONTANEOUS HYPERTENSIVE RATS

    EPA Science Inventory

    In vitro effects of particulate matter on airway epithelial cells isolated from concentrated air particles-exposed spontaneous hypertensive rats

    Ines Pagan, Urmila Kodavanti, Paul Evansky, Daniel L Costa and Janice A Dye. U.S. Environmental Protection Agency, ORD, National...

  13. Responses of well-differentiated nasal epithelial cells exposed to particles: Role of the epithelium in airway inflammation

    SciTech Connect

    Auger, Floriane; Gendron, Marie-Claude; Chamot, Christophe; Marano, Francelyne; Dazy, Anne-Catherine . E-mail: dazy@paris7.jussieu.fr

    2006-09-15

    Numerous epidemiological studies support the contention that ambient air pollution particles can adversely affect human health. To explain the acute inflammatory process in airways exposed to particles, a number of in vitro studies have been performed on cells grown submerged on plastic and poorly differentiated, and on cell lines, the physiology of which is somewhat different from that of well-differentiated cells. In order to obtain results using a model system in which epithelial cells are similar to those of the human airway in vivo, apical membranes of well-differentiated human nasal epithelial (HNE) cells cultured in an air-liquid interface (ALI) were exposed for 24 h to diesel exhaust particles (DEP) and Paris urban air particles (PM{sub 2.5}). DEP and PM{sub 2.5} (10-80 {mu}g/cm{sup 2}) stimulated both IL-8 and amphiregulin (ligand of EGFR) secretion exclusively towards the basal compartment. In contrast, there was no IL-1{beta} secretion and only weak non-reproducible secretion of TNF-{alpha}. IL-6 and GM-CSF were consistently stimulated towards the apical compartment and only when cells were exposed to PM{sub 2.5}. ICAM-1 protein expression on cell surfaces remained low after particle exposure, although it increased after TNF-{alpha} treatment. Internalization of particles, which is believed to initiate oxidative stress and proinflammatory cytokine expression, was restricted to small nanoparticles ({<=} 40 nm). Production of reactive oxygen species (ROS) was detected, and DEP were more efficient than PM{sub 2.5}. Collectively, our results suggest that airway epithelial cells exposed to particles augment the local inflammatory response in the lung but cannot alone initiate a systemic inflammatory response.

  14. Increased frequency of CD4-8-T cells bearing T-cell receptor alpha beta chains in peripheral blood of atomic bomb survivors exposed to high doses.

    PubMed

    Kusunoki, Y; Kyoizumi, S; Hirai, Y; Fujita, S; Akiyama, M

    1994-07-01

    A rare T-cell subpopulation, CD4-8- alpha beta T cells, may be differentiated through a pathway (or pathways) different from the pathway(s) of conventional CD4+ or CD8+ T cells. In the present study, the frequencies of CD4-8-T cells in peripheral-blood alpha beta T cells in 409 atomic bomb survivors (160 estimated to have been exposed to 1.5 Gy or more and 249 controls) were determined to investigate late effects of radiation on the composition of human T-cell subpopulations. The frequency of CD4-8- alpha beta T-cell decreased significantly with the subject's age and was higher in females than males. A significant increase in the frequency was found in the survivors exposed to more than 1.5 Gy, suggesting that the previous radiation exposure altered differentiation and development of T cells.

  15. Nanocrystal-based electrochemiluminescence sensor for cell detection with Au nanoparticles and isothermal circular double-assisted signal amplification.

    PubMed

    Dai, Pan-Pan; Li, Jin-Yi; Yu, Tao; Xu, Jing-Juan; Chen, Hong-Yuan

    2015-08-15

    Here we have developed a sensitive cancer cell amplified detection method which combined Au NPs enhanced electrochemiluminescence (ECL) of CdS nanocrystals (NCs) film, with isothermal circular amplification reaction of polymerase. In DNA circular amplification detection system, hairpin DNA beacon/Au NPs composite modified CdS NCs film was used as an ECL emitter. Messenger DNA is hybridized with the aptamer modified on magnetic beads (MBs) to form MB-Au bioconjugates. In the presence of HL-60 cell, the aptamer would conjugate with the glycoprotein at cell surface and messenger DNA sequence would be released. The released messenger DNA sequence was then introduced into the cycle amplification system to trigger circular polymerizations. This assay allows us to determine the released messenger DNA equivalent to 10 cells and exhibits a significant specificity for HL-60 cells.

  16. Relationship of CD86 surface marker expression and cytotoxicity on dendritic cells exposed to chemical allergen

    SciTech Connect

    Hulette, Ben C.; Ryan, Cindy A.; Gildea, Lucy A.; Gerberick, G. Frank . E-mail: gerberick.gf@pg.com

    2005-12-01

    Human peripheral blood-derived dendritic cells (DC) respond to a variety of chemical allergens by up-regulating expression of the co-stimulatory molecule CD86. It has been postulated that this measure might provide the basis for an in vitro alternative approach for the identification of skin sensitizing chemicals. We recently reported that DC, exposed in culture to the highest non-cytotoxic concentrations of various chemical allergens, displayed marginal up-regulation of membrane CD86 expression; the interpretation being that such changes were insufficiently sensitive for the purposes of hazard identification. For the work presented here, immature DC were derived from human monocytes and treated with the chemical allergens 2,4-dinitrobenzenesulfonic acid (DNBS), nickel sulfate (NiSO{sub 4}), p-phenylenediamine (PPD), Bandrowski's base (BB), hydroquinone (HQ) and propyl gallate (PG) for 48 h at concentrations which induced both no to slight to moderate cytotoxicity. For comparison, DC were treated with the irritants sodium dodecyl sulfate (SDS), benzoic acid (BA), and benzalkonium chloride (BZC) at concentrations resulting in comparable levels of cytotoxicity. CD86 expression, as measured by flow cytometry, was consistently up-regulated (ranging from 162 to 386% control) on DC treated with concentrations of chemical allergens that induced approximately 10-15% cytotoxicity. The irritants BA and BZC did not induce up-regulation of CD86 expression when tested at concentrations that induced similar levels of cytotoxicity. SDS, however, up-regulated CD86 expression to 125-138% of control in 2/4 preparations when tested at concentrations which induced similar toxicity. Our results confirm that chemical allergens up-regulate CD86 expression on blood-derived DC and illustrate further that up-regulation of CD86 surface marker expression is more robust when DC are treated with concentrations of chemical allergen that induce slight to moderate cytotoxicity.

  17. Calcium signalling in human neutrophil cell lines is not affected by low-frequency electromagnetic fields.

    PubMed

    Golbach, Lieke A; Philippi, John G M; Cuppen, Jan J M; Savelkoul, Huub F J; Verburg-van Kemenade, B M Lidy

    2015-09-01

    We are increasingly exposed to low-frequency electromagnetic fields (LF EMFs) by electrical devices and power lines, but if and how these fields interact with living cells remains a matter of debate. This study aimed to investigate the potential effect of LF EMF exposure on calcium signalling in neutrophils. In neutrophilic granulocytes, activation of G-protein coupled receptors leads to efflux of calcium from calcium stores and influx of extracellular calcium via specialised calcium channels. The cytoplasmic rise of calcium induces cytoskeleton rearrangements, modified gene expression patterns, and cell migration. If LF EMF modulates intracellular calcium signalling, this will influence cellular behaviour and may eventually lead to health problems. We found that calcium mobilisation upon chemotactic stimulation was not altered after a short 30 min or long-term LF EMF exposure in human neutrophil-like cell lines HL-60 or PLB-985. Neither of the two investigated wave forms (Immunent and 50 Hz sine wave) at three magnetic flux densities (5 μT, 300 μT, and 500 μT) altered calcium signalling in vitro. Gene-expression patterns of calcium-signalling related genes also did not show any significant changes after exposure. Furthermore, analysis of the phenotypical appearance of microvilli by scanning electron microscopy revealed no alterations induced by LF EMF exposure. The findings above indicate that exposure to 50 Hz sinusoidal or Immunent LF EMF will not affect calcium signalling in neutrophils in vitro.

  18. USING PROTEOMICS TO MONITOR PROTEIN EXPRESSION IN HUMAN CELLS EXPOSED TO CARCINOGENS

    EPA Science Inventory

    People are continuously exposed exogenously to varying amounts of chemicals that have been shown to have carcinogenic properties in experimental systems. It has been estimated that exposure to environmental chemical carcinogens in the environment may contribute significantly to t...

  19. Upregulation of TRPM7 augments cell proliferation and interleukin-8 release in airway smooth muscle cells of rats exposed to cigarette smoke

    PubMed Central

    LIN, XIAOLING; YANG, CHENG; HUANG, LINJIE; CHEN, MING; SHI, JIANTING; OUYANG, LIHUA; TANG, TIANTIAN; ZHANG, WEI; LI, YIQUN; LIANG, RUIYUN; JIANG, SHANPING

    2016-01-01

    Proliferation and synthetic function (i.e. the capacity to release numerous chemokines and cytokines) of airway smooth muscle cells (ASMCs) are important in airway remodeling induced by cigarette smoke exposure. However, the molecular mechanism has not been clarified. Transient receptor potential cation channel subfamily M member 7 (TRPM7) is expressed ubiquitously and is crucial for the cellular physiological function of many cell types. The present study aimed to detect the expression of TRPM7 in ASMCs from smoke-exposed rats and determine the importance of TRPM7 in proliferation and interleukin-8 (IL-8) release. ASMCs were isolated and cultured from smoke-exposed rats. Expression levels of TRPM7 were determined by reverse transcription-polymerase chain reaction, western blot analysis and immunofluorescence. TRPM7 was silenced with TRPM7-short hairpin RNA lentivirus vector. DNA synthesis, cell number and IL-8 release of ASMCs induced by cigarette smoke extract (CSE) and tumor necrosis factor-α (TNF-α) were assessed using [3H]-thymidine incorporation assay, hemocytometer and enzyme-linked immunosorbent assay, respectively. It was determined that mRNA and protein expression levels of TRPM7 were increased in ASMCs from smoke-exposed rats. Stimulation with CSE or TNF-α elevated DNA synthesis, cell number and IL-8 release were more marked in ASMCs from smoke-exposed rats. Silencing of TRPM7 reduced DNA synthesis, cell number and IL-8 release induced by CSE or TNF-α in ASMCs from smoke-exposed rats. In conclusion, expression of TRPM7 increased significantly in ASMCs from smoke-exposed rats and the upregulation of TRPM7 led to augmented cell proliferation and IL-8 release in ASMCs from rats exposed to cigarette smoke. PMID:27108806

  20. Hyperoside enhances the suppressive effects of arsenic trioxide on acute myeloid leukemia cells

    PubMed Central

    Zhang, Feng; Zhu, Fang-Bing; Li, Jia-Jia; Zhang, Ping-Ping; Zhu, Jun-Feng

    2015-01-01

    Hyperoside (Hyp) is the chief component of some Chinese herbs which has anticancer effect and the present study is to identify whether it could enhance the anti leukemic properties of arsenic trioxide (As2O3) in acute myeloid leukemia (AML). We provide evidence on the concomitant treatment of HL-60 human AML cells with hyperoside potentiates As2O3-dependent induction of apoptosis. The activation of caspase-9, Bcl-2-associated agonist of cell death (BAD), p-BAD, p27 was assessed by Western blot. Results showed that hyperoside inhibited BAD from phosphorylating, reactivated caspase-9, and increased p27 levels. Importantly, hyperoside demonstrated its induction of autophagy effect by upregulation of LC-II in HL-60 AML cell line. Taken together, hyperoside may serve as a great candidate of concomitant treatment for leukemia; these effects were probably related to induction of autophagy and enhancing apoptosis-inducing action of As2O3. PMID:26629016

  1. Developing a Novel Indolocarbazole as Histone Deacetylases Inhibitor against Leukemia Cell Lines

    PubMed Central

    Wang, Wenjing; Lv, Maomin; Zhao, Xiong; Zhang, Jingang

    2015-01-01

    A novel indolocarbazole (named as ZW2-1) possessing HDAC inhibition activity was synthesized and evaluated against human leukemia cell lines HL-60 and NB4. ZW2-1 performed anti-population growth effect which was in a concentration-dependent manner (2–12 μM) by inducing both apoptosis and autophagy in cells. The compound also caused differentiation of HL-60 and NB4 cells as shown by increasing expression of CD11b, CD14, and CD38 at moderate concentration (4 μM). At relatively high concentration (8 μM), ZW2-1 significantly decreased intracellular histone deacetylase 1 level which was also observed. All the results indicated that ZW2-1 could be a novel antileukemia lead capable of simultaneously inducing apoptosis, autophagy, and differentiation. PMID:26649226

  2. Increase in DNA damage in lymphocytes and micronucleus frequency in buccal cells in silica-exposed workers

    PubMed Central

    Halder, Ajanta; De, Madhusnata

    2012-01-01

    The alkaline single cell gel electrophoresis (comet assay) was applied to study the genotoxic properties of silica in human peripheral blood lymphocytes (PBL). The study was designed to evaluate the DNA damage of lymphocytes and the end points like micronuclei from buccal smears in a group of 45 workers, occupationally exposed to silica, from small mines and stone quarries. The results were compared to 20 sex and age matched normal individuals. There was a statistically significant difference in the damage levels between the exposed group and the control groups. The types of damages (type I –type 1V) were used to measure the DNA damage. The numbers of micronuclei were higher in the silica-exposed population. The present study suggests that the silica exposure can induce lymphocyte DNA damage and produces significant variation of micronuclei in buccal smear. PMID:23112505

  3. Comparison of hematological alterations and markers of B-cell activation in workers exposed to benzene, formaldehyde and trichloroethylene.

    PubMed

    Bassig, Bryan A; Zhang, Luoping; Vermeulen, Roel; Tang, Xiaojiang; Li, Guilan; Hu, Wei; Guo, Weihong; Purdue, Mark P; Yin, Songnian; Rappaport, Stephen M; Shen, Min; Ji, Zhiying; Qiu, Chuangyi; Ge, Yichen; Hosgood, H Dean; Reiss, Boris; Wu, Banghua; Xie, Yuxuan; Li, Laiyu; Yue, Fei; Freeman, Laura E Beane; Blair, Aaron; Hayes, Richard B; Huang, Hanlin; Smith, Martyn T; Rothman, Nathaniel; Lan, Qing

    2016-07-01

    Benzene, formaldehyde (FA) and trichloroethylene (TCE) are ubiquitous chemicals in workplaces and the general environment. Benzene is an established myeloid leukemogen and probable lymphomagen. FA is classified as a myeloid leukemogen but has not been associated with non-Hodgkin lymphoma (NHL), whereas TCE has been associated with NHL but not myeloid leukemia. Epidemiologic associations between FA and myeloid leukemia, and between benzene, TCE and NHL are, however, still debated. Previously, we showed that these chemicals are associated with hematotoxicity in cross-sectional studies of factory workers in China, which included extensive personal monitoring and biological sample collection. Here, we compare and contrast patterns of hematotoxicity, monosomy 7 in myeloid progenitor cells (MPCs), and B-cell activation biomarkers across these studies to further evaluate possible mechanisms of action and consistency of effects with observed hematologic cancer risks. Workers exposed to benzene or FA, but not TCE, showed declines in cell types derived from MPCs, including granulocytes and platelets. Alterations in lymphoid cell types, including B cells and CD4+ T cells, and B-cell activation markers were apparent in workers exposed to benzene or TCE. Given that alterations in myeloid and lymphoid cell types are associated with hematological malignancies, our data provide biologic insight into the epidemiological evidence linking benzene and FA exposure with myeloid leukemia risk, and TCE and benzene exposure with NHL risk. PMID:27207665

  4. High-throughput downstream process development for cell-based products using aqueous two-phase systems.

    PubMed

    Zimmermann, Sarah; Gretzinger, Sarah; Schwab, Marie-Luise; Scheeder, Christian; Zimmermann, Philipp K; Oelmeier, Stefan A; Gottwald, Eric; Bogsnes, Are; Hansson, Mattias; Staby, Arne; Hubbuch, Jürgen

    2016-09-16

    As the clinical development of cell-based therapeutics has evolved immensely within the past years, downstream processing strategies become more relevant than ever. Aqueous two-phase systems (ATPS) enable the label-free, scalable, and cost-effective separation of cells, making them a promising tool for downstream processing of cell-based therapeutics. Here, we report the development of an automated robotic screening that enables high-throughput cell partitioning analysis in ATPS. We demonstrate that this setup enables fast and systematic investigation of factors influencing cell partitioning. Moreover, we examined and optimized separation conditions for the differentiable promyelocytic cell line HL-60 and used a counter-current distribution-model to investigate optimal separation conditions for a multi-stage purification process. Finally, we show that the separation of CD11b-positive and CD11b-negative HL-60 cells is possible after partial DMSO-mediated differentiation towards the granulocytic lineage. The modeling data indicate that complete peak separation is possible with 30 transfers, and >93% of CD11b-positive HL-60 cells can be recovered with >99% purity. The here described screening platform facilitates faster, cheaper, and more directed downstream process development for cell-based therapeutics and presents a powerful tool for translational research. PMID:27567679

  5. SDF-1/CXCL12 modulates mitochondrial respiration of immature blood cells in a bi-phasic manner.

    PubMed

    Messina-Graham, Steven; Broxmeyer, Hal

    2016-05-01

    SDF-1/CXCL12 is a potent chemokine required for the homing and engraftment of hematopoietic stem and progenitor cells. Previous data from our group has shown that in an SDF-1/CXCL12 transgenic mouse model, lineage(-) Sca-1(+) c-Kit(+) (LSK) bone marrow cells have reduced mitochondrial membrane potential versus wild-type. These results suggested that SDF-1/CXCL12 may function to keep mitochondrial respiration low in immature blood cells in the bone marrow. Low mitochondrial metabolism helps to maintain low levels of reactive oxygen species (ROS), which can influence differentiation. To test whether SDF-1/CXCL12 regulates mitochondrial metabolism, we employed the human leukemia cell line HL-60, that expresses high levels of the SDF-1/CXCL12 receptor, CXCR4, as a model of hematopoietic progenitor cells in vitro. We treated HL-60 cells with SDF-1/CXCL12 for 2 and 24h. Oxygen consumption rates (OCR), mitochondrial-associated ATP production, mitochondrial mass, and mitochondrial membrane potential of HL-60 cells were significantly reduced at 2h and increased at 24h as compared to untreated control cells. These biphasic effects of SDF-1/CXCL12 were reproduced with lineage negative primary mouse bone marrow cells, suggesting a novel function of SDF-1/CXCL12 in modulating mitochondrial respiration by regulating mitochondrial oxidative phosphorylation, ATP production and mitochondrial content. PMID:27067482

  6. Levels of 8-hydroxy-2'-deoxyguanosine in DNA of white blood cells from workers highly exposed to asbestos in Germany.

    PubMed

    Marczynski, B; Rozynek, P; Kraus, T; Schlösser, S; Raithel, H J; Baur, X

    2000-07-10

    Asbestos fibers have genotoxic effects and are a potential carcinogenic hazard to occupationally exposed workers. The ability of inhaled asbestos fibers to induce the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the DNA of white blood cells (WBC) of workers highly exposed at the workplace has been studied. The 8-OHdG adduct level of asbestos-exposed workers was significantly increased (p<0.001) compared to that in the control group in all three years of the study. Asbestos-exposed individuals showed a mean value of 2.61+/-0.91 8-OHdG/10(5) dG (median 2.49, n=496) in 1994-1995, 2.96+/-1.10 8-OHdG/10(5) dG (median 2.76, n=437) in 1995-1996 and 2.55+/-0.56 8-OHdG/10(5) dG (median 2.53, n=447) in 1996-1997. For the control subjects, a mean of 1.52+/-0.39 (median 1.51, n=214) was determined. The results indicate that human DNA samples from exposed individuals contain between 1.7 times and twice the level of oxidative damage relative to that found in control samples in all 3 years of the study. The studies presented here show that asbestos exposure can result in oxidative DNA damage. Our data confirm that oxidative DNA damage occurs in the WBC of workers highly exposed to asbestos fibers, thus supporting the hypothesis that asbestos fibers damage cells through an oxidative mechanism. These in vivo findings underline the importance of oxidative damage in asbestos-induced carcinogenesis and highlight the need for exploring the molecular basis of asbestos-induced diseases, and for more effective diagnosis, prevention and therapy of mesothelioma, lung cancer and pulmonary fibrosis. In addition, preventive and therapeutic approaches using antioxidants may be relevant.

  7. Acute respiratory bronchiolitis: an ultrastructural and autoradiographic study of epithelial cell injury and renewal in rhesus monkeys exposed to ozone.

    PubMed Central

    Castleman, W. L.; Dungworth, D. L.; Schwartz, L. W.; Tyler, W. S.

    1980-01-01

    The pathogenesis of acute respiratory bronchiolitis was examined in rhesus monkeys exposed to 0.8 ppm ozone fpr 4--50 hours. Epithelial injury and renewal was qualitatively and quantitatively characterized by correlated techniques of scanning and transmission electron microscopy as well as by light-microscopic autoradiography following labeling with tritiated thymidine. Extensive degeneration and necrosis of Type 1 epithelial cells occurred on the respiratory bronchiolar wall during the initial 4--12 hours of exposure. Increased numbers of labeled epithelial cells were present in this region after 18 hours of exposure, and the highest labeling index (18% was measured after 50 hours of exposure. Most (67--80%) of the labeled cells and all the mitotic epithelial cells (22) observed ultrastructurally were cuboidal bronchiolar epithelial cells. Of the labeled epithelial cells, 20--33% were Type 2 epithelial cells. After 50 hours of exposure the respiratory bronchiolar epithelium was hyperplastic. The predominant inflammatory cell in respiratory bronchiolar exudate was the alveolar macrophage. Monkeys that were exposed for 50 hours and allowed to recover in unozonized air for 7 days had incomplete resolution of respiratory bronchiolar epithelial hyperplasia. The results indicate that Type 1 epithelial cells lining respiratory bronchioles are the cell type most sensitive to injury and that both cuboidal bronchiolar epithelial cells and Type 2 epithelial cells function as stem cells in epithelial renewal. Images Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 23 Figure 24 Figure 25 Figure 9 Figure 10 Figure 26 Figure 27 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 PMID:6767409

  8. Modulation of endothelial cell network formation in vitro by molecular signaling of head and neck squamous cell carcinoma (HNSCC) exposed to cetuximab.

    PubMed

    Jouan-Hureaux, Valérie; Boura, Cédric; Merlin, Jean-Louis; Faivre, Béatrice

    2012-03-01

    Overexpression of EGFR plays a key-role in head and neck squamous cell carcinoma (HNSCC) and justifies the extensive use of cetuximab, a monoclonal anti-EGFR antibody, as well as EGFR-tyrosine kinase inhibitors (EGFR-TKI), which have been reported to inhibit tumor cell growth and the secretion of pro-angiogenic factors by tumor cells, such as VEGF and IL-8. Moreover, vessel normalization in tumors, suggesting a more complex mediation of endothelial cell growth control has also been observed in vivo. The present study was designed to investigate the angiogenic consequences of exposure of HNSCC tumor cell lines to cetuximab and intercellular signaling between tumor and endothelial cells by secretion of pro- and anti-angiogenic mediators in the conditioned media (CM). The results achieved showed that cetuximab decreased the secretion of VEGF by HNSCC cells and that exposure of human umbilical vein endothelial cells (HUVEC) to CM from HNSCC cells exposed to cetuximab induced an increase in endothelial cell network formation. Angiogenesis proteome profiling showed that cetuximab induced a complex alteration of the secretion of pro- and anti-angiogenic factors by HNSCC cells without enabling to identify a unique molecular marker. Expression of endothelial membrane receptors (VEGFR-2, EGFR, PECAM-1 and Notch-4) was investigated and only EGFR expression was found influenced when HUVEC were exposed to CM from cetuximab-exposed HNSCC cells. These results showed that the decrease in the secretion of pro-angiogenic agents like VEGF by HNSCC cells exposed to cetuximab could not be sufficient to justify its anti-angiogenic activity in vitro. PMID:21820450

  9. Exposure of human cells to electromagnetic fields. Final report, 1 January 1988-31 December 1989

    SciTech Connect

    Henderson, A.S.

    1990-02-27

    This study addressed the following basic question: How does extremely low-level non-ionizing radiation affect human cells, and if there are cellular responses that can be directly related to signal parameters such as frequency, amplitude and time of exposure. The focus of these studies was to identify transcriptional changes in human cultured cells, HL60, which result from exposure of these cells to defined extremely low frequency electromagnetic fields (elf EMFS). Our experiments show a pronounced measurable response observed as transcript increase, with associated changes in protein synthesis. The major findings relative to transcriptional changes are fourfold: (1) transcript changes in human cells correlate with previous findings of transcriptional and translational changes in Drosophila salivary gland cells; (2) the frequency of the signal in the amplitude (with resulting changes in E- and B-fields) in log increments from 0.5 to 500 uV at 60 Hz gives both amplitude and time-dependent windows, and (4) genes not usually expressed in HL-60 are unaffected by exposure to elf EMFs. Changes in the overall protein synthetic pattern have also been observed following exposure of HL60 cells to 60 Hz signals.

  10. Controversial cytogenetic observations in mammalian somatic cells exposed to extremely low frequency electromagnetic radiation: a review and future research recommendations.

    PubMed

    Vijayalaxmi; Obe, Guenter

    2005-07-01

    During the years 1990-2003, a large number of investigations were conducted using animals, cultured rodent and human cells as well as freshly collected human blood lymphocytes to determine the genotoxic potential of exposure to nonionizing radiation emitted from extremely low frequency electromagnetic fields (EMF). Among the 63 peer reviewed scientific reports, the conclusions from 29 studies (46%) did not indicate increased damage to the genetic material, as assessed from DNA strand breaks, incidence of chromosomal aberrations (CA), micronuclei (MN), and sister chromatid exchanges (SCE), in EMF exposed cells as compared with sham exposed and/or unexposed cells, while those from 14 investigations (22%) have suggested an increase in such damage in EMF exposed cells. The observations from 20 other studies (32%) were inconclusive. This study reviews the investigations published in peer reviewed scientific journals during 1990-2003 and attempts to identify probable reason(s) for the conflicting results. Recommendations are made for future research to address some of the controversial observations.

  11. Chromosome-wide aneuploidy study of cultured circulating myeloid progenitor cells from workers occupationally exposed to formaldehyde.

    PubMed

    Lan, Qing; Smith, Martyn T; Tang, Xiaojiang; Guo, Weihong; Vermeulen, Roel; Ji, Zhiying; Hu, Wei; Hubbard, Alan E; Shen, Min; McHale, Cliona M; Qiu, Chuangyi; Liu, Songwang; Reiss, Boris; Beane-Freeman, Laura; Blair, Aaron; Ge, Yichen; Xiong, Jun; Li, Laiyu; Rappaport, Stephen M; Huang, Hanlin; Rothman, Nathaniel; Zhang, Luoping

    2015-01-01

    Formaldehyde (FA) is an economically important industrial chemical to which millions of people worldwide are exposed environmentally and occupationally. Recently, the International Agency for Cancer Research concluded that there is sufficient evidence that FA causes leukemia, particularly myeloid leukemia. To evaluate the biological plausibility of this association, we employed a chromosome-wide aneuploidy study approach, which allows the evaluation of aneuploidy and structural chromosome aberrations (SCAs) of all 24 chromosomes simultaneously, to analyze cultured myeloid progenitor cells from 29 workers exposed to relatively high levels of FA and 23 unexposed controls. We found statistically significant increases in the frequencies of monosomy, trisomy, tetrasomy and SCAs of multiple chromosomes in exposed workers compared with controls, with particularly notable effects for monosomy 1 [P = 6.02E-06, incidence rate ratio (IRR) = 2.31], monosomy 5 (P = 9.01E-06; IRR = 2.24), monosomy 7 (P = 1.57E-05; IRR = 2.17), trisomy 5 (P = 1.98E-05; IRR = 3.40) and SCAs of chromosome 5 (P = 0.024; IRR = 4.15). The detection of increased levels of monosomy 7 and SCAs of chromosome 5 is particularly relevant as they are frequently observed in acute myeloid leukemia. Our findings provide further evidence that leukemia-related cytogenetic changes can occur in the circulating myeloid progenitor cells of healthy workers exposed to FA, which may be a potential mechanism underlying FA-induced leukemogenesis.

  12. Chromosome-wide aneuploidy study of cultured circulating myeloid progenitor cells from workers occupationally exposed to formaldehyde.

    PubMed

    Lan, Qing; Smith, Martyn T; Tang, Xiaojiang; Guo, Weihong; Vermeulen, Roel; Ji, Zhiying; Hu, Wei; Hubbard, Alan E; Shen, Min; McHale, Cliona M; Qiu, Chuangyi; Liu, Songwang; Reiss, Boris; Beane-Freeman, Laura; Blair, Aaron; Ge, Yichen; Xiong, Jun; Li, Laiyu; Rappaport, Stephen M; Huang, Hanlin; Rothman, Nathaniel; Zhang, Luoping

    2015-01-01

    Formaldehyde (FA) is an economically important industrial chemical to which millions of people worldwide are exposed environmentally and occupationally. Recently, the International Agency for Cancer Research concluded that there is sufficient evidence that FA causes leukemia, particularly myeloid leukemia. To evaluate the biological plausibility of this association, we employed a chromosome-wide aneuploidy study approach, which allows the evaluation of aneuploidy and structural chromosome aberrations (SCAs) of all 24 chromosomes simultaneously, to analyze cultured myeloid progenitor cells from 29 workers exposed to relatively high levels of FA and 23 unexposed controls. We found statistically significant increases in the frequencies of monosomy, trisomy, tetrasomy and SCAs of multiple chromosomes in exposed workers compared with controls, with particularly notable effects for monosomy 1 [P = 6.02E-06, incidence rate ratio (IRR) = 2.31], monosomy 5 (P = 9.01E-06; IRR = 2.24), monosomy 7 (P = 1.57E-05; IRR = 2.17), trisomy 5 (P = 1.98E-05; IRR = 3.40) and SCAs of chromosome 5 (P = 0.024; IRR = 4.15). The detection of increased levels of monosomy 7 and SCAs of chromosome 5 is particularly relevant as they are frequently observed in acute myeloid leukemia. Our findings provide further evidence that leukemia-related cytogenetic changes can occur in the circulating myeloid progenitor cells of healthy workers exposed to FA, which may be a potential mechanism underlying FA-induced leukemogenesis. PMID:25391402

  13. Phenotypic malignant changes and untargeted lipidomic analysis of long-term exposed prostate cancer cells to endocrine disruptors

    SciTech Connect

    Bedia, Carmen Dalmau, Núria Jaumot, Joaquim Tauler, Romà

    2015-07-15

    Endocrine disruptors (EDs) are a class of environmental toxic molecules able to interfere with the normal hormone metabolism. Numerous studies involve EDs exposure to initiation and development of cancers, including prostate cancer. In this work, three different EDs (aldrin, aroclor 1254 and chlorpyrifos (CPF)) were investigated as potential inducers of a malignant phenotype in DU145 prostate cancer cells after a chronic exposure. Epithelial to mesenchymal transition (EMT) induction, proliferation, migration, colony formation and release of metalloproteinase 2 (MMP-2) were analyzed in 50-day exposed cells to the selected EDs. As a result, aldrin and CPF exposure led to an EMT induction (loss of 16% and 14% of E-cadherin levels, respectively, compared to the unexposed cells). Aroclor and CPF presented an increased migration (134% and 126%, respectively), colony formation (204% and 144%, respectively) and MMP-2 release (137% in both cases) compared to the unexposed cells. An untargeted lipidomic analysis was performed to decipher the lipids involved in the observed transformations. As general results, aldrin exposure showed a global decrease in phospholipids and sphingolipids, and aroclor and CPF showed an increase of certain phospholipids, glycosphingolipids as well as a remarkable increase of some cardiolipin species. Furthermore, the three exposures resulted in an increase of some triglyceride species. In conclusion, some significant changes in lipids were identified and thus we postulate that some lipid compounds and lipid metabolic pathways could be involved in the acquisition of the malignant phenotype in exposed prostate cancer cells to the selected EDs. - Highlights: • Aldrin, aroclor and chlorpyrifos induced an aggressive phenotype in DU145 cells. • An untargeted lipidomic analysis has been performed on chronic exposed cells. • Lipidomic results showed changes in specific lipid species under chronic exposure. • These lipids may have a role in the

  14. Evaluation of cytotoxicity, morphological alterations and oxidative stress in Chinook salmon cells exposed to copper oxide nanoparticles.

    PubMed

    Srikanth, Koigoora; Pereira, Eduarda; Duarte, Armando C; Rao, Janapala Venkateswara

    2016-05-01

    The current study is aimed to study cytotoxicity and oxidative stress mediated changes induced by copper oxide nanoparticles (CuO NPs) in Chinook salmon cells (CHSE-214). To this end, a number of biochemical responses are evaluated in CHSE-214 cells which are as follows [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] MTT, neutral red uptake (NRU), lactate dehydrogenase (LDH), protein carbonyl (PC), lipid peroxidation (LPO), oxidised glutathione (GSSG), reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione sulfo-transferase (GST), superoxide dismutase (SOD), catalase (CAT), 8-Hydroxy-2'-deoxyguanosine (8-OHdG) and reactive oxygen species (ROS), respectively. The 50% inhibition concentration (IC50) of CuO NPs to CHSE-214 cells after 24 h exposure was found to be 19.026 μg ml(-1). Viability of cells was reduced by CuO NPs, and the decrease was dose dependent as revealed by the MTT and NRU assay. CHSE-214 cells exposed to CuO NPs induced morphological changes. Initially, cells started to detach from the surface (12 h), followed by polyhedric, fusiform appearance (19 h) and finally the cells started to shrink. Later, the cells started losing their cellular contents leading to their death only after 24 h. LDH, PC, LPO, GSH, GPx, GST, SOD, CAT, 8-OHdG and ROS responses were seen significantly increased with the increase in the concentration of CuO NPs when compared to their respective controls. However, significant decrease in GSSG was perceptible in CHSE-214 cells exposed to CuO NPs in a dose-dependent manner. Our data demonstrated that CuO NPs induced cytotoxicity in CHSE-214 cells through the mediation of oxidative stress. The current study provides a baseline for the CuO NPs-mediated cytotoxic assessment in CHSE-214 cells for the future studies. PMID:26115719

  15. A novel PAD4/SOX4/PU.1 signaling pathway is involved in the committed differentiation of acute promyelocytic leukemia cells into granulocytic cells

    PubMed Central

    Song, Guanhua; Shi, Lulu; Guo, Yuqi; Yu, Linchang; Wang, Lin; Zhang, Xiaoyu; Li, Lianlian; Han, Yang; Ren, Xia; Guo, Qiang; Bi, Kehong; Jiang, Guosheng

    2016-01-01

    All-trans retinoic acid (ATRA) treatment yields cure rates > 80% through proteasomal degradation of the PML-RARα fusion protein that typically promotes acute promyelocytic leukemia (APL). However, recent evidence indicates that ATRA can also promote differentiation of leukemia cells that are PML-RARα negative, such as HL-60 cells. Here, gene expression profiling of HL-60 cells was used to investigate the alternative mechanism of impaired differentiation in APL. The expression of peptidylarginine deiminase 4 (PADI4), encoding PAD4, a protein that post-translationally converts arginine into citrulline, was restored during ATRA-induced differentiation. We further identified that hypermethylation in the PADI4 promoter was associated with its transcriptional repression in HL-60 and NB4 (PML-RARα positive) cells. Functionally, PAD4 translocated into the nucleus upon ATRA exposure and promoted ATRA-mediated differentiation. Mechanistic studies using RNAi knockdown or electroporation-mediated delivery of PADI4, along with chromatin immunoprecipitation, helped identify PU.1 as an indirect target and SOX4 as a direct target of PAD4 regulation. Indeed, PAD4 regulates SOX4-mediated PU.1 expression, and thereby the differentiation process, in a SOX4-dependent manner. Taken together, our results highlight an association between PAD4 and DNA hypermethylation in APL and demonstrate that targeting PAD4 or regulating its downstream effectors may be a promising strategy to control differentiation in the clinic. PMID:26673819

  16. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    PubMed

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.

  17. Global transcriptomic analysis of model human cell lines exposed to surface-modified gold nanoparticles: the effect of surface chemistry.

    PubMed

    Grzincic, E M; Yang, J A; Drnevich, J; Falagan-Lotsch, P; Murphy, C J

    2015-01-28

    Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14,000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected.

  18. Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to X-rays.

    PubMed

    Varès, Guillaume; Wang, Bing; Tanaka, Kaoru; Kakimoto, Ayana; Eguchi-Kasai, Kyomi; Nenoi, Mitsuru

    2011-01-10

    The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.

  19. Cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser.

    PubMed

    Alexsandra da Silva Neto Trajano, Larissa; da Silva, Camila Luna; de Carvalho, Simone Nunes; Cortez, Erika; Mencalha, André Luiz; de Souza da Fonseca, Adenilson; Stumbo, Ana Carolina

    2016-07-01

    Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm(2)) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells. PMID:26886589

  20. LASER CAPTURE MICRODISSECTION AND GENE ARRAY ANALYSIS OF PALATAL EPITHELIAL AND MESENCHYMAL CELLS EXPOSED TO TCDD

    EPA Science Inventory

    Palatal shelves from embryos exposed on gestation day (GD) 12 to either retinoic acid (RA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contact but fail to fuse. It is of interest to know if diverse agents that induce clefting via the same etiology also activate the same biochem...

  1. Gene expression profiling and pathway analysis of human bronchial epithelial cells exposed to airborne particulate matter collected from Saudi Arabia

    PubMed Central

    Sun, Hong; Shamy, Magdy; Kluz, Thomas; Muñoz, Alexandra B.; Zhong, Mianhua; Laulicht, Freda; Alghamdi, Mansour A.; Khoder, Mamdouh I.; Chen, Lung-Chi; Costa, Max

    2013-01-01

    Epidemiological studies have established a positive correlation between human mortality and increased concentration of airborne particulate matters (PM). However, the mechanisms underlying PM related human diseases, as well as the molecules and pathways mediating the cellular response to PM, are not fully understood. This study aims to investigate the global gene expression changes in human cells exposed to PM10 and to identify genes and pathways that may contribute to PM related adverse health effects. Human bronchial epithelial cells were exposed to PM10 collected from Saudi Arabia for 1 or 4 days, and whole transcript expression was profiled using the GeneChip human gene 1.0 ST array. A total of 140 and 230 genes were identified that significantly changed more than 1.5 fold after PM10 exposure for 1 or 4 days, respectively. Ingenuity Pathway Analysis revealed that different exposure durations triggered distinct pathways. Genes involved in NRF2-mediated response to oxidative stress were up-regulated after 1 day exposure. In contrast, cells exposed for 4 days exhibited significant changes in genes related to cholesterol and lipid synthesis pathways. These observed changes in cellular oxidative stress and lipid synthesis might contribute to PM related respiratory and cardiovascular disease. PMID:23085030

  2. Evaluation of DNA damage in agricultural workers exposed to pesticides using single cell gel electrophoresis (comet) assay

    PubMed Central

    Kaur, Raminderjeet; Kaur, Satbir; Lata, Mukesh

    2011-01-01

    BACKGROUND: Pesticides are used in agriculture to protect crops, but they pose a potential risk to farmers and environment. The aim of the present study is to investigate the relation between the occupational exposure to various pesticides and the presence of DNA damage. MATERIALS AND METHODS: Blood samples of 210 exposed workers (after a day of intense spraying) and 50 control subjects belonging to various districts of Punjab (India) were evaluated using Comet assay. Sixty workers who showed DNA damage were selected for follow up at 5-6 months after the first sampling during a low or null spraying period. RESULTS: Significant differences were found in DNA damage between freshly exposed workers and controls and freshly exposed and followed up cases. There was significant increase in the comet parameters viz. mean comet tail length and frequency of cells showing migration in exposed workers as compared to controls (72.22 ± 20.76 vs. 46.92 ± 8.17, P<0.001; 31.79 vs. 5.77, P<0.001). In the second samples, followed up cases showed significant decrease in frequency of damaged cells as compared to freshly exposed workers of first sampling (P<0.05). The confounding factors such as variable duration of pesticide exposure, age, smoking, drinking and dietary habits etc which were expected to modulate the damage, were instead found to have no significant effect on DNA fragmentation. CONCLUSION: The evidence of a genetic hazard related to exposure resulting from the intensive use of pesticides stresses the need for educational programs for agricultural workers to reduce the use of chemicals in agriculture. PMID:22345990

  3. Alterations in polyamine levels induced by phorbol diesters and other agents that promote differentiation in human promyelocytic leukemia cells

    SciTech Connect

    Huberman, E.; Weeks, C.; Herrmann, A.; Callaham, M.; Slaga, T.

    1981-02-01

    Polyamine levels were evaluated in human HL-60 promyelocytic leukemia cells after treatment with inducers of terminal differentiation. Differentiation in these cells was determined by increases in the percentage of morphologically mature cells and in lysozyme activity. Treatment of the HL-60 cells with phorbol 12-myristate-13-acetate (PMA), phorbol 12,13-didecanoate or other inducers of terminal differentiation such as dimethylsulfoxide and retinoic acid resulted in increased levels of putrescine. However, no increase in putrescine could be detected after PMA treatment of a HL-60 cell variant that exhibited a decreased susceptibility to PMA-induced terminal differentiation. Similarly, no increase in putrescine was observed with two nontumor-promoters (phorbol 12,13-diacetate and 4-O-methyl-PMA) or with anthralin, a non-phorbol tumor promoter. In addition to enhancing putrescine levels, PMA also increased the amount of spermidine and decreased the amount of spermine. The increase in putrescine and spermidine preceded the expression of the various differentiation markers. Unlike the changes observed in the polyamine levels after PMA treatment, the activities of ornithine and S-adenosylmethionine decarboxylases, which are polyamine biosynthetic enzymes, did not significantly change. ..cap alpha..-Methylornithine and ..cap alpha..-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), which are inhibitors of the polyamine biosynthetic enzymes, did not affect differentiation in control or PMA-treated cells. Because of these observations, we suggest that the change in polyamine levels involve biochemical pathways other than the known biosynthetic ones. By-products of these pathways may perhaps be the controlling factors involved in the induction of terminal differentiation in the HL-60 and other cell types as well.

  4. Signs of Müller cell gliotic response found in the retina of newts exposed to real and simulated microgravity

    NASA Astrophysics Data System (ADS)

    Grigoryan, E. N.; Anton, H. J.; Poplinskaya, V. A.; Aleinikova, K. S.; Domaratskaya, E. I.; Novikova, Y. P.; Almeida, E.

    2012-05-01

    The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Müller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Müller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function.

  5. A new method specifically designed to expose cells isolated in vitro to radon and its decay products.

    PubMed

    Petitot, F; Morlier, J P; Debroche, M; Pineau, J F; Chevillard, S

    2002-06-01

    A system was set up to provide direct exposure of cells cultured in vitro to radon and its decay products. Radon gas emanating from a uranium source was introduced at a measured concentration in a closed 10-m(3) exposure chamber. Cells were cultured on the microporous membrane of an insert that was floating over the culture medium in a six-well cluster plate. Plates with cells were placed in an open thermoregulated bath within the chamber. Under these conditions, cells were irradiated by direct deposition of radon and radon decay products. During exposure, all parameters, including radon gas concentrations, decay product activities, and potential alpha-particle energy concentrations, were determined by periodic air-grab samplings inside the chamber. The energy spectrum of deposited decay products was characterized. An estimation of alpha-particle flux density on the area containing cells was performed using CR-39 detector films that were exposed in cell-free wells during the cell exposure. The number of alpha-particle traversals per cell was deduced both from the mean number of CR-39 tracks per surface unit and from measurements of entire cells or nuclear surfaces. This paper describes the design of experiment, the dosimetry of radon and radon decay product, and the procedures for aerosol measurements. Our preliminary data show the usefulness of the in vitro cell culture approach to the study of the early cellular effects of radon and its decay products.

  6. Cytotoxicity of arsenic trioxide is enhanced by (-)-epigallocatechin-3-gallate via suppression of ferritin in cancer cells

    SciTech Connect

    Lee, Te-Chang; Cheng, I-Cheng; Shue, Jun-Jie; Wang, T.C.

    2011-01-01

    Arsenic trioxide (ATO) treatment is a useful therapy against human acute promyelocytic leukemia (APL), however, it concomitantly brings potential adverse consequences including serious side effect, human carcinogenicity and possible development of resistance. This investigation revealed that those problems might be relaxed by simultaneous application with (-)-epigallocatechin-3-gallate (EGCG), one of the major components from green tea. EGCG significantly lowered down the ATO concentration required for an effective control of APL cells, HL-60. The simultaneous treatment of ATO with EGCG induced a mitochondria-dependent apoptosis in HL-60 cells significantly, which accounted for more than 70% of the cell death in the treatment. The mechanism of apoptosis induction was elucidated. EGCG in HL-60 cells acted as a pro-oxidant enhancing intracellular hydrogen peroxide significantly. ATO, on the other hand, induced heme oxygenase-1 (HO-1) to catalyze heme degradation, thereby provided ferrous iron for EGCG-induced hydrogen peroxide to precede Fenton reaction, which in turn generated deleterious reactive oxygen species to damage cell. In addition, EGCG inhibited expression of ferritin, which supposedly to sequester harmful ferrous iron, thereby augmented the occurrence of Fenton reaction. This investigation also provided evidence that ATO, since mainly acted to induce HO-1 in simultaneous treatment with EGCG, could be replaced by other HO-1 inducer with much less human toxicity. Furthermore, several of our preliminary investigations revealed that the enhanced cytotoxicity induced by combining heme degradation and Fenton reaction is selectively toxic to malignant but not non-malignant cells.

  7. Increased frequency of spontaneous neoplastic transformation in progeny of bystander cells from cultures exposed to densely ionizing radiation.

    PubMed

    Buonanno, Manuela; de Toledo, Sonia M; Azzam, Edouard I

    2011-01-01

    An increased risk of carcinogenesis caused by exposure to space radiation during prolonged space travel is a limiting factor for human space exploration. Typically, astronauts are exposed to low fluences of ionizing particles that target only a few cells in a tissue at any one time. The propagation of stressful effects from irradiated to neighboring bystander cells and their transmission to progeny cells would be of importance in estimates of the health risks of exposure to space radiation. With relevance to the risk of carcinogenesis, we investigated, in model C3H 10T½ mouse embryo fibroblasts (MEFs), modulation of the spontaneous frequency of neoplastic transformation in the progeny of bystander MEFs that had been in co-culture 10 population doublings earlier with MEFs exposed to moderate doses of densely ionizing iron ions (1 GeV/nucleon) or sparsely ionizing protons (1 GeV). An increase (P<0.05) in neoplastic transformation frequency, likely mediated by intercellular communication through gap junctions, was observed in the progeny of bystander cells that had been in co-culture with cells irradiated with iron ions, but not with protons. PMID:21738697

  8. Increased frequency of spontaneous neoplastic transformation in progeny of bystander cells from cultures exposed to densely ionizing radiation.

    PubMed

    Buonanno, Manuela; de Toledo, Sonia M; Azzam, Edouard I

    2011-01-01

    An increased risk of carcinogenesis caused by exposure to space radiation during prolonged space travel is a limiting factor for human space exploration. Typically, astronauts are exposed to low fluences of ionizing particles that target only a few cells in a tissue at any one time. The propagation of stressful effects from irradiated to neighboring bystander cells and their transmission to progeny cells would be of importance in estimates of the health risks of exposure to space radiation. With relevance to the risk of carcinogenesis, we investigated, in model C3H 10T½ mouse embryo fibroblasts (MEFs), modulation of the spontaneous frequency of neoplastic transformation in the progeny of bystander MEFs that had been in co-culture 10 population doublings earlier with MEFs exposed to moderate doses of densely ionizing iron ions (1 GeV/nucleon) or sparsely ionizing protons (1 GeV). An increase (P<0.05) in neoplastic transformation frequency, likely mediated by intercellular communication through gap junctions, was observed in the progeny of bystander cells that had been in co-culture with cells irradiated with iron ions, but not with protons.

  9. NF-κB signaling maintains the survival of cadmium-exposed human renal glomerular endothelial cells

    PubMed Central

    Zhang, Hongyan; Li, Liqun; Wang, Yifan; Dong, Fengyun; Chen, Xiaocui; Liu, Fuhong; Xu, Dongmei; Yi, Fan; Kapron, Carolyn M.; Liu, Ju

    2016-01-01

    The kidney is one of the primary organs targeted by cadmium (Cd), a widely distributed environmental pollutant. The glomerular endothelium is the major component of the glomerular filtration barrier. However, the effects of Cd on glomerular endothelial cells remain largely unknown. For this purpose, we aimed to determine the effects of low dose Cd on the survival of human renal glomerular endothelial cells (HRGECs). Cultured HRGECs were exposed to 4 µM cadmium chloride (CdCl2) and examined at different time-points. We found that Cd activates the nuclear factor-κB (NF-κB) pathway without inducing the apoptosis of HRGECs. Pre-treating the cells with pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, prior to Cd exposure triggered extensive cell death (73.5%). In addition, Cd activates the c-Jun N-terminal kinase (JNK) pathway, and inhibition of the NF-κB pathway significantly elevates Cd-induced JNK phosphorylation in HRGECs (p<0.01). The combination treatment of PDTC and SP600125, a JNK pathway inhibitor, increased the survival of Cd-stimulated HRGECs compared with those cells treated with PDTC alone (p<0.05). Taken together, these findings demonstrate that the NF-κB pathway plays an essential role in maintaining the survival of Cd-exposed HRGECs. PMID:27315281

  10. NF-κB signaling maintains the survival of cadmium-exposed human renal glomerular endothelial cells.

    PubMed

    Zhang, Hongyan; Li, Liqun; Wang, Yifan; Dong, Fengyun; Chen, Xiaocui; Liu, Fuhong; Xu, Dongmei; Yi, Fan; Kapron, Carolyn M; Liu, Ju

    2016-08-01

    The kidney is one of the primary organs targeted by cadmium (Cd), a widely distributed environmental pollutant. The glomerular endothelium is the major component of the glomerular filtration barrier. However, the effects of Cd on glomerular endothelial cells remain largely unknown. For this purpose, we aimed to determine the effects of low dose Cd on the survival of human renal glomerular endothelial cells (HRGECs). Cultured HRGECs were exposed to 4 µM cadmium chloride (CdCl2) and examined at different time-points. We found that Cd activates the nuclear factor-κB (NF-κB) pathway without inducing the apoptosis of HRGECs. Pre-treating the cells with pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, prior to Cd exposure triggered extensive cell death (73.5%). In addition, Cd activates the c-Jun N-terminal kinase (JNK) pathway, and inhibition of the NF-κB pathway significantly elevates Cd-induced JNK phosphorylation in HRGECs (p<0.01). The combination treatment of PDTC and SP600125, a JNK pathway inhibitor, increased the survival of Cd-stimulated HRGECs compared with those cells treated with PDTC alone (p<0.05). Taken together, these findings demonstrate that the NF-κB pathway plays an essential role in maintaining the survival of Cd-exposed HRGECs. PMID:27315281

  11. Cytoprotective effect of kaempferol on paraquat-exposed BEAS-2B cells via modulating expression of MUC5AC.

    PubMed

    Podder, Biswajit; Song, Kyoung Seob; Song, Ho-Yeon; Kim, Yong-Sik

    2014-01-01

    Mucins are highly glycosylated secretary proteins produced by most epithelial cells. Hypersecretion of mucins is one of the prominent symptoms of several airway diseases, including asthma, cystic fibrosis, nasal allergy, rhinitis, and sinusitis. Paraquat (PQ), a common herbicide, has been associated with pulmonary damage and is a potent reactive oxygen species (ROS) producer. However, until now the role of PQ on mucin overproduction has not been studied. The aim of this study is to explore how kaempferol (KM), a widely used dietary flavonoid, affects the protection of human PQ-exposed bronchial epithelium BEAS-2B cells by suppressing Mucin gene expression via nuclear factor-kappa B (NF-κB). We observed that PQ generates intracellular ROS, and also induces lipid peroxidation in BEAS-2B cells. Additionally, we found that PQ effectively induces the expression of the MUC5AC gene; however, co-treatment of PQ with KM drastically reduces its expression. Furthermore, we observed that PQ activates NF-κB, while co-treatment with KM occludes its nuclear translocation, and additionally KM repressed the PQ phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in BEAS-2B cells. Based on our data, we believe that KM can suppress the over-expression of the MUC5AC gene. This would contribute to the protection of PQ cytotoxicity to exposed BEAS-2B cells, and allow further study toward a better understanding of ROS-associated diseases. PMID:25177032

  12. Nobiletin suppresses the proliferation and induces apoptosis involving MAPKs and caspase-8/-9/-3 signals in human acute myeloid leukemia cells.

    PubMed

    Hsiao, Pei-Ching; Lee, Wei-Jiunn; Yang, Shun-Fa; Tan, Peng; Chen, Hui-Yu; Lee, Liang-Ming; Chang, Junn-Liang; Lai, Gi-Ming; Chow, Jyh-Ming; Chien, Ming-Hsien

    2014-12-01

    Nobiletin, a compound isolated from citrus fruits, is a polymethoxylated flavone derivative that was shown to have anti-inflammatory and anticancer activities in various solid tumors. The anticancer effect of nobiletin on nonsolid tumor remains unclear. Herein, the molecular mechanisms by which nobiletin exerts its anticancer effects on acute myeloid leukemia (AML) cells were investigated. The results showed that nobiletin suppressed cell proliferation in various types of AML cell lines. Moreover, nobiletin induced cell-cycle arrest of HL-60 AML cells at the G0/G1 phase by suppressing extracellular signal-regulated kinase (ERK) activity. Furthermore, nobiletin effectively induced apoptosis of HL-60 cells through caspase-8, caspase-9, and caspases-3 activation concomitantly with a marked induction of p38 mitogen-activated protein kinase (MAPK) activation, but without affecting expression levels of Bcl-2, Bax, or Bid. Taken together, our results suggest that nobiletin inhibited HL-60 cell proliferation through inducing cell-cycle arrest and apoptosis and could serve as a potential additional chemotherapeutic agent for treating AML.

  13. Synergistic cytotoxicity and DNA strand breaks in cells and plasmid DNA exposed to uranyl acetate and ultraviolet radiation

    PubMed Central

    Wilson, Janice; Zuniga, Mary C.; Yazzie, Filbert; Stearns, Diane M.

    2015-01-01

    Depleted uranium (DU) has a chemical toxicity that is independent of its radioactivity. The purpose of this study was to explore the photoactivation of uranyl ion by ultraviolet (UV) radiation as a chemical mechanism of uranium genotoxicity. The ability of UVB (302 nm) and UVA (368 nm) radiation to photoactivate uranyl ion to produce single strand breaks was measured in pBR322 plasmid DNA, and the presence of adducts and apurinic/apyrimidinic sites that could be converted to single strand breaks by heat and piperidine was analyzed. Results showed that DNA lesions in plasmid DNA exposed to UVB- or UVA-activated DU were only slightly heat reactive, but were piperidine sensitive. The cytotoxicity of UVB-activated uranyl ion was measured in repair-proficient and repair-deficient Chinese hamster ovary cells and human keratinocyte HaCaT cells. The cytotoxicity of co-exposures of uranyl ion and UVB radiation was dependent on the order of exposure and was greater than co-exposures of arsenite and UVB radiation. Uranyl ion and UVB radiation were synergistically cytotoxic in cells, and cells exposed to photoactivated DU required different DNA repair pathways than cells exposed to non-photoactivated DU. This study contributes to our understanding of the DNA lesions formed by DU, as well as their repair. Results suggest that excitation of uranyl ion by UV radiation can provide a pathway for uranyl ion to be chemically genotoxic in populations with dermal exposures to uranium and UV radiation, which would make skin an overlooked target organ for uranium exposures. PMID:24832689

  14. Synergistic cytotoxicity and DNA strand breaks in cells and plasmid DNA exposed to uranyl acetate and ultraviolet radiation.

    PubMed

    Wilson, Janice; Zuniga, Mary C; Yazzie, Filbert; Stearns, Diane M

    2015-04-01

    Depleted uranium (DU) has a chemical toxicity that is independent of its radioactivity. The purpose of this study was to explore the photoactivation of uranyl ion by ultraviolet (UV) radiation as a chemical mechanism of uranium genotoxicity. The ability of UVB (302 nm) and UVA (368 nm) radiation to photoactivate uranyl ion to produce single strand breaks was measured in pBR322 plasmid DNA, and the presence of adducts and apurinic/apyrimidinic sites that could be converted to single strand breaks by heat and piperidine was analyzed. Results showed that DNA lesions in plasmid DNA exposed to UVB- or UVA-activated DU were only slightly heat reactive, but were piperidine sensitive. The cytotoxicity of UVB-activated uranyl ion was measured in repair-proficient and repair-deficient Chinese hamster ovary cells and human keratinocyte HaCaT cells. The cytotoxicity of co-exposures of uranyl ion and UVB radiation was dependent on the order of exposure and was greater than co-exposures of arsenite and UVB radiation. Uranyl ion and UVB radiation were synergistically cytotoxic in cells, and cells exposed to photoactivated DU required different DNA repair pathways than cells exposed to non-photoactivated DU. This study contributes to our understanding of the DNA lesions formed by DU, as well as their repair. Results suggest that excitation of uranyl ion by UV radiation can provide a pathway for uranyl ion to be chemically genotoxic in populations with dermal exposures to uranium and UV radiation, which would make skin an overlooked target organ for uranium exposures.

  15. Modulation by retinoic acid of cellular, surface-exposed, and secreted glycoconjugates in cultured human sarcoma cells.

    PubMed

    Meromsky, L; Lotan, R

    1984-02-01

    The effect of beta-all-trans-retinoic acid (RA) on the synthesis of cellular, cell surface, and secreted glycoconjugates by human Hs705 chondrosarcoma and Hs791 osteosarcoma cells was investigated in vitro. Untreated and RA-treated cells were labeled either metabolically with radioactive precursors or by oxidation of externally exposed cell membrane glycoprotein(s) (GP) by treatment with NalO4 or neuraminidase and galactose oxidase followed by reduction with NaB[3H]4. The cells were solubilized and analyzed by polyacrylamide gel electrophoresis followed by fluorography. RA enhanced the labeling of sialic acid and galactose residues on the GP of relative molecular weight(s) (Mr) in the range 95,000-300,000 on the surfaces of both cell types. [3H]glycosamine incorporation into GP with Mr of 100,000, 150,000, and 190,000 in both cell lines was also stimulated. In the Hs705 cells there was also an increase in the labeling of a 290,000-Mr GP. In contrast, [3H]glucosamine incorporation into glycoconjugates greater than 400,000 Mr in both the cells and the conditioned medium of Hs705 cells decreased. The latter glycoconjugates were susceptible to hyaluronidase and chondroitinases. [3H]glucosamine incorporation into a secreted 230,000-Mr GP, identified as fibronectin, was also reduced. Analyses of conditioned media of cells labeled with [35S]methionine or [14C]proline demonstrated that RA decreased the secretion of procollagen chains and fibronectin. Immunofluorescence revealed that RA alters the distribution of cell-associated fibronectin. These results demonstrated that RA increases the glycosylation of specific cellular and cell surface GP and decreases the production of secreted GP and glycosaminoglycans by the sarcoma cells.

  16. Mast cells in the intestine and gills of the sea bream, Sparus aurata, exposed to a polychlorinated biphenyl, PCB 126.

    PubMed

    Lauriano, Eugenia Rita; Calò, Margherita; Silvestri, Giuseppa; Zaccone, Daniele; Pergolizzi, Simona; Lo Cascio, Patrizia

    2012-02-01

    The presence of mast cells has been reported in all classes of vertebrates, including many teleost fish families. The mast cells of teleosts, both morphologically and functionally, show a close similarity to the mast cells of mammals. Mast cells of teleosts, localized in the vicinity of blood vessels of the intestine, gills and skin, may play an important role in the mechanisms of inflammatory response, because they express a number of functional proteins, including piscidins, which are antimicrobical peptides that act against a broad-spectrum of pathogens. An increase in the number of mast cells in various tissues and organs of teleosts seems to be linked to a wide range of stressful conditions, such as exposure to heavy metals (cadmium, copper, lead and mercury), exposure to herbicides and parasitic infections. This study analyzed the morphological localization and abundance of mast cells in the intestine and gills of sea bream, Sparus aurata, after a 12, 24 or 72 h exposure to PCB 126, a polychlorinated biphenyl, which is a potent immunotoxic agent. In the organs of fish exposed to PCB 126, it was observed that in addition to congestion of blood vessels, there was extravasation of red blood cells, infiltration of lymphocytes, and a progressive increase in numbers of mast cells. These data confirm the immunotoxic action of PCB, and the involvement of mast cells in the inflammatory response. PMID:21565388

  17. Immunostimulatory Activity of the Cytokine-Based Biologic, IRX-2, on Human Papillomavirus-Exposed Langerhans Cells

    PubMed Central

    Da Silva, Diane M.; Woodham, Andrew W.; Naylor, Paul H.; Egan, James E.; Berinstein, Neil L.

    2016-01-01

    Langerhans cells (LCs) are the antigen-presenting cells of the epithelial layer and are responsible for initiating immune responses against skin and mucosa-invading viruses. Human papillomavirus (HPV)-mediated suppression of LC function is a crucial mechanism of HPV immune evasion, which can lead to persistent infection and development of several human cancers, including cervical, anal, and head and neck cancers. The cell-derived cytokine-based biologic, IRX-2, consists of multiple well-defined cytokines and is broadly active on various immune cell subsets. In this study, we investigated primary human LC activation after exposure to HPV16, followed by treatment with IRX-2 in vitro, and evaluated their subsequent ability to induce HPV16-specific T cells. In contrast to its activity on dendritic cells, HPV16 alone is not sufficient to induce phenotypic and functional activation of LCs. However, IRX-2 induces a significant upregulation of antigen presentation and costimulatory molecules, T helper 1 (Th1)-associated cytokine release, and chemokine-directed migration of LCs pre-exposed to HPV16. Furthermore, LCs treated with IRX-2 after HPV16 exposure induced CD8+ T-cell responses against specific HLA-A*0201-binding HPV16 T-cell epitopes. The present study suggests that IRX-2 is an attractive immunomodulator for assisting the immune response in eradication of HPV-infected cells, thereby potentially preventing HPV-induced cancers. PMID:26653678

  18. Abnormal regulation of DNA replication and increased lethality in ataxia telangiectasia cells exposed to carcinogenic agents

    SciTech Connect

    Jaspers, N.G.; de Wit, J.; Regulski, M.R.; Bootsma, D.

    1982-01-01

    The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells.

  19. Proliferation, migration and invasion of human glioma cells exposed to paclitaxel (Taxol) in vitro.

    PubMed Central

    Terzis, A. J.; Thorsen, F.; Heese, O.; Visted, T.; Bjerkvig, R.; Dahl, O.; Arnold, H.; Gundersen, G.

    1997-01-01

    Paclitaxel (Taxol), an anti-cancer drug derived from Taxus species, was tested for its anti-migrational, anti-invasive and anti-proliferative effect on two human glioma cell lines (GaMg and D-54Mg) grown as multicellular tumour spheroids. In addition, the direct effect of paclitaxel on glioma cells was studied using flow cytometry and scanning confocal microscopy. Both cell lines showed a dose-dependent growth and migratory response to paclitaxel. The GaMg cells were found to be 5-10 times more sensitive to paclitaxel than D-54Mg cells. Paclitaxel also proved to be remarkably effective in preventing invasion in a co-culture system in which tumour spheroids were confronted with fetal rat brain cell aggregates. Control experiments with Cremophor EL (the solvent of paclitaxel for clinical use) in this study showed no effect on tumour cell migration, cell proliferation or cell invasion. Scanning confocal microscopy of both cell lines showed an extensive random organization of the microtubules in the cytoplasm. After paclitaxel exposure, the GaMg and the D-54Mg cells exhibited a fragmentation of the nuclear material, indicating a possible induction of apoptosis. In line with this, flow cytometric DNA histograms showed an accumulation of cells in the G2/M phase of the cell cycle after 24 h of paclitaxel exposure. After 48 h, a deterioration of the DNA histograms was observed indicating nuclear fragmentation. Images Figure 3 Figure 6 PMID:9192976

  20. Lycopene supplementation prevents reactive oxygen species mediated apoptosis in Sertoli cells of adult albino rats exposed to polychlorinated biphenyls

    PubMed Central

    Krishnamoorthy, Gunasekaran; Selvakumar, Kandaswamy; Venkataraman, Prabhu; Elumalai, Perumal

    2013-01-01

    Sertoli cell proliferation is attenuated before attaining puberty and the number is fixed in adult testes. Sertoli cells determine both testis size and daily sperm production by providing physical and metabolic support to spermatogenic cells. Polychlorinated biphenyls (PCBs) exposure disrupts functions of Sertoli cells causing infertility with decreased sperm count. On the other hand, lycopene is improving sperm count and motility by reducing oxidative stress in humans and animals. Hence we hypothesized that PCBs-induced infertility might be due to Sertoli cell apoptosis mediated by oxidative stress and lycopene might prevent PCBs-induced apoptosis by acting against oxidative stress. To test this hypothesis, animals were treated with vehicle control, lycopene, PCBs and PCBs + lycopene for 30 days. After the experimental period, the testes and cauda epididymidis were removed for isolation of Sertoli cells and sperm, respectively. We observed increased levels of oxidative stress markers (H2O2 and LPO) levels, increased expression of apoptotic molecules (caspase-8, Bad, Bid, Bax, cytochrome C and caspase-3), decreased anti-apoptotic (Bcl2) molecule and elevated apoptotic marker activity (caspase-3) in Sertoli cells of PCBs-exposed animals. These results were associated with decreased sperm count and motility in PCBs exposed animals. On the other hand, lycopene prevented the elevation of Sertoli cellular apoptotic parameters and prevented the reduction of sperm parameters (count and motility). The data confirmed that lycopene as an antioxidant scavenged reactive oxygen substances, prevented apoptosis, maintained normal function in Sertoli cells and helped to provide physical and metabolic support for sperm production, thereby treating infertility in men. PMID:24179434

  1. Pulmonary function and symptoms of Nigerian workers exposed to carbon black in dry cell battery and tire factories

    SciTech Connect

    Oleru, U.G.; Elegbeleye, O.O.; Enu, C.C.; Olumide, Y.M.

    1983-02-01

    The pulmonary function and symptoms of 125 workers exposed to carbon black in dry cell battery and tire manufacturing plants were investigated. There was no significant difference in the pulmonary function of the subjects in the two plants. There was good agreement in the symptoms reported in the two different factories: cough with phlegm production, tiredness, chest pain, catarrh, headache, and skin irritation. The symptoms also corroborate those reported in the few studies on the pulmonary effects of carbon black. The suspended particulate levels in the dry cell battery plant ranged from 25 to 34 mg/m/sup 3/ and the subjects with the highest probable exposure level had the most impaired pulmonary function. The pulmonary function of the exposed subjects was significantly lower than that of a control, nonindustrially exposed population. The drop in the lung function from the expected value per year of age was relatively constant for all the study subgroups but the drop per year of duration of employment was more severe in the earlier years of employment. This study has underscored the need for occupational health regulations in the industries of developing countries.

  2. Induction of Apoptosis by [8]-shogaol via Reactive Oxygen Species Generation, Glutathione Depletion and Caspase Activation in Human Leukemia Cells

    PubMed Central

    Shieh, Po-Chuen; Chen, Yi-Own; Kuo, Daih-Huang; Chen, Fu-An; Tsai, Mei-Ling; Chang, Ing-Shing; Wu, Hou; Sang, Shengmin; Ho, Chi-Tang; Pan, Min-Hsiung

    2010-01-01

    Ginger, the rhizome of Zingiber officinale, is a traditional medicine with carminative effect, anti-nausea, anti-inflammatory, and anti-carcinogenic properties. This study examined the growth inhibitory effects of [8]-shogaol, one of pungent phenolic compounds in ginger, on human leukemia HL-60 cells. It demonstrated that [8]-shogaol was able to induce apoptosis in a time- and concentration-dependent manner. Treatment with [8]-shogaol caused a rapid loss of mitochondrial transmembrane potential, stimulation of reactive oxygen species (ROS) production, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 and procaspase-3 processing. Taken together, these results suggest for the first time that ROS production and depletion of the glutathione that committed to [8]-shogaol-induced apoptosis in HL-60 cells. PMID:20163181

  3. Nanotopography guides and directs cell migration in amoeboid and epithelial cells</