Science.gov

Sample records for jurkat cells effects

  1. Normal Untreated Jurkat Cells

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. The objective of the research was to define a way to differentiate between effects due to microgravity and those due to possible stress from non-optimal spaceflight conditions. These Jurkat cells, a human acute T-cell leukemia was obtained to evaluate three types of potential experimental stressors: a) Temperature elevation; b) Serum starvation; and c) Centrifugal force. The data from previous spaceflight experiments showed that actin filaments and cell shape are significantly different for the control. These normal cells serve as the baseline for future spaceflight experiments.

  2. Antiproliferative and proapoptotic effects of a pyrrole containing arylthioindole in human Jurkat leukemia cell line and multidrug-resistant Jurkat/A4 cells

    PubMed Central

    Philchenkov, Alex A; Zavelevich, Michael P; Tryndyak, Volodymyr P; Kuiava, Ludmila M; Blokhin, Dmitry Yu; Miura, Koh; Silvestri, Romano; Pogribny, Igor P

    2015-01-01

    Recently, a series of novel arylthioindole compounds, potent inhibitors of tubulin polymerization and cancer cell growth, were synthesized. In the present study the effects of 2-(1H-pyrrol-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (ATI5 compound) on cell proliferation, cell cycle progression, and induction of apoptosis in human T-cell acute leukemia Jurkat cells and their multidrug resistant Jurkat/A4 subline were investigated. Treatment of the Jurkat cells with the ATI5 compound for 48 hrs resulted in a strong G2/M cell cycle arrest and p53-independent apoptotic cell death accompanied by the induction of the active form of caspase-3 and poly(ADP-ribose) polymerase-1 (PARP-1) cleavage. ATI5 treatment also caused non-cell death related mitotic arrest in multidrug resistant Jurkat/A4 cells after 48 hrs of treatment suggesting promising opportunities for the further design of pyrrole-containing ATI compounds as anticancer agents. Cell death resistance of Jurkat/A4 cells to ATI5 compound was associated with alterations in the expression of pro-survival and anti-apoptotic protein-coding and microRNA genes. More importantly, findings showing that ATI5 treatment induced p53-independent apoptosis are of great importance from a therapeutic point of view since p53 mutations are common genetic alterations in human neoplasms. PMID:26785947

  3. Effects of canola and corn oil mimetic on Jurkat cells

    PubMed Central

    2011-01-01

    Background The Western diet is high in omega-6 fatty acids and low in omega-3 fatty acids. Canola oil contains a healthier omega 3 to omega 6 ratio than corn oil. Jurkat T leukemia cells were treated with free fatty acids mixtures in ratios mimicking that found in commercially available canola oil (7% α-linolenic, 30% linoleic, 54% oleic) or corn oil (59% linoleic, 24% oleic) to determine the cell survival or cell death and changes in expression levels of inflammatory cytokines and receptors following oil treatment. Methods Fatty acid uptake was assessed by gas chromatography. Cell survival and cell death were evaluated by cell cycle analyses, propidium-iodide staining, trypan blue exclusion and phosphatidylserine externalization. mRNA levels of inflammatory cytokines and receptors were assessed by RT-PCR. Results There was a significant difference in the lipid profiles of the cells after treatment. Differential action of the oils on inflammatory molecules, following treatment at non-cytotoxic levels, indicated that canola oil mimetic was anti-inflammatory whereas corn oil mimetic was pro-inflammatory. Significance These results indicate that use of canola oil in the diet instead of corn oil might be beneficial for diseases promoted by inflammation. PMID:21631947

  4. Cytotoxic effect of mistletoe (Viscum album L.) extract on Jurkat cells and its interaction with doxorubicin.

    PubMed

    Sabová, Lucia; Pilátová, Martina; Szilagyi, Katarína; Sabo, Rastislav; Mojzis, Ján

    2010-03-01

    Mistletoe preparations are frequently used by cancer patients because of their ability to stimulate the immunity and to improve the quality of life. Moreover mistletoe and its active substances (especially lectins) possess cytotoxic effect on various cancer cell lines. However, only little is known about its interaction with anticancer drugs. Therefore the cytotoxic and apoptosis-inducing effects of aqueous mistletoe extract (VA) and its interaction with doxorubicin (DOXO) were investigated in Jurkat cells. The results show that VA extract as well as DOXO exert cytotoxic effects on Jurkat cells in a dose-dependent manner. Cytotoxicity of DOXO was much stronger (LC(50) = 11.68 ng/mL) than that of VA extract (LC(50) = 35.67 microg/mL). Their combination led to synergism only at those concentrations that were highly cytotoxic alone. Both substances (alone and in combination) induced DNA fragmentation in Jurkat cells. In conclusion, an aqueous extract prepared from mistletoe tops exerted cytotoxic and apoptosis-inducing effects on Jurkat cells alone as well as in combination with DOXO.

  5. Effect of direct albumin binding to sphingosylphosphorylcholine in Jurkat T cells.

    PubMed

    Han, Mijin; Kim, Yu-Lee; Sacket, Santosh J; Kim, Kyeok; Kim, Hyo-Lim; Jo, Ji-Yeong; Ha, Nam-Chul; Im, Dong-Soon

    2007-11-01

    We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca(2+)](i), and decreased MMP. However, those effects were only observed at concentrations higher than 10 microM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca(2+)](i), and decreased MMP in Jurkat T cells.

  6. The effect of miR-146a on STAT1 expression and apoptosis in acute lymphoblastic leukemia Jurkat cells

    PubMed Central

    Yan, Weihong; Guo, Hua; Suo, Feng; Han, Chunling; Zheng, Hua; Chen, Tong

    2017-01-01

    The effect of miR-146a-dependent regulation of STAT1 on apoptosis in acute lymphoblastic leukemia (ALL) Jurkat cells was investigated. The miR-146a mimic and miR-146a inhibitor vectors were constructed in vitro, and experimental grouping was as follows: Control group (untreated Jurkat cells), empty vector group (Jurkat cells transfected with empty vector), agonist group (Jurkat cells transfected with miR-146a mimic) and the inhibitor group (Jurkat cells transfected with miR-146a inhibitor). Western blot analysis was used to observe the expression, respectively, of STAT1, p-STAT1 and Bcl-xL, and flow cytometry was used to test apoptosis in Jurkat cells. STAT1 and p-STAT1 expression in the agonist group was higher than that in the control and empty vector groups, but lower in the inhibitor group, and differences were statistically significant (P<0.05). The rate of apoptosis in the agonist group was significantly higher than that of the control group and blank vector group, and it was significantly lower in the inhibitor group (P<0.05). As a tumor suppressor, miR-146a can regulate expression of apoptosis-promoting factor STAT1, and anti-apoptosis factor Bcl-xL, and is able to promote apoptosis of ALL Jurkat cells. PMID:28123535

  7. The Effect and Mechanisms of Proliferative Inhibition of Crocin on Human Leukaemia Jurkat Cells

    PubMed Central

    Sun, Y; Wang, Z; Wang, L; Wang, L-Z; Zang, C; Sun, L-R

    2015-01-01

    ABSTRACT Targeted therapy is a potentially useful approach for the treatment of T-lineage acute lymphoblastic leukaemia. This study aimed to find a highly effective, low toxic anti-tumour drug and further investigate its mechanisms. Jurkat cells were used as the object and were stimulated by different concentrations of crocin. By cell count, growth curve, methyl thiazolyl tetrazolium (MTT) method for the detection of cell proliferation, annexin V/propidium iodide (PI) method for the apoptosis rates, and reverse transcription-polymerase chain reaction (RT-PCR) for Bcl-2 and Bax gene expression, the effect and mechanisms of proliferative inhibition of crocin on Jurkat cells were further explored. Crocin promoted Jurkat cell apoptosis and inhibited cell growth, in a dose-time-dependent manner. The mechanism might be related to the inhibition of Bcl-2 gene expression and the promotion of Bax gene expression. These results suggest that crocin can be used as a suitable clinical agent for the treatment of T-lineage acute lymphoblastic leukaemia. PMID:27398676

  8. Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and Jurkat cells

    NASA Astrophysics Data System (ADS)

    Mahlke, Megan A.; Navara, Christopher; Ibey, Bennett L.

    2014-03-01

    Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to

  9. Effect of UV irradiation on the apoptosis and necrosis of Jurkat cells using UV LEDs

    NASA Astrophysics Data System (ADS)

    Inada, Shunko A.; Amano, Hiroshi; Akasaki, Isamu; Morita, Akimichi; Kobayashi, Keiko

    2009-02-01

    Phototherapy is a very effective method for treating most of the incurable skin diseases. A fluorescent light bulb is used as a conventional UV light source for this type of therapy. However, infrared radiation from the light source sometimes causes serious problems on patient's health. In addition, the normal part of the skin is irradiated when a large fluorescent light bulb is used. Moreover, a conventional UV irradiation system is heavy and has a short lifetime and a high electrical power consumption. Therefore, a new UV light source for solving the problems of phototherapy is required. To realize low-power-consumption, lightweight and long-lifetime systems, group III nitride-based UV-A1 light-emitting diodes (LEDs) were investigated. We examined the UV LED irradiation of Jurkat cell, which is a tumor cell and more sensitive to UV light than a healthy cell. The numbers of apoptotic and necrotic cells were confirmed to be the same using a UV LED and a conventional lamp system. The UV LED showed the possibility of realizing a new UV light source for phototherapy.

  10. Effect of silencing HOXA5 gene expression using RNA interference on cell cycle and apoptosis in Jurkat cells.

    PubMed

    Huang, Hui-Ping; Liu, Wen-Jun; Guo, Qu-Lian; Bai, Yong-Qi

    2016-03-01

    Acute lymphocytic leukemia (ALL) is a common malignant tumor with a high morbidity rate among children, accounting for approximately 80% of leukemia cases. Although there have been improvements in the treatment of patients frequent relapse lead to a poor prognosis. The aim of the present study was to determine whether HOXA5 may be used as a target for gene therapy in leukemia in order to provide a new treatment. Mononuclear cells were extracted from the bone marrow according to the clinical research aims. After testing for ALL in the acute stage, the relative mRNA and protein expression of HOXA5 was detected in the ALL remission groups (n=25 cases per group) and the control group [n=20 cases, immune thrombocytopenia (ITP)]. Gene silencing by RNA interference (RNAi) was used to investigate the effect of silencing HOXA5 after small interfering RNA (siRNA) transfection to Jurkat cells. The HOXA5-specific siRNA was transfected to Jurkat cells using lipofectamine. The experiment was divided into the experimental group (liposomal transfection of HOXA5 targeting siRNA), the negative control group (liposomal transfection of cells with negative control siRNA) and the control group (plus an equal amount of cells and culture media only). Western blotting and quantitative fluorescent polymerase chain reaction (QF‑PCR) were used to detect the relative HOXA5 mRNA expression and protein distribution in each cell group. Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined using flow cytometry. The expression of HOXA5 at the mRNA and protein levels in the acute phase of ALL was significantly higher than that in ALL in the remission and control groups. In cells transfected with HOXA5-specific siRNA, the expression of HOXA5 at the mRNA and protein levels decreased significantly (P<0.05). The distribution of cells in the cell cycle was also altered. Specifically, more cells were present in the G0/G1 phase compared to the S phase (P<0.05). In

  11. Pro-apoptotic effect of Persea americana var. Hass (avocado) on Jurkat lymphoblastic leukemia cells.

    PubMed

    Bonilla-Porras, Angelica R; Salazar-Ospina, Andrea; Jimenez-Del-Rio, Marlene; Pereañez-Jimenez, Andres; Velez-Pardo, Carlos

    2013-11-05

    Abstract Context: Therapy for leukemia has a limited efficacy. There is a need to search for alternative anti-leukemia therapies. Persea americana Mill var. Hass (Lauraceae) is a tropical fruit (avocado) that might be used against cancer. Objective: To investigate whether P. americana induces death in Jurkat lymphoblastic leukemia cells. Materials and methods: Four ethanol extracts (0.1, 0.5, 1, 2 and 5 mg/mL) from avocado fruit (endocarp, whole seed, seed and leaves) were analyzed against Jurkat cells. Hydrogen peroxide generation by oxidation of 2',7'-dichlorodihydrofluorescein diacetate to the fluorescent compound 2',7'-dichlorfluorescein assay, acridine orange/ethidium bromide staining, flow cytometry analysis of annexin-V/7-amino-actinomycin, mitochondrial membrane potential and immunocytochemistry detection of transcription factor p53, caspase-3 and apoptosis-inducing factor (AIF) were evaluated. Results: Endocarp, seed, whole seed, and leaf (0.1 mg/mL) extracts induced significant apoptosis in Jurkat cells (p < 0.001) in an oxidative stress-dependent fashion via mitochondrial membrane depolarization (52-87%), activation of transcription factor p53 (6.3-25.4%), protease caspase-3 (8.3-20%) and predominance of AIF reactivity (20.6-36%) in all extracts. Similar results were obtained with 0.5 mg/mL extracts. However, extract ≥1 mg/mL concentration induced necrosis (100%). Conclusions: P. americana extracts function as a pro-apoptotic compound. Leukemic cells are eliminated through an oxidative stress mechanism. This study contributes to the understanding of the molecular mechanism of the avocado and its therapeutic action on leukemia.

  12. Calcium ionophoretic and apoptotic effects of ferutinin in the human Jurkat T-cell line.

    PubMed

    Macho, Antonio; Blanco-Molina, Magdalena; Spagliardi, Paola; Appendino, Giovanni; Bremner, Paul; Heinrich, Michael; Fiebich, Bernd L; Muñoz, Eduardo

    2004-09-01

    We have investigated the ionophoretic and apoptotic properties of the daucane sesquiterpene ferutinin and three related compounds, ferutidin, 2-alpha-hydroxyferutidin and teferin, all isolated from various species of plants from the genus Ferula. Ferutinin induced a biphasic elevation of intracellular Ca2+ in the leukemia T-cell line, Jurkat. First, a rapid calcium peak was observed and inhibited by BAPTA-AM. This initial calcium mobilization was followed by a sustained elevation, mediated by the entry of extracellular calcium through L-type calcium channels and sensitive to inhibition by EGTA. Moreover, ferutinin-induced apoptosis in Jurkat cells, and this event was preceded, in a cyclosporine-A sensitive manner, by a loss of mitochondrial transmembrane potential (DeltaPsim) and by an increase in intracellular reactive oxygen species. Ferutinin-induced DNA fragmentation was mediated by a caspase-3-dependent pathway, and was initiated independently of any specific phase of the cell cycle. The evaluation of ferutinin analogs in calcium mobilization and apoptosis assays showed strict structure-activity relationships, with p-hydroxylation of the benzoyl moiety being requested for activity.

  13. Effect of ultraviolet B radiation and 100 Hz electromagnetic fields on proliferation and DNA synthesis of Jurkat cells.

    PubMed

    Nindl, G; Hughes, E F; Johnson, M T; Spandau, D F; Vesper, D N; Balcavage, W X

    2002-09-01

    The use of ultraviolet B light (UVB) has been proven to be highly effective for treatment of various inflammatory skin diseases, but UVB phototherapy is limited by its carcinogenic side effects. It is necessary to uncover effectors that augment UVB so that similar or improved efficacy can be obtained with lower UVB doses. We found that low frequency, low intensity electromagnetic fields (EMFs) can act as such an effector and synergistically inhibit T lymphocyte proliferation. We first characterized the effects of UVB on Jurkat cells, a model for cutaneous T lymphocytes, and determined UVB's dose dependent inhibition of cell proliferation and induction of apoptosis. Cells exposed to a sublethal UVB dose retained their sensitivity to UVB, but repetitive irradiation seemed to cause accumulation of delayed DNA damage. We then exposed cells to combinations of UVB plus EMFs and found that 100 Hz, 1 mT EMFs decrease DNA synthesis of UVB-activated Jurkat cells by 34 +/- 13% compared to UVB alone. The decrease is, however, most effective when relatively high UVB doses are employed. Since EMFs alone had only a very weak inhibitory effect (10 +/- 2%), the data suggest that EMFs augment the cell killing effects of UVB in a synergistic way. These findings could provide the basis for development of new and improved clinical phototherapy protocols.

  14. Evaluation of the Effect of PEGylated Single-Walled Carbon Nanotubes on Viability and Proliferation of Jurkat Cells

    PubMed Central

    Hadidi, Naghmeh; Hosseini Shirazi, Seyed Farshad; Kobarfard, Farzad; Nafissi-Varchehd, Nastaran; Aboofazeli, Reza

    2012-01-01

    Among the numerous nanosized drug delivery systems currently under investigation, carbon nanotubes (CNTs), regardless of being single or multiple-walled, offer several advantages and are considered as promising candidates for drug targeting. Despite the valuable potentials of CNTs in drug delivery, their toxicity still remains an important issue. After the PEGylation of single-walled CNTs (SWCNTs) with phospholipid-PEG (Pl-PEG) conjugates to prepare water-dispersible nanostructures, the present study was designed to evaluate whether the functionalization with Pl-PEG derivatives could alter the cytotoxic response of cells in culture, affect their viability and proliferation. In-vitro cytotoxicity screens were performed on cultured Jurkat cells. The SWCNTs samples used in this exposure were pristine SWCNTs, Pl-PEG 2000/5000-SWCNTs at various concentrations. Jurkat cells were first incubated for 3 h at 37°C with test materials and seeded in 6-well culture plates at a given concentration. The plates were then incubated for 24, 48 and 72 h at 37°C in a 5% CO2 humidified incubator. Cell Viability and proliferation assay were performed using trypan blue exclusion test and the cell cycle kinetic status of Jurkat cells was analyzed by flow cytometry. Cell morphology was finally studied using double staining technique and a fluorescence microscope. We found that, regardless of the duration of exposure, functionalized SWCNTs were substantially less toxic, compared to pure SWCNTs and that the molecular weight of Pl-PEGs played an important role at higher concentrations. In conclusion, our noncovalent protocol seemed to be effective for increasing SWCNTs biocompatibility. PMID:25317182

  15. Chlamydia trachomatis induces anti-inflammatory effect in human macrophages by attenuation of immune mediators in Jurkat T-cells.

    PubMed

    Azenabor, Anthony A; Cintrón-Cuevas, Jenniffer; Schmitt, Heather; Bumah, Violet

    2011-12-01

    The chronic course of Chlamydia trachomatis infection is subtle with no obvious unusual inflammatory change. The reason for this is not clear. The data reported here explain how macrophage usual inflammatory response switches to anti-inflammatory response during C. trachomatis infection of mixed culture of macrophages and Jurkat T-cells. We assessed the establishment of productive infection in individual or mixed cell culture models, determined the status of C. trachomatis in the cells by monitoring HSP-60:MOMP or the proportions of the estimated IFUs that shed HSP-60 or MOMP. Also, the specific time-course expression of IL-12, IL-10 and IFN-γ or IL-12R, IL-10R, and IFN-γ-R during infection of cell models was assessed. Finally, the early events in cytokine elaboration in circumstances of varying intracellular Ca²⁺ levels were determined. There was evidence of productive infection in all individual and mixed cell culture models. The shedding of HSP-60 was highest in THP-1/Jurkat mixed cell culture model. The proportions of IFU that shed HSP-60 was heightened in infected THP-1/Jurkat mixed culture model, while the proportion of IFU that shed MOMP was higher in infected macrophage/Jurkat mixed culture and infected macrophages only. There was profound early elaboration of IL-10, varying significantly from IL-12 and IFN-γ in all infected individual or mixed cell culture models except in the case of Jurkat; where all cytokine elaboration was downregulated. The receptor to IL-10 was upregulated in infected macrophage/Jurkat cells and THP-1/Jurkat cells compared with other models in which IL-12 and IFN-γ receptors were more expressed. There was no observed significant change in cytokine in any model following the impairment of intracellular Ca²⁺ except in the case of macrophage/Jurkat cell model in which IL-12 and IL-10 were upregulated in 1h or 3 h, respectively. The implication of these findings is that C. trachomatis mediates a switch from inflammatory to anti

  16. New and cost effective cell-based assay for Dialyzed Leukocyte Extract (DLE)-induced Jurkat cells proliferation under azathioprine treatment.

    PubMed

    Cardoso, F M; Tomkova, M; Petrovajova, D; Bubanova, M; Ragac, O; Hornakova, T

    2017-05-10

    The human Dialyzed Leukocyte Extract (DLE) is a heterogeneous mix of oligopeptides of <10kDa, extracted from leukocytes of healthy donors. There is significant clinical evidence of improvement using DLE during treatment of allergies, cancer,immunodeficiencies, and in mycotic and viral infections. Nevertheless, the DLE exact nature and mechanism of action have been elusive for more than 50 years. DLE biological activity testing is necessary in DLE production and quality control. Both in vitro and in vivo assays exist: E-rosette test, induction of delayed type hypersensitivity in mice, leukocyte migration and IFN-γ secretion. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic burden, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. Here we are reporting a new DLE biological activity cell-based assay. The A20 and Jurkat cell lines were treated with (+Aza) or without (-Aza) azathioprine, DLE (+DLE) or both (+Aza/+DLE). After 72h, the cell proliferation was analyzed by the MTT or BrdU incorporation assays. In +Aza/+DLE treated cells, we observed a significant higher proliferation, when compared with +Aza/-DLE. In the absence of Aza, cells did not present any proliferation difference between -DLE or +DLE treatments. Both assays, MTT and BrdU showed similar results, being the MTT test more cost effective and we select it for validation as DLE biological assay using Jurkat cells only. We tested three different lyophilized DLE batches and we found consistent results with acceptable assay reproducibility and linearity. The DLE capacity for rescuing Jurkat cell proliferation during +Aza treatment was consistent using different liquid and lyophilized DLE batches, presenting also consistent chromatographic profiles. Finally, DLE treatment in Jurkat cells did not result into significant IL-2 of IFN-γ secretion, and known lymphocyte proliferative drugs

  17. The effect of Amaryllidaceae alkaloids haemanthamine and haemanthidine on cell cycle progression and apoptosis in p53-negative human leukemic Jurkat cells.

    PubMed

    Havelek, Radim; Seifrtova, Martina; Kralovec, Karel; Bruckova, Lenka; Cahlikova, Lucie; Dalecka, Marketa; Vavrova, Jirina; Rezacova, Martina; Opletal, Lubomir; Bilkova, Zuzana

    2014-03-15

    Plants from the Amaryllidaceae family have been shown to be a promising source of biologically active natural compounds of which some selected are currently in pre-clinical development. Regardless of interesting pioneer works, little is known about Amaryllidaceae alkaloids that have shown promising anti-cancer activities. The crinane group of the Amaryllidaceae, including haemanthamine and haemanthidine, was amongst the first of these compounds to exhibit an interesting cytotoxic potential against cancer cell lines. However, the mechanism of cytotoxic and anti-proliferative activity is not yet entirely clear. The primary objectives of the current study were to investigate the effects of haemanthamine and haemanthidine on the induction of apoptosis and the cell cycle regulatory pathway in p53-null Jurkat cells. Results indicate that haemanthamine and haemanthidine treatment decreases cell viability and mitochondrial membrane potential, leads to a decline in the percentage of cells in the S phase of the cell cycle, induces apoptosis detected by Annexin V staining and increases caspase activity. Dose dependent apoptosis was cross verified by fluorescence and bright field microscopy through Annexin V/propidium iodine staining and morphological changes which characteristically attend programmed cell death. The apoptotic effect of haemanthamine and haemanthidine on leukemia cells is more pronounced than that of gamma radiation. Contrary to gamma radiation, Jurkat cells do not completely halt the cell cycle 24h upon haemanthamine and haemanthidine exposure. Both Amaryllidaceae alkaloids accumulate cells preferentially at G1 and G2 stages of the cell cycle with increased p16 expression and Chk1 Ser345 phosphorylation. Concerning the pro-apoptotic effect, haemanthidine was more active than haemanthamine in the Jurkat leukemia cell line.

  18. [Mechanisms of gamma-inducible death of Jurkat cells line].

    PubMed

    Gamkrelidze, M M; Bezhitashvili, N D; Pavliashvili, A T; Mchedlishvili, T V; Sanikidze, T V

    2008-06-01

    Mechanisms of radio-inducible death of Jurkat cells were investigated. Human lymphoblastoid T-cell line Jurkat is widely established model for studying apoptosis mechanisms. The cell was radiated by "Teragam" (Czech Republic) by dose 2 g during 1 minute. After radiation cells were incubated at standard conditions during 24 hours. After gamma radiation in cell population amount of cells in gaplois (apoptotic G 0) stage was increased 8,2 folds, in diplois (G 0/G1) stage - by 17%, in synthetic (S) stage decreased by 35% and tetraploid (G2/M) stage by 73% in comparison to control group. It was revealed intensive production of free radicals of oxygen and nitric oxide and decreasing activity of antioxidant enzymes (superoxidismutasa, catalasa and glutathione peroxidase). Revealed dependence between intensification of apoptosis and radiation-induced arrest of cell cycle G2/M phase may be determined by excess amount of free oxygen and nitrogen radicals generated in Jurkat cells as a result of nondirect effects of low doses of gamma radiation.

  19. CD147 and CD98 complex-mediated homotypic aggregation attenuates the CypA-induced chemotactic effect on Jurkat T cells.

    PubMed

    Guo, Na; Zhang, Kui; Lv, Minghua; Miao, Jinlin; Chen, Zhinan; Zhu, Ping

    2015-02-01

    Homotypic cell aggregation plays important roles in physiological and pathological processes, including embryogenesis, immune responses, angiogenesis, tumor cell invasion and metastasis. CD147 has been implicated in most of these phenomena, and it was identified as a T cell activation-associated antigen due to its obvious up-regulation in activated T cells. However, the explicit function and mechanism of CD147 in T cells have not been fully elucidated. In this study, large and compact aggregates were observed in Jurkat T cells after treatment with the specific CD147 monoclonal antibody HAb18 or after the expression of CD147 was silenced by RNA interference, which indicated an inhibitory effect of CD147 in T cell homotypic aggregation. Knocking down CD147 expression resulted in a significant decrease in CD98, along with prominent cell aggregation, similar to that treated by CD98 and CD147 monoclonal antibodies. Furthermore, decreased cell chemotactic activity was observed following CD147- and CD98-mediated cell aggregation, and increased aggregation was correlated with a decrease in the chemotactic ability of the Jurkat T cells, suggesting that CD147- and CD98-mediated homotypic cell aggregation plays a negative role in T cell chemotaxis. Our data also showed that p-ERK, p-ZAP70, p-CD3ζ and p-LCK were significantly decreased in the CD147- and CD98-knocked down Jurkat T cells, which suggested that decreased CD147- and/or CD98-induced homotypic T cell aggregation and aggregation-inhibited chemotaxis might be associated with these signaling pathways. A role for CD147 in cell aggregation and chemotaxis was further indicated in primary CD4(+) T cells. Similarly, low expression of CD147 in primary T cells induced prominent cell aggregation and this aggregation attenuated primary T cell chemotactic ability in response to CypA. Our results have demonstrated the correlation between homotypic cell aggregation and the chemotactic response of T cells to CypA, and these data

  20. Antiproliferative effect of T/Tn specific Artocarpus lakoocha agglutinin (ALA) on human leukemic cells (Jurkat, U937, K562) and their imaging by QD-ALA nanoconjugate.

    PubMed

    Chatterjee, Urmimala; Bose, Partha Pratim; Dey, Sharmistha; Singh, Tej P; Chatterjee, Bishnu P

    2008-11-01

    T/Tn specificity of Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha (Moraceae) fruit and a heterodimer (16 kD and 12 kD) of molecular mass 28 kD, was further confirmed by SPR analysis using T/Tn glycan containing mammalian glycoproteins. N-terminal amino acid sequence analysis of ALA showed homology at 15, 19-21, 24-27, and 29 residues with other lectin members of Moraceae family viz., Artocarpus integrifolia (jacalin) lectin, Artocarpus hirsuta lectin, and Maclura pomifera agglutinin. It is mitogenic to human PBMC and the maximum proliferation was observed at 1 ng/ml. It showed an antiproliferative effect on leukemic cells, with the highest effect toward Jurkat cells (IC(50) 13.15 ng/ml). Synthesized CdS quantum dot-ALA nanoconjugate was employed to detect the expression of T/Tn glycans on Jurkat, U937, and K562 leukemic cells surfaces as well as normal lymphocytes by fluorescence microscopy. No green fluorescence was observed with normal lymphocytes indicating that T/Tn determinants, which are recognized as human tumor associated structures were cryptic on normal lymphocyte surfaces, whereas intense green fluorescent dots appeared during imaging of leukemic cells, where such determinants were present in unmasked form. The above results indicated that QD-ALA nanoconjugate is an efficient fluorescent marker for identification of leukemic cell lines that gives rise to high quality images.

  1. Galectin-1 and Galectin-3 induce mitochondrial apoptotic pathway in Jurkat cells

    NASA Astrophysics Data System (ADS)

    Vasil'eva, O. A.; Isaeva, A. V.; Prokhorenko, T. S.; Zima, A. P.; Novitsky, V. V.

    2016-08-01

    Cellular malignant transformation is often accompanied by increased gene expression of low-molecular proteins of lectins family-galectins. But it is unknown how galectins promote tumor growth and malignization. Galectins-1 and galectin-3 are thought to be possible immunoregulators exerting their effects by regulating the balance of CD4+ lymphocytes. In addition it is known that tumor cells overexpressing galectins are capable of escaping immunological control, causing apoptosis of lymphocytes. The aim of the study is to investigate the role of galectin-1 and galectin-3 in the implementation of mitochondrial apoptotic pathway in Jurkat cells. Methods: Jurkat cells were used as a model for the study of T-lymphocytes. Jurkat cells were activated with antibodies to CD3 and CD28 and cultured with recombinant galectin-1 and -3. Apoptosis of Jurkat cells and depolarization of the mitochondrial membrane were assessed by flow cytometry. It was found that galectin-1 and galectin-3 have a dose-dependent pro-apoptotic effect on Jurkat cells in vitro and enlarge the number of cells with decreased mitochondrial membrane potential compared with intact cells.

  2. Antiproliferative Activity of T. welwitschii Extract on Jurkat T Cells In Vitro

    PubMed Central

    Moyo, Batanai; Mukanganyama, Stanley

    2015-01-01

    Triumfetta welwitschii is a plant used traditionally for the treatment of fever and diarrhoea. Previous work has shown that T. welwitschii has antibacterial activity. The purpose of this study was to investigate T. welwitschii extract for anticancer activity against Jurkat T cells. The Jurkat T cell line is used to study acute T cell leukaemia. An antiproliferation assay, determination of induction of apoptosis, the determination of the effect of the combination of the extract and GSH, and effects of the extract on DNA leakage were conducted. T. welwitschii was found to decrease cell viability in a dose- and time-dependent manner. T. welwitschii caused apoptosis in the Jurkat T cells as shown by DNA fragmentation. When T. welwitschii was combined with reduced GSH, it was found that the growth of the Jurkat T cells was significantly reduced compared to untreated cells after 72 h of treatment. This was unexpected, as cancer cells have elevated levels of GSH compared to normal cells. The results of this study show that T. welwitschii is a potential source of compounds that may serve as leads for anticancer compounds. PMID:26557698

  3. Reactive oxygen species-dependent necroptosis in Jurkat T cells induced by pathogenic free-living Naegleria fowleri.

    PubMed

    Song, K-J; Jang, Y S; Lee, Y A; Kim, K A; Lee, S K; Shin, M H

    2011-07-01

    Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N. fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N. fowleri induces ROS-dependent necroptosis in Jurkat T cells.

  4. MIP-1α enhances Jurkat cell transendothelial migration by up-regulating endothelial adhesion molecules VCAM-1 and ICAM-1.

    PubMed

    Ma, Yi-Ran; Ma, Ying-Huan

    2014-11-01

    The aim of this study is to evaluate the expression of macrophage inflammatory protein-1α (MIP-1α) in Jurkat cells and its effect on transendothelial migration. In the present study, human acute lymphoblastic leukemia Jurkat cells (Jurkat cells) were used as a model of T cells in human T-cell acute lymphoblastic leukemia (T-ALL), which demonstrated significantly higher MIP-1α expression compared with that in normal T-cell controls. The ability of Jurkat cells to cross a human brain microvascular endothelial cell (HBMEC) monolayer was almost completely abrogated by MIP-1α siRNA. In addition, the overexpression of MIP-1α resulted in the up-regulated expression of endothelial adhesion molecules, which enhanced the migration of Jurkat cells through a monolayer of HBMEC. MIP-1α levels in Jurkat cells appeared to be an important factor for its transendothelial migration, which may provide the theoretical basis to understand the mechanisms of brain metastases of T-ALL at cellular and molecular levels.

  5. n-Hexane toxicity in Jurkat T-cells is mediated by reactive oxygen species.

    PubMed

    McDermott, Catherine; O'Donoghue, Maria Hutch; Heffron, James J A

    2008-03-01

    Here we assess the role of reactive oxygen species (ROS) formation in the manifestation of n-hexane toxicity in Jurkat T-cells and the chemo-protective potential of the antioxidants epigallocatechin-3-gallate (EGCG) and thymoquinone (TQ) against n-hexane toxicity in vitro. n-Hexane is an important industrial solvent and ambient air pollutant. Subchronic exposure to n-hexane results in a concentration-dependent increase in ROS formation with a corresponding decrease in Jurkat T-cell proliferation. Results from time-course studies indicate that ROS formation plays a causal role in n-hexane induced alterations in Jurkat T-cell proliferation and membrane integrity. Treatment of cells with EGCG, at a concentration reached in plasma, reduced the ROS formation caused by exposure to n-hexane and inhibited the decrease in cell proliferation. Similar effects were obtained with TQ. Both EGCG and TQ significantly reduced n-hexane-induced LDH leakage to control levels. The combined results show that oxidative stress plays a role in the development of n-hexane toxicity.

  6. Constituents of French Marigold (Tagetes patula L.) Flowers Protect Jurkat T-Cells against Oxidative Stress

    PubMed Central

    Chkhikvishvili, Irakli; Sanikidze, Tamar; Gogia, Nunu; Enukidze, Maia; Machavariani, Marine; Kipiani, Nana; Vinokur, Yakov; Rodov, Victor

    2016-01-01

    The flowers of French marigold (Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μM H2O2. Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10) in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers. PMID:27433287

  7. Constituents of French Marigold (Tagetes patula L.) Flowers Protect Jurkat T-Cells against Oxidative Stress.

    PubMed

    Chkhikvishvili, Irakli; Sanikidze, Tamar; Gogia, Nunu; Enukidze, Maia; Machavariani, Marine; Kipiani, Nana; Vinokur, Yakov; Rodov, Victor

    2016-01-01

    The flowers of French marigold (Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μM H2O2. Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10) in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers.

  8. Carvacrol induces mitochondria-mediated apoptosis in HL-60 promyelocytic and Jurkat T lymphoma cells.

    PubMed

    Bhakkiyalakshmi, Elango; Suganya, Natarajan; Sireesh, Dornadula; Krishnamurthi, Kannan; Saravana Devi, Sivanesan; Rajaguru, Palanisamy; Ramkumar, Kunka Mohanram

    2016-02-05

    The aim of the present study was to investigate the effect of carvacrol, a phenolic monoterpenoid on the induction of apoptosis in HL-60 (Human acute promyelocytic leukemia cells) and Jurkat (human T lymphocyte cells) cells. Carvacrol showed a potent cytotoxic effect on both cells with dose-dependent increase in the level of free radical formation as measured by an oxidation sensitive fluorescent dye, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) levels. The reduction in the level of antioxidants such as catalase (CAT) and superoxide dismutase (SOD) (P<0.05) was observed in carvacrol-treated cells. The major cytotoxic effect appears to be intervened by the induction of apoptotic cell death as assessed by annexin-V labeling assay using flow cytometry. Western blot analysis showed that Bax expression was increased, whereas Bcl-2 expression was significantly decreased in carvacrol exposed HL-60 cells and Jurkat cells. Further studies revealed that the dissipation of mitochondrial membrane potential of intact cells was accompanied by the activation of caspase-3. Our results found that the potential mechanism of cellular apoptosis induced by carvacrol is mediated by caspase-3 and is associated with the collapse of mitochondrial membrane potential, generation of free radicals, and depletion of the intracellular antioxidant pool.

  9. Inhibition of CXCL12-mediated chemotaxis of Jurkat cells by direct immunotoxicants.

    PubMed

    Shao, Jia; Stout, Inge; Volger, Oscar L; Hendriksen, Peter J M; van Loveren, Henk; Peijnenburg, Ad A C M

    2016-07-01

    Directional migration of cells to specific locations is required in tissue development, wound healing, and immune responses. Immune cell migration plays a crucial role in both innate and adaptive immunity. Chemokines are small pro-inflammatory chemoattractants that control the migration of leukocytes. In addition, they are also involved in other immune processes such as lymphocyte development and immune pathology. In a previous toxicogenomics study using the Jurkat T cell line, we have shown that the model immunotoxicant TBTO inhibited chemotaxis toward the chemokine CXCL12. In the present work, we aimed at assessing a novel approach to detecting chemicals that affect the process of cell migration. For this, we first evaluated the effects of 31 chemicals on mRNA expression of genes that are known to be related to cell migration. With this analysis, seven immunotoxicants were identified as potential chemotaxis modulators, of which five (CoCl2 80 µM, MeHg 1 µM, ochratoxin A 10 µM, S9-treated ochratoxin A 10 µM, and TBTO 100 nM) were confirmed as chemotaxis inhibitor in an in vitro trans-well chemotaxis assay using the chemokine CXCL12. The transcriptome data of the five compounds together with previously obtained protein phosphorylation profiles for two out of five compounds (i.e., ochratoxin A and TBTO) revealed that the mechanisms behind the chemotaxis inhibition are different for these immunotoxicants. Moreover, the mTOR inhibitor rapamycin had no effect on the chemotaxis of Jurkat cells, indicating that the mTOR pathway is not involved in CXCL12-mediated chemotaxis of Jurkat cells, which is opposite to the findings on human primary T cells (Munk et al. in PLoS One 6(9):e24667, 2011). Thus, the results obtained from the chemotaxis assay conducted with Jurkat cells might not fully represent the results obtained with human primary T cells. Despite this difference, the present study indicated that some compounds may exert their immunotoxic effects through

  10. [The mbr-FPGS efficient expression plasmid enhances the sensitivity of Jurkat cells to methotrexate].

    PubMed

    Hu, Wen-Jia; Sun, Yu-Jie

    2012-06-01

    Folylpolyglutamate synthetase (FPGS) is the key enzyme that converts chemotherapy drug Methotrexate (MTX) into MTXPG. The expression level of FPGS directly influences MTX-sensitivity of tumor cells. Compared with B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL) cells express a lower level of FPGS, which results in insensitivity of the cells to MTX. Our previous work has demonstrated that 279 bp mbr element located within the 3'-UTR of the BCL2 gene possesses enhancer function. In this study, FPGS expression plasmid containing mbr element at the 5' upstream of the gene was constructed and transfected into Jurkat cells to sensitize the cells to MTX. Western blotting and MTT assay were applied to detect the FPGS expression level and suppression rate of the cells treated by MTX, respectively. We found that the mbr enhanced the expression of FPGS significantly and increased sensitivity of Jurkat cells to MTX efficiently, while FPGS expression plasmid without mbr element had less effect. Our data provides a new clue for the clinical application of mbr regulatory element and may contribute to improvement of MTX treatment in T-ALL.

  11. Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by downregulation of PI3K/AKT and upregulation of p38 MAPK pathways.

    PubMed

    Yang, Tingfang; Yao, Shuluan; Zhang, Xianfeng; Guo, Yan

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) as a prevalent hematologic malignancy is one of the most common malignant tumors worldwide in children. Andrographolide (Andro), the major active component from Andrographis paniculata, has been shown to possess antitumor activities in several types of cancer cells. However, whether Andro would inhibit T-ALL cell growth remains unclear. In this study, we investigated the cytotoxic effect of Andro on human T-ALL Jurkat cells and explored the mechanisms of cell death. Cell apoptosis was assayed by flow cytometry, and the signaling transduction for Andro was analyzed by Western blotting. The results indicated 10 μg/mL Andro could significantly induce Jurkat cells' apoptosis, depending on the inhibition of PI3K/AKT pathway. Moreover, Andro-induced apoptosis is enhanced by AKT-selective inhibitor LY294002. ERK- or JNK-selective inhibitors PD98059 and SP600125 had no effect on Andro-induced apoptosis. In addition, p38 inhibitor SB203580 could reverse Andro-induced apoptosis in Jurkat cells. We also found that the protein expression of p-p53 and p-p38 were increased after Andro treatments. The result of an in vivo study also demonstrated Andro's dose-dependent inhibition in subcutaneous Jurkat xenografts. In conclusion, our findings explained a novel mechanism of drug action by Andro in Jurkat cells and suggested that Andro might be developed into a new candidate therapy for T-ALL patients in the coming days.

  12. Inhibition of TNF-alpha induced cell death in human umbilical vein endothelial cells and Jurkat cells by protocatechuic acid.

    PubMed

    Zhou-Stache, J; Buettner, R; Artmann, G; Mittermayer, C; Bosserhoff, A K

    2002-11-01

    The Chinese herb radix Salviae miltiorrhizae (RSM) is used in traditional Chinese medicine as a treatment for cardiovascular and cerebrovascular diseases. Several components of the plant extract from salvia mitorrhiza bunge have been determined previously, one of which is protocatechuic acid (PAC). It has been found, in the study, that PAC inhibited TNF-alpha-induced cell death of human umbilical vein endothelial cells (HUVECs) and Jurkat cells in a concentration of 100 microM when applied 2 h prior to TNF-alpha exposure. Molecular studies revealed that PAC activated NF-kappaB with a maximum effect after 30 min of treatment. Inhibition of NF-kappaB action by MG132 and NF-kappaB inhibitory peptide suppressed the cell-protective effect of PAC. Further, degradation of IkBalpha occurred in response to PAC treatment. The results provide evidence that activation of NF-kappaB plays an important role in mediating the cell-protecting effect of PAC on HUVECs and Jurkat cells. Further studies are required to test whether PAC, a component of radix salviae miltiorrhizae, could be useful in preventing in vivo cell death resulting from cardiovascular or cerebrovascular diseases.

  13. Cell retention by encapsulation for the cultivation of Jurkat cells in fixed and fluidized bed reactors.

    PubMed

    Kaiser, P; Werner, M; Jérôme, V; Hübner, H; Buchholz, R; Freitag, R

    2014-12-01

    Jurkat cells are accepted model cells for primary human T lymphocytes, for example, in medical research. Their growth to tissue-like cell densities (up to 100 × 10(6)  cells/mLcapsule ) in semi-permeable (molecular weight cut off <10,000 Da) sodium cellulose sulfate/poly(diallyldimethylammonium chloride) polyelectrolyte capsules has previously been shown by us [Werner et al. (2013). Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells. Biotechnol Prog 29(4): 986-993]. Herein, we demonstrate that encapsulation can be used to retain the cells in continuously operated bioreactors, which opens new possibilities for research, for example, the use of Jurkat cells in pulse response experiments under steady state conditions. Two reactor concepts are presented, a fluidized and a fixed bed reactor. A direct comparison of the growth kinetics in batch and repeated batch spinner cultivations, that is, under conditions where both encapsulated and non-encapsulated cells can be cultivated under otherwise identical conditions, showed that maximum specific growth rates were higher for the encapsulated than for the non-encapsulated cells. In the subsequent batch and repeated batch bioreactor experiments (only encapsulated cells), growth rates were similar, with the exception of the fixed bed batch reactor, where growth kinetics were significantly slower. Concomitantly, a significant fraction of the cells towards the bottom of the bed were no longer metabolically active, though apparently not dead. In the repeated batch fluidized bed reactor cellular division could be maintained for more than two weeks, albeit with a specific growth rate below the maximum one, leading to final cell densities of approximately 180 × 10(6)  cell/gcapsule . At the same time, the cell cycle distribution of the cells was shifted to the S and G2/M phases.

  14. Combined treatment with fenretinide and indomethacin induces AIF-mediated, non-classical cell death in human acute T-cell leukemia Jurkat cells

    SciTech Connect

    Hojka-Osinska, Anna; Ziolo, Ewa; Rapak, Andrzej

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer The combination of fenretinide and indomethacin induces a high level of cell death. Black-Right-Pointing-Pointer Apoptotic pathway is caspase-independent. Black-Right-Pointing-Pointer Jurkat cells undergo AIF-mediated cell death. -- Abstract: Currently used cytotoxic drugs in cancer therapy have a similar mechanism of action and low specificity. Applied simultaneously, they show an additive effect with strong side effects. Clinical trials with the use of different agents in cancer therapy show that the use of these compounds alone is not very effective in fighting cancer. An alternative solution could be to apply a combination of these agents, because their combination has a synergistic effect on some cancer cells. Therefore, in our investigations we examined the effects of a synthetic retinoid-fenretinide when combined with a non-steroidal anti-inflammatory drug-indomethacin on the process of apoptosis in the acute human T-cell leukemia cell line Jurkat. We demonstrate that treatment with the combination of the tested compounds induces the death of cells, that is peculiar and combines features of apoptosis as well as non-apoptotic cell death. In detail we observed, cell membrane permeabilization, phosphatydylserine exposure, no oligonucleosomal DNA fragmentation, no caspase-3 activation, but apoptosis inducing factor (AIF) nuclear translocation. Taken together these results indicate, that Jurkat cells after treatment with a combination of fenretinide and indomethacin undergo AIF-mediated programmed cell death.

  15. Lithium induces ER stress and N-glycan modification in galactose-grown Jurkat cells.

    PubMed

    Nagy, Tamás; Frank, Dorottya; Kátai, Emese; Yahiro, Rikki K K; Poór, Viktor S; Montskó, Gergely; Zrínyi, Zita; Kovács, Gábor L; Miseta, Attila

    2013-01-01

    We previously reported that lithium had a significant impact on Ca(2+) regulation and induced unfolded protein response (UPR) in yeast cells grown on galactose due to inhibition of phosphoglucomutase (PGM), however the exact mechanism has not been established yet. In this study, we analysed lithium's effect in galactose-fed cells to clarify whether these ER-related changes are the result of a relative hypoglycemic state. Furthermore, we investigated whether the alterations in galactose metabolism impact protein post-translational modifications. Thus, Jurkat cells were incubated in glucose or galactose containing media with or without lithium treatment. We found that galactose-fed and lithium treated cells showed better survivability than fasting cells. We also found higher UDP-Hexose and glycogen levels in these cells compared to fasting cells. On the other hand, the UPR (X-box binding protein 1 mRNA levels) of galactose-fed and lithium treated cells was even greater than in fasting cells. We also found increased amount of proteins that contained N-linked N-acetyl-glucosamine, similar to what was reported in fasting cells by a recent study. Our results demonstrate that lithium treatment of galactose-fed cells can induce stress responses similar to hypoglycemia, however cell survival is still secured by alternative pathways. We propose that clarifying this process might be an important addition toward the better understanding of the molecular mechanisms that regulate ER-associated stress response.

  16. Fe2O3 nanoparticles suppress Kv1.3 channels via affecting the redox activity of Kvβ2 subunit in Jurkat T cells

    NASA Astrophysics Data System (ADS)

    Yan, Li; Liu, Xiao; Liu, Wei-Xia; Tan, Xiao-Qiu; Xiong, Fei; Gu, Ning; Hao, Wei; Gao, Xue; Cao, Ji-Min

    2015-12-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are promising nanomaterials in medical practice due to their special magnetic characteristics and nanoscale size. However, their potential impacts on immune cells are not well documented. This study aims to investigate the effects of Fe2O3 nanoparticles (Fe2O3-NPs) on the electrophysiology of Kv1.3 channels in Jurkat T cells. Using the whole-cell patch-clamp technique, we demonstrate that incubation of Jurkat cells with Fe2O3-NPs dose- and time-dependently decreased the current density and shifted the steady-state inactivation curve and the recovery curve of Kv1.3 channels to a rightward direction. Fe2O3-NPs increased the NADP level but decreased the NADPH level of Jurkat cells. Direct induction of NADPH into the cytosole of Jurkat cells via the pipette abolished the rightward shift of the inactivation curve. In addition, transmission electron microscopy showed that Fe2O3-NPs could be endocytosed by Jurkat cells with relatively low speed and capacity. Fe2O3-NPs did not significantly affect the viability of Jurkat cells, but suppressed the expressions of certain cytokines (TNFα, IFNγ and IL-2) and interferon responsive genes (IRF-1 and PIM-1), and the time courses of Fe2O3-NPs endocytosis and effects on the expressions of cytokines and interferon responsive genes were compatible. We conclude that Fe2O3-NPs can be endocytosed by Jurkat cells and act intracellularly. Fe2O3-NPs decrease the current density and delay the inactivation and recovery kinetics of Kv1.3 channels in Jurkat cells by oxidizing NADPH and therefore disrupting the redox activity of the Kvβ2 auxiliary subunit, and as a result, lead to changes of the Kv1.3 channel function. These results suggest that iron oxide nanoparticles may affect T cell function by disturbing the activity of Kv1.3 channels. Further, the suppressing effects of Fe2O3-NPs on the expressions of certain inflammatory cytokines and interferon responsive genes suggest that iron

  17. Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by downregulation of PI3K/AKT and upregulation of p38 MAPK pathways

    PubMed Central

    Yang, Tingfang; Yao, Shuluan; Zhang, Xianfeng; Guo, Yan

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) as a prevalent hematologic malignancy is one of the most common malignant tumors worldwide in children. Andrographolide (Andro), the major active component from Andrographis paniculata, has been shown to possess antitumor activities in several types of cancer cells. However, whether Andro would inhibit T-ALL cell growth remains unclear. In this study, we investigated the cytotoxic effect of Andro on human T-ALL Jurkat cells and explored the mechanisms of cell death. Cell apoptosis was assayed by flow cytometry, and the signaling transduction for Andro was analyzed by Western blotting. The results indicated 10 μg/mL Andro could significantly induce Jurkat cells’ apoptosis, depending on the inhibition of PI3K/AKT pathway. Moreover, Andro-induced apoptosis is enhanced by AKT-selective inhibitor LY294002. ERK- or JNK-selective inhibitors PD98059 and SP600125 had no effect on Andro-induced apoptosis. In addition, p38 inhibitor SB203580 could reverse Andro-induced apoptosis in Jurkat cells. We also found that the protein expression of p-p53 and p-p38 were increased after Andro treatments. The result of an in vivo study also demonstrated Andro’s dose-dependent inhibition in subcutaneous Jurkat xenografts. In conclusion, our findings explained a novel mechanism of drug action by Andro in Jurkat cells and suggested that Andro might be developed into a new candidate therapy for T-ALL patients in the coming days. PMID:27114702

  18. Polarization imaging and classification of Jurkat T and Ramos B cells using a flow cytometer.

    PubMed

    Feng, Yuanming; Zhang, Ning; Jacobs, Kenneth M; Jiang, Wenhuan; Yang, Li V; Li, Zhigang; Zhang, Jun; Lu, Jun Q; Hu, Xin-Hua

    2014-09-01

    Label-free and rapid classification of cells can have awide range of applications in biology. We report a robust method of polarization diffraction imaging flow cytometry (p-DIFC) for achieving this goal. Coherently scattered light signals are acquired from single cells excited by a polarized laser beam in the form of two cross-polarized diffraction images. Image texture and intensity parameters are extracted with a gray level co-occurrence matrix (GLCM) algorithm to obtain an optimized set of feature parameters as the morphological "fingerprints" for automated cell classification. We selected the Jurkat T cells and Ramos B cells to test the p-DIFC method's capacity for cell classification. After detailed statistical analysis, we found that the optimized feature vectors yield accuracies of classification between the Jurkat and Ramos ranging from 97.8% to 100% among different cell data sets. Confocal imaging and three-dimensional reconstruction were applied to gain insights on the ability of p-DIFC method for classifying the two cell lines of highly similar morphology. Based on these results we conclude that the p-DIFC method has the capacity to discriminate cells of high similarity in their morphology with "fingerprints" features extracted from the diffraction images, which may be attributed to subtle but statistically significant differences in the nucleus-to-cell volume ratio in the case of Jurkat and Ramos cells.

  19. Inhibition of Kv1.3 Channels in Human Jurkat T Cells by Xanthohumol and Isoxanthohumol.

    PubMed

    Gąsiorowska, Justyna; Teisseyre, Andrzej; Uryga, Anna; Michalak, Krystyna

    2015-08-01

    Using whole-cell patch-clamp technique, we investigated influence of selected compounds from groups of prenylated chalcones and flavonoids: xanthohumol and isoxanthohumol on the activity of Kv1.3 channels in human leukemic Jurkat T cells. Obtained results provide evidence that both examined compounds were inhibitors of Kv1.3 channels in these cells. The inhibitory effects occurred in a concentration-dependent manner. The estimated value of the half-blocking concentration (EC50) was about 3 μM for xanthohumol and about 7.8 μM for isoxanthohumol. The inhibition of Kv1.3 channels by examined compounds was not complete. Upon an application of the compounds at the maximal concentrations equal to 30 μM, the activity of Kv1.3 channels was inhibited to about 0.13 of the control value. The inhibitory effect was reversible. The application of xanthohumol and isoxanthohumol did not change the currents' activation and inactivation rate. These results may confirm our earlier hypothesis that the presence of a prenyl group in a molecule is a factor that facilitates the inhibition of Kv1.3 channels by compounds from the groups of flavonoids and chalcones. The inhibition of Kv1.3 channels might be involved in antiproliferative and proapoptotic effects of the compounds observed in cancer cell lines expressing these channels.

  20. Enhanced histamine production through the induction of histidine decarboxylase expression by phorbol ester in Jurkat cells.

    PubMed

    Nagashima, Yusuke; Kako, Koichiro; Kim, Jun-Dal; Fukamizu, Akiyoshi

    2012-11-01

    Histamine (HA), a mediator of inflammation, type I allergic responses and neurotransmission, is synthesized from L-histidine, the reaction of which is catalyzed by histidine decarboxylase (HDC). HDC has been reported to be induced by various stimuli, not only in mast cells and basophils, but also in T lymphocytes and macrophages. Although its mRNA has been shown to be increased in Jurkat cells when treated with phorbol 12-myristate 13-acetate (TPA), little is known concerning the induced production of HA by HDC. The present study quantified the trace amounts of intracellular HA using ultra-high liquid chromatography in combination with the 6-aminoquinoline carbamate-derivatization technique. To test whether the cellular level of HA is elevated by the induction of HDC in Jurkat cells treated with TPA, the peak corresponding to authentic HA in the cell lysate was fractioned and its molecular weight determined by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry. The results of this study show that the HA level is increased by the induction of HDC expression by TPA in Jurkat cells. Therefore, this method is useful in elucidating the physiological significance of HA production.

  1. Anti-proliferative properties of commercial Pelargonium sidoides tincture, with cell-cycle G0/G1 arrest and apoptosis in Jurkat leukaemia cells.

    PubMed

    Pereira, Andreia; Bester, Megan; Soundy, Puffy; Apostolides, Zeno

    2016-09-01

    Context Pelargonium sidoides DC (Geraniaceae) is an important medicinal plant indigenous to South Africa and Lesotho. Previous studies have shown that root extracts are rich in polyphenolic compounds with antibacterial, antiviral and immunomodulatory activities. Little is known regarding the anticancer properties of Pelargonium sidoides extracts. Objective This study evaluates the anti-proliferative effects of a Pelargonium sidoides radix mother tincture (PST). Materials and methods The PST was characterized by LC-MS/MS. Anti-proliferative activity was evaluated in the pre-screen panel of the National Cancer Institute (NCI-H460, MCF-7 and SF-268) and the Jurkat leukaemia cell line at concentrations of 0-150 μg/mL. The effect on cell growth was determined with sulphorhodamine B and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays after 72 h. The effect on cell cycle and apoptosis induction in Jurkat cells was determined by flow cytometry with propidium iodide and Annexin V: fluorescein isothiocyanate staining. Results Dihydroxycoumarin sulphates, gallic acid as well as gallocatechin dimers and trimers were characterized in PST by mass spectrometry. Moderate anti-proliferative effects with GI50 values between 40 and 80 μg/mL were observed in the NCI-pre-screen panel. Strong activity observed with Jurkat cells with a GI50 value of 6.2 μg/mL, significantly better than positive control 5-fluorouracil (GI50 value of 9.7 μg/mL). The PST arrested Jurkat cells at the G0/G1 phase of the cell cycle and increased the apoptotic cells from 9% to 21%, while the dead cells increased from 4% to 17%. Conclusion We present evidence that P. sidoides has cancer cell type-specific anti-proliferative effects and may be a source of novel anticancer molecules.

  2. Diversity and Complexity of Ceramide Generation After Exposure of Jurkat Leukemia Cells to Irradiation

    SciTech Connect

    Ardail, Dominique Maalouf, Mira; Boivin, Anthony; Chapet, Olivier; Bodennec, Jacques; Rousson, Robert; Rodriguez-Lafrasse, Claire

    2009-03-15

    Purpose: To define which intracellular pools of sphingomyelin and ceramide are involved in the triggering of apoptosis of Jurkat leukemia cells in response to {gamma}-ray exposure. Methods and Materials: We examined the kinetics of ceramide generation at the whole-cell level and in different subcellular compartments (plasma membrane rafts, mitochondria, and endoplasmic reticulum) after irradiation with photons. Ceramide was measured by high-performance liquid chromatography or after pulse labeling experiments, and the presence of sphingomyelinase within mitochondria was assessed by electron microscopy. Results: Irradiation of Jurkat leukemia cells resulted in the sequential triggering of sphingomyelin hydrolysis, followed by de novo synthesis that led to a late ceramide response (from 24 h) correlated with the triggering of apoptosis. At the subcellular level, pulse-label experiments, using [{sup 3}H]-palmitate as a precursor, strengthened the involvement of the radiation-induced sphingomyelin breakdown and revealed a very early peak (15 min) of ceramide in plasma membrane rafts. A second peak in mitochondria was measured 4 h after irradiation, resulting from an increase of the sphingomyelin content relating to the targeting of acid sphingomyelinase toward this organelle. Conclusion: These data confirm that ceramide is a major determinant in the triggering of radiation-induced apoptosis and highlight the complexity of the sequential compartment-specific ceramide-mediated response of Jurkat leukemia cells to {gamma}-rays.

  3. Cell-cycle-dependent regulation of Ca2+-activated K+ channel in Jurkat T-lymphocyte.

    PubMed

    Morimoto, Takashi; Ohya, Susumu; Hayashi, Hidetoshi; Onozaki, Kikuo; Imaizumi, Yuji

    2007-05-01

    Small-conductance Ca2+-activated K+ (SK2) channel plays an important role in the activation of Jurkat T-lymphocytes by maintaining electrical gradients for the sustained Ca2+ influx. Apamin-sensitive K+ current was significantly decreased with cell-cycle progression from G0/G1 into G2/M phases, and protein expression of SK2 channels showed parallel down-regulation, with its highest expression at early G0/G1 phase. In the G0/G1 phase, the apamin-sensitive component of thapsigargin-induced Ca2+ influx was significantly larger than that in the G2/M phase. These observations suggest that SK2-channel activation may largely contribute to the sustained Ca2+ influx in the G0/G1 phase in comparison of that in the G2/M phase in Jurkat T-lymphocytes.

  4. Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells

    PubMed Central

    Gessi, Stefania; Varani, Katia; Merighi, Stefania; Morelli, Anna; Ferrari, Davide; Leung, Edward; Baraldi, Pier Giovanni; Spalluto, Giampiero; Borea, Pier Andrea

    2001-01-01

    The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line. The A3 subtype was found by means of RT-PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 1.9±0.2 nM and Bmax of 1.3±0.1 pmol mg−1 of protein. The pharmacological profile of [3H]-MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype. Thermodynamic data indicated that [3H]-MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy- and enthalpy-driven, according with that found in cells expressing the recombinant human A3 subtype. In functional assays the high affinity A3 agonists Cl-IB-MECA and IB-MECA were able to inhibit cyclic AMP accumulation and stimulate Ca2+ release from intracellular Ca2+ pools followed by Ca2+ influx. The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]-DPCPX binding with a KD of 0.9±0.1 nM and Bmax of 42±3 fmol mg−1 of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a KD of 2.5±0.3 nM and a Bmax of 1.4±0.2 pmol mg−1 of protein. In conclusion, by means of the first antagonist radioligand [3H]-MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells. PMID:11522603

  5. Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells

    PubMed Central

    Suauam, Pitipreya; Yingyongnarongkul, Boon-ek; Palaga, Tanapat; Miyakawa, Tokichi; Yompakdee, Chulee

    2015-01-01

    Small-molecule inhibitors of Ca2+-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca2+-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl2 in the growth medium of Ca2+-sensitive Δzds1 strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca2+ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δmpk1 (lacking the Mpk1 MAP kinase pathway) but not Δcnb1 (lacking the calcineurin pathway) strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant. PMID:26313553

  6. Amoebic PI3K and PKC is required for Jurkat T cell death induced by Entamoeba histolytica.

    PubMed

    Lee, Young Ah; Kim, Kyeong Ah; Min, Arim; Shin, Myeong Heon

    2014-08-01

    The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.

  7. 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.

  8. Transcriptome analysis of the human T lymphocyte cell line Jurkat and human peripheral blood mononuclear cells exposed to deoxynivalenol (DON): New mechanistic insights

    SciTech Connect

    Katika, Madhumohan R.; Hendriksen, Peter J.M.; Shao, Jia; Loveren, Henk van; Peijnenburg, Ad

    2012-10-01

    Deoxynivalenol (DON) or vomitoxin is a commonly encountered type-B trichothecene mycotoxin, produced by Fusarium species predominantly found in cereals and grains. DON is known to exert toxic effects on the gastrointestinal, reproductive and neuroendocrine systems, and particularly on the immune system. Depending on dose and exposure time, it can either stimulate or suppress immune function. The main objective of this study was to obtain a deeper insight into DON-induced effects on lymphoid cells. For this, we exposed the human T-lymphocyte cell line Jurkat and human peripheral blood mononuclear cells (PBMCs) to various concentrations of DON for various times and examined gene expression changes by DNA microarray analysis. Jurkat cells were exposed to 0.25 and 0.5 μM DON for 3, 6 and 24 h. Biological interpretation of the microarray data indicated that DON affects various processes in these cells: It upregulates genes involved in ribosome structure and function, RNA/protein synthesis and processing, endoplasmic reticulum (ER) stress, calcium-mediated signaling, mitochondrial function, oxidative stress, the NFAT and NF-κB/TNF-α pathways, T cell activation and apoptosis. The effects of DON on the expression of genes involved in ER stress, NFAT activation and apoptosis were confirmed by qRT-PCR. Other biochemical experiments confirmed that DON activates calcium-dependent proteins such as calcineurin and M-calpain that are known to be involved in T cell activation and apoptosis. Induction of T cell activation was also confirmed by demonstrating that DON activates NFATC1 and induces its translocation from the cytoplasm to the nucleus. For the gene expression profiling of PBMCs, cells were exposed to 2 and 4 μM DON for 6 and 24 h. Comparison of the Jurkat microarray data with those obtained with PBMCs showed that most of the processes affected by DON in the Jurkat cell line were also affected in the PBMCs. -- Highlights: ► The human T cell line Jurkat and human

  9. Changes in protein expression of U937 and Jurkat cells exposed to nanosecond pulsed electric fields

    NASA Astrophysics Data System (ADS)

    Moen, Erick K.; Roth, Caleb C.; Cerna, Caesar; Estalck, Larry; Wilmink, Gerald; Ibey, Bennett L.

    2013-02-01

    Application of nanosecond pulsed electric fields (nsPEF) to various biological cell lines has been to shown to cause many diverse effects, including poration of the plasma membrane, depolarization of the mitochondrial membrane, blebbing, apoptosis, and intracellular calcium bursts. The underlying mechanism(s) responsible for these diverse responses are poorly understood. Of specific interest in this paper are the long-term effects of nsPEF on cellular processes, including the regulation of genes and production of proteins. Previous studies have reported transient activation of select signaling pathways involving mitogen-activated protein kinases (MAPKs), protein phosphorylation and downstream gene expression following nsPEF application. We hypothesize that nsPEF represents a unique stimulus that could be used to externally modulate cellular processes. To validate our hypothesis, we performed a series of cuvette-based exposures at 10 and 600ns pulse widths using a custom Blumlien line pulser system. We measured acute changes in the plasma membrane structure using flow cytometry by tracking phosphatidylserine externalization via FITC-Annexin V labeling and poration via propidium iodide uptake. We then compared these results to viability of the cells at 24 hours post exposure using MTT assay and changes in the MAPK family of proteins at 8 hours post-exposure using Luminex assay. By comparing exposures at 10 and 600ns duration, we found that most MAPK family-protein expression increased in Jurkat and U937 cell lines following exposure and compared well with drops in viability and changes in plasma membrane asymmetry. What proved interesting is that some MAPK family proteins (e.g. p53, STAT1), were expressed in one cell line, but not the other. This difference may point to an underlying mechanism for observed difference in cellular sensitivity to nsPEFinduced stresses.

  10. Galectin-8 binds specific beta1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells.

    PubMed

    Cárcamo, Claudia; Pardo, Evelyn; Oyanadel, Claudia; Bravo-Zehnder, Marcela; Bull, Paulina; Cáceres, Mónica; Martínez, Jorge; Massardo, Loreto; Jacobelli, Sergio; González, Alfonso; Soza, Andrea

    2006-02-15

    Integrin-mediated encounters of T cells with extracellular cues lead these cells to adhere to a variety of substrates and acquire a spread phenotype needed for their tissue incursions. We studied the effects of galectin-8 (Gal-8), a beta-galactoside binding lectin, on Jurkat T cells. Immobilized Gal-8 bound alpha1beta1, alpha3beta1 and alpha5beta1 but not alpha2beta1 and alpha4beta1 and adhered these cells with similar kinetics to immobilized fibronectin (FN). Function-blocking experiments with monoclonal anti-integrin antibodies suggested that alpha5beta1 is the main mediator of cell adhesion to this lectin. Gal-8, but not FN, induced extensive cell spreading frequently leading to a polarized phenotype characterized by an asymmetric lamellipodial protrusion. These morphological changes involved actin cytoskeletal rearrangements controlled by PI3K, Rac-1 and ERK1/2 activity. Gal-8-induced Rac-1 activation and binding to alpha1 and alpha5 integrins have not been described in any other cellular system. Strikingly, Gal-8 was also a strong stimulus on Jurkat cells in suspension, triggering ERK1/2 activation that in most adherent cells is instead dependent on cell attachment. In addition, we found that patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disorder, produce Gal-8 autoantibodies that impede both its binding to integrins and cell adhesion. These are the first function-blocking autoantibodies reported for a member of the galectin family. These results indicate that Gal-8 constitutes a novel extracellular stimulus for T cells, able to bind specific beta1 integrins and to trigger signaling pathways conducive to cell spreading. Gal-8 could modulate a wide range of T cell-driven immune processes that eventually become altered in autoimmune disorders.

  11. Nickel and vanadium metal ions induce apoptosis of T-lymphocyte Jurkat cells.

    PubMed

    Au, Angela; Ha, Jinny; Hernandez, Mauro; Polotsky, Anna; Hungerford, David S; Frondoza, Carmelita G

    2006-12-01

    Metal alloys are used as prosthetic components in the orthopaedic and dental field. However, there is growing concern over the reported leaching of metal ions from implants. Ions released from metals have been thought to be associated with local immune dysfunction, inflammation, and tissue cell death. The objective of our study was to investigate whether nickel(II) and vanadium(V), present at a smaller percentage in most alloys, are cytotoxic to T-lymphocyte cell models. Jurkat T cells possess characteristics similar to human T-lymphocytes and proliferate at a faster rate. Jurkat T cells were incubated with control media alone or with concentrations of 1, 10, and 100 microg/mL of Ni(II) or V(V) for 24 h. Both types of metal ions reduced cell viability and proliferation in a dose-dependent manner. Ni(II) at 10 microg/mL and V(V) at 100 microg/mL activated Caspase-3 expression. Hoechst 33258 staining and transmission electron microscopy revealed chromatin condensation, as well as nuclear blebbing and fragmentation. Induction of DNA fragmentation by Ni(II) at 100 microg/mL was also indicated by agarose electrophoresis. Our observations indicate that Ni and V ions kill T cells via apoptotic and nonapoptotic pathways.

  12. Characterization of arsenic trioxide resistant clones derived from Jurkat leukemia T cell line: focus on PI3K/Akt signaling pathway.

    PubMed

    Roszak, Joanna; Smok-Pieniążek, Anna; Nocuń, Marek; Stępnik, Maciej

    2013-10-05

    In this study the role of PI3K/Akt signaling pathway in arsenic trioxide (ATO)-treated parental Jurkat cells and also in derived ATO-resistant clones grown in the presence of given ATO concentration was investigated. ATO-resistant clones (cultured for 8-12weeks in the presence of 1, 2.5 and 5μM ATO) were characterized by high viability in the presence of ATO but slower growth rate compared to the parental cells. Morphological and functional characterization of derived ATO-resistant clones revealed that they did not differ fundamentally from parental Jurkat cells in terms of cell size, level of GSH, the lysosomal fluorescence or CD95/Fas surface antigen expression. However, a slight increase in the mitochondrial potential (JC-1 staining) was detected in the clones compared to parental Jurkat cells. Side population analysis (Vybrant DyeCycle Violet™ staining) in ATO resistant clones did not indicate any enrichment withcancer stem cells. Akt1/2, AktV or wortmannin inhibitors decreased viability of ATO-resistant clones grown in the presence of ATO, with no effect on ATO-treated parental cells. Flow cytometry analysis showed that ATO decreased the level of p-Akt in ATO-treated parental cells, while the resistant clones exhibited higher levels of p-Akt immunostaining than parental Jurkat cells. Expression analysis of 84 genes involved in the PI3K/Akt pathway revealed that this pathway was predominantly active in ATO-resistant clones. c-JUN seems to play a key role in the induction of cell death in ATO-treated parental Jurkat cells, as dose-dependent strong up-regulation of JUN was specific for the ATO-treated parental Jurkat cells. On the other hand, changes in expression of cyclin D1 (CCND1), insulin receptor substrate 1 (IRS1) and protein kinase C isoforms (PRKCZ,PRKCB and PRKCA) may be responsible for the induction of resistance to ATO. The changes in expression of growth factor receptor-bound protein 10 (GRB10) observed in ATO-resistant clones suggest a

  13. Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro.

    PubMed

    Schmeits, Peter C J; Shao, Jia; van der Krieken, Danique A; Volger, Oscar L; van Loveren, Henk; Peijnenburg, Ad A C M; Hendriksen, Peter J M

    2015-07-01

    Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.

  14. Exposure of Jurkat cells to bis (tri-n-butyltin) oxide (TBTO) induces transcriptomics changes indicative for ER- and oxidative stress, T cell activation and apoptosis

    SciTech Connect

    Katika, Madhumohan R.; Hendriksen, Peter J.M.; Loveren, Henk van; Peijnenburg, Ad

    2011-08-01

    Tributyltin oxide (TBTO) is an organotin compound that is widely used as a biocide in agriculture and as an antifouling agent in paints. TBTO is toxic for many cell types, particularly immune cells. The present study aimed to identify the effects of TBTO on the human T lymphocyte cell line Jurkat. Cells were treated with 0.2 and 0.5 {mu}M TBTO for 3, 6, 12 and 24 h and then subjected to whole genome gene expression microarray analysis. The biological interpretation of the gene expression profiles revealed that endoplasmic reticulum (ER) stress is among the earliest effects of TBTO. Simultaneously or shortly thereafter, oxidative stress, activation of NFKB and NFAT, T cell activation, and apoptosis are induced. The effects of TBTO on genes involved in ER stress, NFAT pathway, T cell activation and apoptosis were confirmed by qRT-PCR. Activation and nuclear translocation of NFATC1 and the oxidative stress response proteins NRF2 and KEAP1 were confirmed by immunocytology. Taking advantage of previously published microarray data, we demonstrated that the induction of ER stress, oxidative stress, T cell activation and apoptosis by TBTO is not unique for Jurkat cells but does also occur in mouse thymocytes both ex vivo and in vivo and rat thymocytes ex vivo. We propose that the induction of ER stress leading to a T cell activation response is a major factor in the higher sensitivity of immune cells above other types of cells for TBTO. - Research Highlights: > The human T lymphocyte cell line Jurkat was exposed to TBTO. > Whole-genome microarray experiments were performed. > Data analysis revealed the induction of ER stress and activation of NFAT and NFKB. > Exposure to TBTO also led to T cell activation, oxidative stress and apoptosis.

  15. Influence of zinc deficiency on cell-membrane fluidity in Jurkat, 3T3 and IMR-32 cells.

    PubMed Central

    Verstraeten, Sandra V; Zago, M Paola; MacKenzie, Gerardo G; Keen, Carl L; Oteiza, Patricia I

    2004-01-01

    We investigated whether zinc deficiency can affect plasma membrane rheology. Three cell lines, human leukaemia T-cells (Jurkat), rat fibroblasts (3T3) and human neuroblastoma cells (IMR-32), were cultured for 48 h in control medium, in zinc-deficient medium (1.5 microM zinc; 1.5 Zn), or in the zinc-deficient medium supplemented with 15 microM zinc (15 Zn). The number of viable cells was lower in the 1.5 Zn group than in the control and 15 Zn groups. The frequency of apoptosis was higher in the 1.5 Zn group than in the control and 15 Zn groups. Membrane fluidity was evaluated using the 6-(9-anthroyloxy)stearic acid and 16-(9-anthroyloxy)palmitic acid probes. Membrane fluidity was higher in 1.5 Zn cells than in the control cells; no differences were observed between control cells and 15 Zn cells. The effect of zinc deficiency on membrane fluidity at the water/lipid interface was associated with a higher phosphatidylserine externalization. The higher membrane fluidity in the hydrophobic region of the bilayer was correlated with a lower content of arachidonic acid. We suggest that the increased fluidity of the membrane secondary to zinc deficiency is in part due to a decrease in arachidonic acid content and the apoptosis-related changes in phosphatidylserine distribution. PMID:14629198

  16. Caspase-resistant vimentin suppresses apoptosis after photodynamic treatment with a silicon phthalocyanine in Jurkat cells.

    PubMed

    Belichenko, I; Morishima, N; Separovic, D

    2001-06-01

    Oxidative stress, such as photodynamic therapy, is an apoptosis inducer. Apoptosis, as well as photosensitization, have been associated with disruption of the cytoskeletal network. The purpose of the present study was to assess the role of vimentin, a major cytoskeletal protein, in apoptosis after photodynamic treatment (PDT) with the silicon phthalocyanine Pc 4 in human Jurkat T cells. Here we show for the first time that photosensitization with Pc 4 initiates vimentin cleavage and that this event precedes poly(ADP-ribose) polymerase (PARP) degradation. Similar findings were obtained in the presence of C2-ceramide, an inducer of oxidative stress and apoptosis. In the presence of benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone, a pan-caspase inhibitor, Pc 4-PDT-induced vimentin and PARP cleavage were abolished. In Jurkat cells transfected with a caspase-resistant vimentin apoptosis was partly suppressed and delayed post-Pc 4-PDT. We suggest that the full-length vimentin confers resistance to nuclear apoptosis after PDT with Pc 4.

  17. Up-regulation of FOXP3 and induction of suppressive function in CD4+ Jurkat T-cells expressing hepatitis C virus core protein.

    PubMed

    Dominguez-Villar, Margarita; Fernandez-Ponce, Cecilia; Munoz-Suano, Alba; Gomez, Esperanza; Rodríguez-Iglesias, Manuel; Garcia-Cozar, Francisco

    2012-07-01

    HCV (hepatitis C virus) infection is a serious health care problem that affects more than 170 million people worldwide. Viral clearance depends on the development of a successful cellular immune response against the virus. Interestingly, such a response is altered in chronically infected patients, leading to chronic hepatitis that can result in liver fibrosis, cirrhosis and hepatocellular carcinoma. Among the mechanisms that have been described as being responsible for the immune suppression caused by the virus, Treg-cells (regulatory T-cells) are emerging as an essential component. In the present work we aim to study the effect of HCV-core protein in the development of T-cells with regulatory-like function. Using a third-generation lentiviral system to express HCV-core in CD4+ Jurkat T-cells, we describe that HCV-core-expressing Jurkat cells show an up-regulation of FOXP3 (forkhead box P3) and CTLA-4 (cytotoxic T-lymphocyte antigen-4). Moreover, we show that HCV-core-transduced Jurkat cells are able to suppress CD4+ and CD8+ T-cell responses to anti-CD3 plus anti-CD28 stimulation.

  18. Micromanipulation of adhesion of a Jurkat cell to a planar bilayer membrane containing lymphocyte function-associated antigen 3 molecules

    PubMed Central

    1992-01-01

    Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl- phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape. PMID:1370839

  19. Mössbauer Study and Modeling of Iron Import and Trafficking in Human Jurkat Cells

    PubMed Central

    Jhurry, Nema D.; Chakrabarti, Mrinmoy; McCormick, Sean P.; Gohil, Vishal M.; Lindahl, Paul A.

    2014-01-01

    The Fe content of Jurkat cells grown on transferrin-bound iron (TBI) and FeIII citrate (FC) was characterized using Mössbauer, EPR, and UV-vis spectroscopies, electron microscopy, and ICP-MS. Isolated mitochondria were similarly characterized. Fe-limited cells contained ∼ 100 μM of essential Fe, mainly as mitochondrial Fe and non-mitochondrial nonheme high-spin (NHHS) FeII. Fe-replete cells also contained ferritin-bound Fe and FeIII oxyhydroxide nanoparticles. Only 400 ± 100 Fe ions were loaded per ferritin complex, regardless of the growth medium Fe concentration. Ferritin regulation thus appears more complex than is commonly assumed. The magnetic/structural properties of Jurkat nanoparticles differed from those in yeast mitochondria. They were smaller and may be located in the cytosol. The extent of nanoparticle formation scaled nonlinearly with the concentration of Fe in the medium. Nanoparticle formation was not strongly correlated with ROS damage. Cells could utilize nanoparticle Fe, converting such aggregates into essential Fe forms. Cells grown on galactose rather than glucose respired faster, grew slower, exhibited more ROS damage and generally contained more nanoparticles. Cells grown with TBI rather than FC contained lower Fe concentrations, more ferritin and fewer nanoparticles. Cells in which transferrin receptor expression was increased contained more ferritin Fe. Frataxin-deficient cells contained more nanoparticles than comparable WT cells. Data were analyzed by a chemically-based mathematical model. Although simple, it captured essential features of Fe import, trafficking and regulation. TBI import was highly regulated but FC import was not. Nanoparticle formation was not regulated but the rate was third-order in cytosolic Fe. PMID:24180611

  20. Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase activation in Jurkat T-cell leukemia tumor cells.

    PubMed

    Comin-Anduix, Begona; Boros, Laszlo G; Marin, Silvia; Boren, Joan; Callol-Massot, Carles; Centelles, Josep J; Torres, Josep L; Agell, Neus; Bassilian, Sara; Cascante, Marta

    2002-11-29

    The fermented extract of wheat germ, trade name Avemar, is a complex mixture of biologically active molecules with potent anti-metastatic activities in various human malignancies. Here we report the effect of Avemar on Jurkat leukemia cell viability, proliferation, cell cycle distribution, apoptosis, and the activity of key glycolytic/pentose cycle enzymes that control carbon flow for nucleic acid synthesis. The cytotoxic IC(50) concentration of Avemar for Jurkat tumor cells is 0.2 mg/ml, and increasing doses of the crude powder inhibit Jurkat cell proliferation in a dose-dependent fashion. At concentrations higher than 0.2 mg/ml, Avemar inhibits cell growth by more than 50% (72 h of incubation), which is preceded by the appearance of a sub-G(1) peak on flow histograms at 48 h. Laser scanning cytometry of propidium iodide- and annexin V-stained cells indicated that the growth-inhibiting effect of Avemar was consistent with a strong induction of apoptosis. Inhibition by benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone of apoptosis but increased proteolysis of poly(ADP-ribose) indicate caspases mediate the cellular effects of Avemar. Activities of glucose-6-phosphate dehydrogenase and transketolase were inhibited in a dose-dependent fashion, which correlated with decreased (13)C incorporation and pentose cycle substrate flow into RNA ribose. This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. Avemar exhibits about a 50-fold higher IC(50) (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.

  1. Down-regulation of 11β-hydroxysteroid dehydrogenase type 2 by bortezomib sensitizes Jurkat leukemia T cells against glucocorticoid-induced apoptosis.

    PubMed

    Tao, Yi; Gao, Lu; Wu, Xiaosong; Wang, Hongmei; Yang, Guang; Zhan, Fenghuang; Shi, Jumei

    2013-01-01

    11β-Hydroxysteroid dehydrogenases type 2 (11β-HSD2), a key regulator for pre-receptor metabolism of glucocorticoids (GCs) by converting active GC, cortisol, to inactive cortisone, has been shown to be present in a variety of tumors. But its expression and roles have rarely been discussed in hematological malignancies. Proteasome inhibitor bortezomib has been shown to not only possess antitumor effects but also potentiate the activity of other chemotherapeutics. In this study, we demonstrated that 11β-HSD2 was highly expressed in two GC-resistant T-cell leukemic cell lines Jurkat and Molt4. In contrast, no 11β-HSD2 expression was found in two GC-sensitive non-hodgkin lymphoma cell lines Daudi and Raji as well as normal peripheral blood T cells. Inhibition of 11β-HSD2 by 11β-HSD inhibitor 18β-glycyrrhetinic acid or 11β-HSD2 shRNA significantly increased cortisol-induced apoptosis in Jurkat cells. Additionally, pretreatment of Jurkat cells with low-dose bortezomib resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis, more cells arrested at G1 stage and up-regulation of GC-induced leucine zipper which is an important mediator of GC action. Furthermore, we clarified that bortezomib could dose-dependently inhibit 11β-HSD2 messenger RNA and protein levels as well as activity (cortisol-cortisone conversion) through p38 mitogen-activated protein kinase signaling pathway. Therefore, we suggest 11β-HSD2 is, at least partially if not all, responsible for impaired GC suppression in Jurkat cells and also indicate a novel mechanism by which proteasome inhibitor bortezomib may influence GC action.

  2. Lectin interactions with the Jurkat leukemic T-cell line: quantitative binding studies and interleukin-2 production

    SciTech Connect

    Dupuis, G.; Bastin, B.

    1988-03-01

    Phytohemagglutinin (PHA), concanavalin A (Con A), pea lectin, and wheat germ agglutinin (WGA) have been used to investigate their binding properties to Jurkat 77 6.8 leukemic human T cells and their ability to induce these cells to produce interleukin-2 (IL-2). Binding studies showed that the Jurkat cells fixed 0.82 +/- 0.11 microgram pea lectin, 2.02 +/- 0.17 micrograms Con A, 1.85 +/- 0.07 micrograms PHA and 8.88 +/- 0.61 micrograms WGA. Scatchard plots were linear, indicating that the binding process was homogeneous with respect to the binding constant. PHA and Con A bound with the highest affinity (Kass (apparent) approximately equal to 9 x 10(9) M-1), followed by pea lectin and WGA (Kass (apparent) approximately equal to 3 x 10(9) M-1). The number of lectin binding sites was in agreement with the results of saturation experiments. We also evaluated the effect of the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the binding process. Results show that there were no gross alterations in the value of (apparent) Kass in the case of PHA and WGA. In contrast, the presence of TPA decreased the affinity of Con A and modified the Scatchard profile for pea lectin, which was curvilinear with a concavity turned upward. In this case, data were (apparent) K1 = 17.7 x 10(9) M-1 (high-affinity sites) and (apparent) K2 = 2.6 x 10(9) M-1 (low-affinity sites). The four lectins shared the ability to stimulate Jurkat 77 6.8 cells to secrete IL-2. Optimal lectin concentrations were 20 micrograms/ml (PHA) and 50 micrograms/ml (WGA and Con A). Pea lectin failed to display a dose-response relationship, and IL-2 production increased proportionally with lectin concentration. Con A was the most efficient stimulator (250 U/ml), followed by WGA (160 U/ml) and PHA (108 U/ml).

  3. Oxygen dependence of two-photon activation of zinc and copper phthalocyanine tetrasulfonate in Jurkat cells.

    PubMed

    Mir, Youssef; van Lier, Johan E; Paquette, Benoit; Houde, Daniel

    2008-01-01

    Photodynamic therapy (PDT), the use of light-activated drugs, is a promising treatment of cancer as well as several nonmalignant conditions. However, the efficacy of one-photon (1-gamma) PDT is limited by hypoxia, which can prevent the production of the cytotoxic singlet oxygen ((1)O(2)) species, leading to tumor resistance to PDT. To solve this problem, we propose an irradiation protocol based on a simultaneous, two-photon (2-gamma) excitation of the photosensitizer (Ps). Excitation of the Ps triplet state leads to an upper excited triplet state T(n) with distinct photochemical properties, which could inflict biologic damage independent of the presence of molecular oxygen. To determine the potential of a 2-gamma excitation process, Jurkat cells were incubated with zinc or copper phthalocyanine tetrasulfonate (ZnPcS(4) or CuPcS(4)). ZnPcS(4) is a potent (1)O(2) generator in 1-gamma PDT, while CuPcS(4) is inactive under these conditions. Jurkat cells incubated with either ZnPcS(4) or CuPcS(4) were exposed to a 670 nm continuous laser (1-gamma PDT), 532 nm pulsed-laser light (2-gamma PDT), or a combination of 532 and 670 nm (2-gamma PDT). The efficacy of ZnPcS(4) to photoinactivate the Jurkat cells decreased as the concentration of oxygen decreased for both the 1-gamma and 2-gamma protocols. In the case of CuPcS(4), cell phototoxicity was measured only following 2-gamma irradiation, and its efficacy also decreased at a lower oxygen concentration. Our results suggest that for CuPcS(4) the T(n) excited state can be populated after 2-gamma irradiation at 532 nm or the combination of 532 and 670 nm light. Dependency of phototoxicity upon aerobic conditions for both 1-gamma and 2-gamma PDT suggests that reactive oxygen species play an important role in 1-gamma and 2-gamma PDT.

  4. The pan-ErbB tyrosine kinase inhibitor canertinib induces caspase-mediated cell death in human T-cell leukemia (Jurkat) cells

    SciTech Connect

    Trinks, Cecilia; Severinsson, Emelie A.; Holmlund, Birgitta; Green, Anna; Green, Henrik; Joensson, Jan-Ingvar; Hallbeck, Anna-Lotta; Walz, Thomas M.

    2011-07-08

    Highlights: {yields} Canertinib induces caspase-mediated apoptosis in T-cell leukemia cells in vitro. {yields} Canertinib mediates activation of the intrinsic apoptotic pathway. {yields} Canertinib induces apoptosis in an ErbB receptor independent manner. {yields} Lymphocyte specific proteins as well as survival kinases are inhibited. {yields} Canertinib may act as a multi-kinase inhibiting drug in human T-cell malignancies. -- Abstract: Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects are however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 {mu}M caused accumulation of Jurkat cells in the G{sub 1} cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells.

  5. Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

    SciTech Connect

    Mader, Jamie S.; Richardson, Angela; Salsman, Jayme; Top, Deniz; Antueno, Roberto de; Duncan, Roy; Hoskin, David W. . E-mail: d.w.hoskin@dal.ca

    2007-07-15

    Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.

  6. Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

    SciTech Connect

    Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J.

    2011-11-01

    The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.

  7. Quantitative Secretome Analysis of Activated Jurkat Cells Using Click Chemistry-Based Enrichment of Secreted Glycoproteins.

    PubMed

    Witzke, Kathrin E; Rosowski, Kristin; Müller, Christian; Ahrens, Maike; Eisenacher, Martin; Megger, Dominik A; Knobloch, Jürgen; Koch, Andrea; Bracht, Thilo; Sitek, Barbara

    2017-01-06

    Quantitative secretome analyses are a high-performance tool for the discovery of physiological and pathophysiological changes in cellular processes. However, serum supplements in cell culture media limit secretome analyses, but serum depletion often leads to cell starvation and consequently biased results. To overcome these limiting factors, we investigated a model of T cell activation (Jurkat cells) and performed an approach for the selective enrichment of secreted proteins from conditioned medium utilizing metabolic marking of newly synthesized glycoproteins. Marked glycoproteins were labeled via bioorthogonal click chemistry and isolated by affinity purification. We assessed two labeling compounds conjugated with either biotin or desthiobiotin and the respective secretome fractions. 356 proteins were quantified using the biotin probe and 463 using desthiobiotin. 59 proteins were found differentially abundant (adjusted p-value ≤0.05, absolute fold change ≥1.5) between inactive and activated T cells using the biotin method and 86 using the desthiobiotin approach, with 31 mutual proteins cross-verified by independent experiments. Moreover, we analyzed the cellular proteome of the same model to demonstrate the benefit of secretome analyses and provide comprehensive data sets of both. 336 proteins (61.3%) were quantified exclusively in the secretome. Data are available via ProteomeXchange with identifier PXD004280.

  8. 17{alpha}-Estradiol arrests cell cycle progression at G{sub 2}/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

    SciTech Connect

    Jun, Do Youn; Park, Hae Sun; Kim, Jun Seok; Kim, Jong Sik; Park, Wan; Song, Bang Ho; Kim, Hee-Sook; Taub, Dennis; Kim, Young Ho

    2008-09-15

    A pharmacological dose (2.5-10 {mu}M) of 17{alpha}-estradiol (17{alpha}-E{sub 2}) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17{alpha}-E{sub 2} was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G{sub 2}/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17{alpha}-E{sub 2}-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G{sub 2}/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17{alpha}-E{sub 2}-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G{sub 1}/S boundary, 17{alpha}-E{sub 2} failed to induce the G{sub 2}/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17{alpha}-E{sub 2} toward Jurkat T cells is attributable to apoptosis mainly induced in G{sub 2}/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.

  9. Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression in Jurkat Cells.

    PubMed

    Pan, Yao; Wei, Xuetao; Hao, Weidong

    2015-08-28

    Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells.

  10. Trichloroethylene and Its Oxidative Metabolites Enhance the Activated State and Th1 Cytokine Gene Expression inJurkat Cells

    PubMed Central

    Pan, Yao; Wei, Xuetao; Hao, Weidong

    2015-01-01

    Trichloroethylene (TCE) is an occupational and ubiquitous environmental contaminant, and TCE exposure will increase the risk of autoimmune diseases and allergic diseases. T cells play an important role in the pathogenesis of TCE-related immune disorders, but the effect of TCE and its oxidative metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), on the activation of human T cells is still unknown. In this study, Jurkat cells were pre-treated with TCE, TCA and DCA overnight and then stimulated with phorbol 12-myristate 13-acetate and ionomycin for another 4, 8 and 24 hours. IL-2 secretion was detected by ELISA; the expressions of CD25 and CD69 were tested by flow cytometry; and IFN-γ and IL-2 mRNA expression levels were investigated by real-time PCR. The results showed that TCE and its oxidative metabolites, TCA and DCA, significantly enhanced IL-2 releasing and the expression of T cell activation markers, CD25 and CD69. Consistent with this result, these compounds markedly up-regulated the expression levels of IFN-γ and IL-2 mRNA. Collectively, these findings suggest that TCE and its metabolites, TCA and DCA, might enhance the activation of T cells and disrupt various activities of peripheral T cells. PMID:26343699

  11. DNA Electrophoretic Migration Patterns Change after Exposure of Jurkat Cells to a Single Intense Nanosecond Electric Pulse

    PubMed Central

    Romeo, Stefania; Zeni, Luigi; Sarti, Maurizio; Sannino, Anna; Scarfì, Maria Rosaria; Vernier, P. Thomas; Zeni, Olga

    2011-01-01

    Intense nanosecond pulsed electric fields (nsPEFs) interact with cellular membranes and intracellular structures. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of nsPEFs, including cancer therapy, and b) better understanding of the mechanisms underlying such bioelectrical effects. In this work, we explored relatively mild exposure conditions to provide insight into weak, reversible effects, laying a foundation for a better understanding of the interaction mechanisms and kinetics underlying nsPEF bio-effects. In particular, we report changes in the nucleus of Jurkat cells (human lymphoblastoid T cells) exposed to single pulses of 60 ns duration and 1.0, 1.5 and 2.5 MV/m amplitudes, which do not affect cell growth and viability. A dose-dependent reduction in alkaline comet-assayed DNA migration is observed immediately after nsPEF exposure, accompanied by permeabilization of the plasma membrane (YO-PRO-1 uptake). Comet assay profiles return to normal within 60 minutes after pulse delivery at the highest pulse amplitude tested, indicating that our exposure protocol affects the nucleus, modifying DNA electrophoretic migration patterns. PMID:22164287

  12. Inability of 50 Hz magnetic fields to regulate PKC- and Ca(2+)-dependent gene expression in Jurkat cells.

    PubMed

    Still, Maria; Lindström, Ewa; Ekstrand, A Jonas; Mild, Kjell Hansson; Mattsson, Mats-Olof; Lundgren, Erik

    2002-01-01

    We have previously reported that the T cell line Jurkat registers the exposure of a sinusoidal extremely low frequency magnetic field at the level of the plasma membrane, resulting in activation of the tyrosine kinase p56(lck), increase in inositol-3-phosphate levels and increase in intracellular calcium concentration within minutes. To elucidate if these events associated with changes in intracellular calcium ion levels were biologically significant, transient transfections of Jurkat cells were performed with calcium-ion dependent reporter constructs. Three different enhancer/promoter constructs were studied coupled to the luciferase reporter gene. The luciferase activity of each construct was measured after treatment of transfected cells to EMF exposure alone, or in combination with ionomycin, phorbol ester or cross-linking anti-CD3 antibodies. There was no indication that the used EMFs could influence any of these reporter constructs.

  13. Pandemic Influenza A (H1N1) Virus Infection Increases Apoptosis and HIV-1 Replication in HIV-1 Infected Jurkat Cells.

    PubMed

    Wang, Xue; Tan, Jiying; Biswas, Santanu; Zhao, Jiangqin; Devadas, Krishnakumar; Ye, Zhiping; Hewlett, Indira

    2016-02-02

    Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.

  14. Regulation of heat shock protein message in Jurkat cells cultured under serum-starved and gravity-altered conditions

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Hughes-Fulford, M.

    2000-01-01

    Although our understanding of effects of space flight on human physiology has advanced significantly over the past four decades, the potential contribution of stress at the cellular and gene regulation level is not characterized. The objective of this ground-based study was to evaluate stress gene regulation in cells exposed to altered gravity and environmentally suboptimal conditions. We designed primers to detect message for both the constitutive and inducible forms of the heat shock protein, HSP-70. Applying the reverse transcriptase-polymerase chain reaction (RT-PCR), we probed for HSP-70 message in human acute T-cell leukemia cells, Jurkat, subjected to three types of environmental stressors: (1) altered gravity achieved by centrifugation (hypergravity) and randomization of the gravity vector in rotating bioreactors, (2) serum starvation by culture in medium containing 0.05% serum, and (3) temperature elevation (42 degrees C). Temperature elevation, as the positive control, significantly increased HSP-70 message, while centrifugation and culture in rotating bioreactors did not upregulate heat shock gene expression. We found a fourfold increase in heat shock message in serum-starved cells. Message for the housekeeping genes, actin and cyclophilin, were constant and comparable to unstressed controls for all treatments. We conclude that gravitational perturbations incurred by centrifugal forces, exceeding those characteristic of a Space Shuttle launch (3g), and culture in rotating bioreactors do not upregulate HSP-70 gene expression. In addition, we found RT-PCR useful for evaluating stress in cultured cells. Copyright 2000 Wiley-Liss, Inc.

  15. Semi-purified extracts of Commelina benghalensis (Commelinaceae) induce apoptosis and cell cycle arrest in Jurkat-T cells

    PubMed Central

    2014-01-01

    Background Commelina benghalensis (CB) is a small plant whose fleshy stems are used in South Africa to treat skin conditions (e.g., cancerous skin outgrowths). This study was aimed at evaluating the effect of sub-fractions of acetone extracts of CB stems on growth-associated molecular events of apoptosis and cell division cycle of Jurkat-T (JT) cells. Methods Acetone extract of CB stems were subfractioned into n-hexane (F1) and dichloromethane (F2) fractions. After treatment of JT cells with these subfractions, cell proliferation, viability and apoptosis were determined using a haemocytometer, the trypan blue dye exclusion assay, and Hoechst 33258 staining, respectively. Cell division cycle distribution profiles were analysed using an Epics Alba Flow Cytometer and the expression of cell division cycle regulatory genes was analysed using RT-PCR, while immunoreactive proteins were detected on western blots. Results The F1 and F2 fractions inhibited the proliferation and viability of JT cells in a concentration- and time-dependent manner, with IC50 values of 32.5 μg/mℓ and 56 μg/mℓ, respectively. The observed cytotoxicity was established to be a consequence of apoptosis. as verified using Hoechst staining method. Both fractions induced a G1/S interphase arrest of the cell division cycle of JT cells. RT-PCR analyses showed an up-regulatory effect by the F1 fraction in the expression of cyclin B1, cdc2 and bax, with a down-regulatory effect in the expression levels of bcl-2. Fraction F1 also increased the protein expression levels of p53 and its downstream regulators, p21 and Cdc2. However, protein Bax and p21 and p53 transcripts were undetectable under the same experimental conditions. On the other hand, fraction F2 increased the mRNA expression levels of bax, bcl-2, cyclin B1 and cdc2. Concomitantly, fraction F2 showed an up-regulation in the protein expression levels of Cdc2, Bcl-2, Cyclin B1 and p21. Despite the up-regulation in protein expression levels by

  16. Integration of conventional quantitative and phospho-proteomics reveals new elements in activated Jurkat T-cell receptor pathway maintenance.

    PubMed

    Jouy, Florent; Müller, Stephan A; Wagner, Juliane; Otto, Wolfgang; von Bergen, Martin; Tomm, Janina M

    2015-01-01

    Recent years have seen a constant development of tools for the global assessment of phosphoproteins. Here, we outline a concept for integrating approaches for quantitative proteomics and phosphoproteomics. The strategy was applied to the analysis of changes in signalling and protein synthesis occurring after activation of the T-cell receptor (TCR) pathway in a T-cell line (Jurkat cells). For this purpose, peptides were obtained from four biological replicates of activated and control Jurkat T-cells and phosphopeptides enriched via a TiO2-based chromatographic step. Both phosphopeptide-enriched and flow-through fractions were analyzed by LC-MS. We observed 1314 phosphopeptides in the enriched fraction whereas 19 were detected in the flow-through, enabling the quantification of 414 and eight phosphoproteins in the respective fractions. Pathway analysis revealed the differential regulation of many metabolic pathways. Among the quantified proteins, 11 kinases with known TCR-related function were detected. A kinase-substrate database search for the phosphosites identified also confirmed the activity of a further ten kinases. In total, these two approaches provided evidence of 19 unique TCR-related kinases. The combination of phosphoproteomics and conventional quantitative shotgun analysis leads to a more comprehensive assessment of the signalling networks needed for the maintenance of the activated status of Jurkat T-cells.

  17. Biophysical Investigation of the Ironome of Human Jurkat Cells and Mitochondria

    PubMed Central

    Jhurry, Nema D.; Chakrabarti, Mrinmoy; McCormick, Sean P.; Holmes-Hampton, Gregory P.; Lindahl, Paul A.

    2012-01-01

    The speciation of iron in intact human Jurkat leukemic cells and their isolated mitochondria was assessed using biophysical methods. Large-scale cultures were grown in medium enriched with 57Fe citrate. Mitochondria were isolated anaerobically to prevent oxidation of iron centers. 5 K Mössbauer spectra of cells were dominated by a sextet due to ferritin. They also exhibited an intense central quadrupole doublet due to S = 0 [Fe4S4]2+ clusters and low-spin (LS) FeII heme centers. Spectra of isolated mitochondria were largely devoid of ferritin but contained the central doublet and features arising from what appear to be FeIII oxyhydroxide (phosphate) nanoparticles. Spectra from both cells and mitochondria contained a low-intensity doublet from non-heme high-spin (NHHS) FeII species. A portion of these species may constitute the ‘labile iron pool’ (LIP) proposed in cellular Fe trafficking. Such species might engage in Fenton chemistry to generate reactive oxygen species. EPR spectra of cells and mitochondria exhibited signals from reduced Fe/S clusters, and HS FeIII heme and non-heme species. The basal redox state of mitochondria within cells is reduced as monitored by heme redox states; this redox poise is unaltered during the anaerobic isolation of the organelle. Contributions from heme a, b and c centers were quantified using electronic absorption spectroscopy. Metal concentrations in cells and mitochondria were measured using ICP-MS. Results were collectively assessed to estimate the concentrations of various Fe-containing species in mitochondria and whole cells – the first “ironome” profile of a human cell. PMID:22726227

  18. In-vitro assessment of Jurkat T-cells response to 1966 MHz electromagnetic fields in a GTEM cell.

    PubMed

    Moraitis, Nektarios; Christopoulou, Maria; Nikita, Konstantina S; Voulgaridou, Georgia-Persephoni; Anestopoulos, Ioannis; Panagiotidis, Mihalis I; Pappa, Aglaia

    2015-01-01

    This paper presents the experimental configuration and procedure as well as the in-vitro assessment of Jurkat T-cells response to 1966 MHz exposure of modulated and unmodulated electromagnetic signals within a Gigahertz Transverse Electro-Magnetic (GTEM) cell. Different combinations of electric field intensity, exposure duration and modulation schemes were applied. Exposures at continuous wave (CW) signal at low intensity levels (3 V/m) did not induce any significant DNA damage, but a slight increase was observed for extreme stress levels (76.4 V/m). On the other hand, the results indicate that, at both, low and high electric field intensity UMTS (Universal Mobile Telecommunications System) signal could be statistically related to DNA damage in-vitro. Nevertheless, further experiments are required, increasing the statistical number of samples and recruiting more DNA damage endpoints before conclusive statements are drawn.

  19. The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells.

    PubMed Central

    Hamaia, S; Cassé, H; Gazzolo, L; Duc Dodon, M

    1997-01-01

    The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may

  20. Apoptosis induction in Jurkat cells and sCD95 levels in women's sera are related with the risk of developing cervical cancer

    PubMed Central

    Aguilar-Lemarroy, Adriana; Romero-Ramos, Jose E; Olimon-Andalon, Vicente; Hernandez-Flores, Georgina; Lerma-Diaz, Jose M; Ortiz-Lazareno, Pablo C; Morgan-Villela, Gilberto; del Toro-Arreola, Susana; Bravo-Cuellar, Alejandro; Jave-Suarez, Luis F

    2008-01-01

    Background Currently, there is clear evidence that apoptosis plays an important role in the development and progression of tumors. One of the best characterized apoptosis triggering systems is the CD95/Fas/APO-1 pathway; previous reports have demonstrated high levels of soluble CD95 (sCD95) in serum of patients with some types of cancer. Cervical cancer is the second most common cancer among women worldwide. As a first step in an attempt to design a minimally invasive test to predict the risk of developing cervical cancer in patients with precancerous lesions, we used a simple assay based on the capacity of human serum to induce apoptosis in Jurkat cells. We evaluated the relationship between sCD95 levels and the ability to induce apoptosis in Jurkat cells in cervical cancer patients and controls. Methods Jurkat cells were exposed to serum from 63 women (20 healthy volunteers, 21 with cervical intraepithelial neoplasia grade I [CIN 1] and 22 with cervical-uterine carcinoma). The apoptotic rate was measured by flow cytometry using Annexin-V-Fluos and Propidium Iodide as markers. Serum levels of sCD95 and soluble CD95 ligand (sCD95L) were measured by ELISA kits. Results We found that serum from almost all healthy women induced apoptosis in Jurkat cells, while only fifty percent of the sera from women with CIN 1 induced cell death in Jurkat cells. Interestingly, only one serum sample from a patient with cervical-uterine cancer was able to induce apoptosis, the rest of the sera protected Jurkat cells from this killing. We were able to demonstrate that elimination of Jurkat cells was mediated by the CD95/Fas/Apo-1 apoptotic pathway. Furthermore, the serum levels of sCD95 measured by ELISA were significantly higher in women with cervical cancer. Conclusion Our results demonstrate that there is a strong correlation between low levels of sCD95 in serum of normal women and higher apoptosis induction in Jurkat cells. We suggest that an analysis of the apoptotic rate induced

  1. Polysaccharide Isolated from Zizyphus jujuba (紅棗 Hóng Zǎo) Inhibits Interleukin-2 Production in Jurkat T Cells

    PubMed Central

    Hsu, Bo-Yang; Kuo, Yuh-Chi; Chen, Bing-Huei

    2014-01-01

    Zizyphus jujuba (紅棗 Hóng Zǎo), a traditional Chinese herb widely used in many Asian countries, has been shown to possess vital biological activities such as anti-cancer activity. The objective of this study was to evaluate the immunomodulatory effect of deproteinated polysaccharide (DP) isolated from Z. jujuba. The DP isolated from Z. jujuba consisted of two polysaccharide fractions and their molecular weights (MWs) were found to be 143,108 and 67,633 Da, respectively. The DP could significantly decrease interleukin (IL)-2 production in phytohemagglutinin (PHA)-activated Jurkat T cells in a dose-dependent manner after 48 h of incubation, with the inhibition being 47.5%, 61.2%, and 81.7% for DP concentrations of 0.75, 1.75, and 2.5 mg/ml, respectively. Thus, our study showed that DP isolated from Z. jujuba may possess anti-inflammatory activity as it could significantly reduce IL-2 production in activated Jurkat T cells. PMID:24860737

  2. The content of DNA and RNA in microparticles released by Jurkat and HL-60 cells undergoing in vitro apoptosis

    SciTech Connect

    Reich, Charles F.; Pisetsky, David S.

    2009-03-10

    Microparticles are small membrane-bound vesicles that are released from apoptotic cells during blebbing. These particles contain DNA and RNA and display important functional activities, including immune system activation. Furthermore, nucleic acids inside the particle can be analyzed as biomarkers in a variety of disease states. To elucidate the nature of microparticle nucleic acids, DNA and RNA released in microparticles from the Jurkat T and HL-60 promyelocytic cell lines undergoing apoptosis in vitro were studied. Microparticles were isolated from culture media by differential centrifugation and characterized by flow cytometry and molecular approaches. In these particles, DNA showed laddering by gel electrophoresis and was present in a form that allowed direct binding by a monoclonal anti-DNA antibody, suggesting antigen accessibility even without fixation. Analysis of RNA by gel electrophoresis showed intact 18s and 28s ribosomal RNA bands, although lower molecular bands consistent with 28s ribosomal RNA degradation products were also present. Particles also contained messenger RNA as shown by RT-PCR amplification of sequences for {beta}-actin and GAPDH. In addition, gel electrophoresis showed the presence of low molecular weight RNA in the size range of microRNA. Together, these results indicate that microparticles from apoptotic Jurkat and HL-60 cells contain diverse nucleic acid species, indicating translocation of both nuclear and cytoplasmic DNA and RNA as particle release occurs during death.

  3. Comparative assessment of therapeutic safety of norcantharidin, N-farnesyloxy-norcantharimide, and N-farnesyl-norcantharimide against Jurkat T cells relative to human normal lymphoblast

    PubMed Central

    Chang, Ming-Che; Wu, Jin-Yi; Liao, Hui-Fen; Chen, Yu-Jen; Kuo, Cheng-Deng

    2016-01-01

    Abstract The therapeutic safety of an anticancer drug is one of the most important concerns of the physician treating the cancer patient. Half maximal inhibitory concentration (IC50) and hillslope are usually used to represent the strength and sensitivity of an anticancer drug on cancer cells. The therapeutic safety of the anticancer drug can be assessed by comparing the IC50 and hillslope of anticancer drugs on cancer cells relative to normal cells. Since there are situations where “more anticancer activity” implies “more toxicity,” the safety of an anticancer drug in these situations is hard to evaluate by using IC50 and hillslope alone. In a previous study, the “net effect” index was devised to represent the net therapeutic effects of one anticancer drug relative to the other. However, the therapeutic safety of one specific anticancer drug alone was not defined in the “net effect” index. This study introduced the “safety index (SI)” to quantify the degree of safety of an anticancer drug by using 4-parameter logistic model on cancer cells relative to normal cells. The therapeutic safety of norcantharidin (NCTD), N-farnesyloxy-norcantharimide (NOC15), and N-farnesyl-norcantharimide (NC15) in the treatment of Jurkat T cells relative to human normal lymphoblast was compared using the newly defined SI. We found that the SI of NOC15 and NC15 was significantly higher than that of NCTD, suggesting that both NOC15 and NC15 can damage more cancer cells and less normal cells than NCTD. We conclude that both NOC15 and NC15 are safer anticancer drugs than NCTD in the treatment of Jurkat T cells relative to human normal lymphoblast. The SI can be further applied to the screening, developments, and applications of anticancer drugs in the future. PMID:27495082

  4. Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein

    PubMed Central

    Sanclemente, Manuel; Iturralde, María; Naval, Javier; Alava, María Angeles; Martínez-Lostao, Luis; Thierse, Hermann-Josef; Anel, Alberto

    2016-01-01

    We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion. PMID:27086912

  5. Comparative proteomics of exosomes secreted by tumoral Jurkat T cells and normal human T cell blasts unravels a potential tumorigenic role for valosin-containing protein.

    PubMed

    Bosque, Alberto; Dietz, Lisa; Gallego-Lleyda, Ana; Sanclemente, Manuel; Iturralde, María; Naval, Javier; Alava, María Angeles; Martínez-Lostao, Luis; Thierse, Hermann-Josef; Anel, Alberto

    2016-05-17

    We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion.

  6. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

    PubMed

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

    2009-12-01

    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  7. Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

    PubMed

    Holland, Tai C; Killilea, David W; Shenvi, Swapna V; King, Janet C

    2015-12-01

    A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

  8. Human T-cell line (Jurkat cell) encapsulation by nano-organized polyelectrolytes and their response assessment in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Pandey, Siddharth; Afrin, Fahrat; Tripathi, R. P.; Gangenahalli, Gurudutta

    2013-07-01

    Single living Jurkat cells have been encapsulated through polyelectrolytes nanoparticles poly(allyl amine hydrochloride) size 15.6 nm and poly(styrene- co-sulfonic acid sodium salt) size 30.2 nm, through layer by layer coating of oppositely charged plasma membrane. Confocal microscopy and scanning electron microscopy results showed complete shielding of Jurkat cells, and no changes in cell surface morphology of encapsulated cells were observed. Cell viability was not affected after encapsulation and no toxicity was found. In vivo studies demonstrated no significant changes in hematological and biochemical parameters of blood serum at day 1 and 7 in mice. Histopathological analyses of liver and spleen tissues showed nontoxic nature of prepared formulations.

  9. High ACSL5 Transcript Levels Associate with Systemic Lupus Erythematosus and Apoptosis in Jurkat T Lymphocytes and Peripheral Blood Cells

    PubMed Central

    2011-01-01

    Background Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which increased apoptosis and decreased apoptotic cells removal has been described as most relevant in the pathogenesis. Long-chain acyl-coenzyme A synthetases (ACSLs) have been involved in the immunological dysfunction of mouse models of lupus-like autoimmunity and apoptosis in different in vitro cell systems. The aim of this work was to assess among the ACSL isoforms the involvement of ACSL2, ACSL4 and ACSL5 in SLE pathogenesis. Findings With this end, we determined the ACSL2, ACSL4 and ACSL5 transcript levels in peripheral blood mononuclear cells (PBMCs) of 45 SLE patients and 49 healthy controls by quantitative real time-PCR (q-PCR). We found that patients with SLE had higher ACSL5 transcript levels than healthy controls [median (range), healthy controls = 16.5 (12.3–18.0) vs. SLE = 26.5 (17.8–41.7), P = 3.9×10 E-5] but no differences were found for ACSL2 and ACSL4. In in vitro experiments, ACSL5 mRNA expression was greatly increased when inducing apoptosis in Jurkat T cells and PBMCs by Phorbol-Myristate-Acetate plus Ionomycin (PMA+Io). On the other hand, short interference RNA (siRNA)-mediated silencing of ACSL5 decreased induced apoptosis in Jurkat T cells up to the control levels as well as decreased mRNA expression of FAS, FASLG and TNF. Conclusions These findings indicate that ACSL5 may play a role in the apoptosis that takes place in SLE. Our results point to ACSL5 as a potential novel functional marker of pathogenesis and a possible therapeutic target in SLE. PMID:22163040

  10. An Integrated Microfluidic Device for Monitoring Changes in Nitric Oxide Production in Single T-Lymphocyte (Jurkat) Cells

    PubMed Central

    Metto, Eve C.; Evans, Karsten; Barney, Patrick; Culbertson, Anne H.; Gunasekara, Dulan B.; Caruso, Giuseppe; Hulvey, Matthew K.; da Silva, Jose Alberto Fracassi; Lunte, Susan M.; Culbertson, Christopher T.

    2014-01-01

    A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a twofold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO. PMID:24010877

  11. Rho-ROCK-dependent ezrin-radixin-moesin phosphorylation regulates Fas-mediated apoptosis in Jurkat cells.

    PubMed

    Hébert, Marylise; Potin, Sophie; Sebbagh, Michaël; Bertoglio, Jacques; Bréard, Jacqueline; Hamelin, Jocelyne

    2008-11-01

    Upon engagement by its ligand, the Fas receptor (CD95/APO-1) is oligomerized in a manner dependent on F-actin. It has been shown that ezrin, a member of the ERM (ezrin-radixin-moesin) protein family can link Fas to the actin cytoskeleton. We show herein that in Jurkat cells, not only ezrin but also moesin can associate with Fas. The same observation was made in activated human peripheral blood T cells. Fas/ezrin or moesin (E/M) association increases in Jurkat cells following Fas triggering and occurs concomitantly with the formation of SDS- and 2-ME-stable high molecular mass Fas aggregates. Ezrin and moesin have to be present together for the formation of Fas aggregates since down-regulation of either ezrin or moesin expression with small interfering RNAs completely inhibits Fas aggregate formation. Although FADD (Fas-associated death domain protein) and caspase-8 associate with Fas in the absence of E/M, subsequent events such as caspase-8 activation and sensitivity to apoptosis are decreased. During the course of Fas stimulation, ezrin and moesin become phosphorylated, respectively, on T567 and on T558. This phosphorylation is mediated by the kinase ROCK (Rho-associated coiled coil-containing protein kinase) I subsequently to Rho activation. Indeed, inhibition of either Rho or ROCK prevents ezrin and moesin phosphorylation, abrogates the formation of Fas aggregates, and interferes with caspase-8 activation. Thus, phosphorylation of E/M by ROCK is involved in the early steps of apoptotic signaling following Fas triggering and regulates apoptosis induction.

  12. Activation of the STAT6 transcription factor in Jurkat T-cells by the herpesvirus saimiri Tip protein

    PubMed Central

    Kim, Yuri; Kwon, Eun-Kyung; Jeon, Ju-Hong; So, Insuk; Kim, In-Gyu; Choi, Myung-Sik; Kim, Ik-Sang; Choi, Joong-Kook; Jung, Jae Ung

    2012-01-01

    Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr127 residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS. PMID:22012462

  13. The N-terminal Arg2, Arg3 and Arg4 of human lactoferrin interact with sulphated molecules but not with the receptor present on Jurkat human lymphoblastic T-cells.

    PubMed Central

    Legrand, D; van Berkel, P H; Salmon, V; van Veen, H A; Slomianny, M C; Nuijens, J H; Spik, G

    1997-01-01

    We previously characterized a 105 kDa receptor for human lactoferrin (hLf) on Jurkat human lymphoblastic T-cells. To delineate the role of the basic cluster Arg2-Arg3-Arg4-Arg5 of hLf in the interaction with Jurkat cells, we isolated N-terminally deleted hLf species of molecular mass 80 kDa lacking two, three or four N-terminal residues (hLf-2N, hLf-3N and hLf-4N) from native hLf that had been treated with trypsin. Native hLf bound to 102000 sites on Jurkat cells with a dissociation constant (Kd) of 70 nM. Consecutive removal of N-terminal arginine residues from hLf progressively increased the binding affinity but decreased the number of binding sites on the cells. A recombinant hLF mutant lacking the first five N-terminal residues (rhLf-5N) bound to 17000 sites with a Kd of 12 nM. The binding parameters of bovine lactoferrin (Lf) and native hLf did not significantly differ, whereas the binding parameters of murine Lf (8000 sites; Kd 30 nM) resembled those of rhLf-5N. Culture of Jurkat cells in the presence of chlorate, which inhibits sulphation, decreased the number of binding sites for both native hLf and hLf-3N but not for rhLf-5N, indicating that the hLf-binding sites include sulphated molecules. We propose that the interaction of hLf with a large number of binding sites (approx. 80000 per cell) on Jurkat cells is dependent on Arg2-Arg3-Arg4, but not on Arg5. Interaction with approx. 20000 binding sites per cell, presumably the hLf receptor, does not require the first N-terminal basic cluster of hLf. Moreover, the affinity of hLf for the latter binding site is enhanced approx. 6-fold after removal of the first basic cluster. Thus N-terminal proteolysis of hLf in vivo might serve to modulate the nature of its binding to cells and thereby its effects on cellular physiology. PMID:9581564

  14. Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP)

    PubMed Central

    Glickman, Randolph D.; Tolstykh, Gleb P.; Estlack, Larry E.; Moen, Erick K.; Echchgadda, Ibtissam; Beier, Hope T.; Barnes, Ronald A.; Ibey, Bennett L.

    2016-01-01

    Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress. PMID:27135944

  15. Benzo[a]pyrene affects Jurkat T cells in the activated state via the antioxidant response element dependent Nrf2 pathway leading to decreased IL-2 secretion and redirecting glutamine metabolism

    SciTech Connect

    Murugaiyan, Jayaseelan; Rockstroh, Maxie; Wagner, Juliane; Baumann, Sven; Schorsch, Katrin; Trump, Saskia; Lehmann, Irina; Bergen, Martin von; Tomm, Janina M.

    2013-06-15

    There is a clear evidence that environmental pollutants, such as benzo[a]pyrene (B[a]P), can have detrimental effects on the immune system, whereas the underlying mechanisms still remain elusive. Jurkat T cells share many properties with native T lymphocytes and therefore are an appropriate model to analyze the effects of environmental pollutants on T cells and their activation. Since environmental compounds frequently occur at low, not acute toxic concentrations, we analyzed the effects of two subtoxic concentrations, 50 nM and 5 μM, on non- and activated cells. B[a]P interferes directly with the stimulation process as proven by an altered IL-2 secretion. Furthermore, B[a]P exposure results in significant proteomic changes as shown by DIGE analysis. Pathway analysis revealed an involvement of the AhR independent Nrf2 pathway in the altered processes observed in unstimulated and stimulated cells. A participation of the Nrf2 pathway in the change of IL-2 secretion was confirmed by exposing cells to the Nrf2 activator tBHQ. tBHQ and 5 μM B[a]P caused similar alterations of IL-2 secretion and glutamine/glutamate metabolism. Moreover, the proteome changes in unstimulated cells point towards a modified regulation of the cytoskeleton and cellular stress response, which was proven by western blotting. Additionally, there is a strong evidence for alterations in metabolic pathways caused by B[a]P exposure in stimulated cells. Especially the glutamine/glutamate metabolism was indicated by proteome pathway analysis and validated by metabolite measurements. The detrimental effects were slightly enhanced in stimulated cells, suggesting that stimulated cells are more vulnerable to the environmental pollutant model compound B[a]P. - Highlights: • B[a]P affects the proteome of Jurkat T cells also at low concentrations. • Exposure to B[a]P (50 nM, 5 μM) did not change Jurkat T cell viability. • Both B[a]P concentrations altered the IL-2 secretion of stimulated cells.

  16. Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c-Jun N-terminal kinase.

    PubMed

    Du, J; Suzuki, H; Nagase, F; Akhand, A A; Yokoyama, T; Miyata, T; Kurokawa, K; Nakashima, I

    2000-03-01

    Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG-induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT-4, HeLa, or COS-7 cells. A caspase inhibitor, Z-DEVD-fmk, completely blocked MG-induced poly(ADP-ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG-induced caspase-3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential.

  17. AMP-activated protein kinase mediates T cell activation-induced expression of FasL and COX-2 via protein kinase C theta-dependent pathway in human Jurkat T leukemia cells.

    PubMed

    Lee, Jung Yeon; Choi, A-Young; Oh, Young Taek; Choe, Wonchae; Yeo, Eui-Ju; Ha, Joohun; Kang, Insug

    2012-06-01

    AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, is known to be activated during T cell activation. T cell activation by T cell receptor (TCR) engagement or its pharmacological mimics, PMA plus ionomycin (PMA/Io), induces immunomodulatory FasL and cyclooxygenase-2 (COX-2) expression. In this study, we examined the role and mechanisms of AMPK in PMA/Io-induced expression of FasL and COX-2 in Jurkat T human leukemic cells. Inhibition of AMPK by a pharmacological agent, compound C, or AMPKα1 siRNA suppressed expression of FasL and COX-2 mRNAs and proteins in PMA/Io-activated Jurkat cells. It also reduced secretion of FasL protein and prostaglandin E2, a main product of COX-2, in Jurkat cells and peripheral blood lymphocytes activated with PMA/Io or monoclonal anti-CD3 plus anti-CD28. Consistently, inhibition of AMPK blocked promoter activities of FasL and COX-2 in activated Jurkat cells. As protein kinase C theta (PKCθ) is a central molecule for TCR signaling, we examined any possible cross-talk between AMPK and PKCθ in activated T cells. Of particular importance, we found that inhibition of AMPK blocked phosphorylation and activation of PKCθ, suggesting that AMPK is an upstream kinase of PKCθ. Moreover, we showed that AMPK was directly associated with PKCθ and phosphorylated Thr538 of PKCθ in PMA/Io-stimulated Jurkat cells. We also showed that inhibition of PKCθ by rottlerin or dominant negative PKCθ reduced AMPK-mediated transcriptional activation of NF-AT and AP-1 in activated Jurkat cells. Taken together, these results suggest that AMPK regulates expression of FasL and COX-2 via the PKCθ and NF-AT and AP-1 pathways in activated Jurkat cells.

  18. Synthesis of some Mannich bases with dimethylamine and their hydrazones and evaluation of their cytotoxicity against Jurkat cells.

    PubMed

    Kucukoglu, Kaan; Gul, Mustafa; Atalay, Mustafa; Mete, Ebru; Kazaz, Cavit; Hanninen, Osmo; Gul, Halise Inci

    2011-01-01

    1-Aryl-3-dimethylamino-1-propanone hydrochlorides type mono Mannich bases, D series, and corresponding hydrazone derivatives, K series, were synthesized and their cytotoxicity was tested against Jurkat cells (transformed human T-lymphocytes). The aryl part was changed as phenyl in D1 and K1, 4-methylphenyl in D2 and K2, 4-methoxyphenyl in D3 and K3, 4-hydroxyphenyl in D4 and K4, 4-chlorophenyl in D5 and K5, 3-methoxyphenyl in D6 and K6, 4-fluorophenyl in D7 and K7, 4-bromophenyl in D8 and K8, 3-hydroxyphenyl in D9 and K9, and 2-acetylthiophene in D10 and K10. Of the compounds synthesized, K2, K3, K5, K6, K7, K8, K9, and K10 are reported for the first time. Cytotoxic activities of the D and K series were compared with each other to see alterations in bioactivity depending on the chemical structures in Jurkat cells. Cytotoxicities of the compounds synthesized were also compared with the reference compound, 5-fluorouracil (CAS 148-82-3). Mono Mannich bases, D1 (3.60 times), D2 (4.45 times), D3 (2.46 times), D4 (3.52 times), D5 (5.18 times), D6 (3.20 times), D7 (3.23 times), D8 (3.95 times), D9 (3.36 times) and D10 (3.99 times) had 2.46-5.18 times higher cytotoxic potency than the reference compound 5-fluorouracil against Jurkat cells, while hydrazones K1 (4.92 times), K2 (4.65 times), K3 (6.04 times), K4 (6.34 times), K5 (4.67 times), K6 (5.12 times), K7 (5.39 times), K8 (8.31 times), K9 (4.65 times) and K10 (8.65 times) had 4.65-8.65 times higher cytotoxic potency than the reference compound 5-fluorouracil against the same cell line. On the other hand, hydrazone compounds K1 (1.37 times), K3 (2.46 times), K4 (1.80 times), K6 (1.60 times), K7 (1.67 times), K8 (2.11 times), K9 (1.38 times), and K10 (2.17 times) had 1.37-2.46 times higher cytotoxic potency than their corresponding mono Mannich bases. The results of this study suggest that hydrazones were better compounds compared with the corresponding mono Mannich bases in terms of cytotoxicity, and they may serve as

  19. Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation*

    PubMed Central

    Norambuena, Andrés; Metz, Claudia; Vicuña, Lucas; Silva, Antonia; Pardo, Evelyn; Oyanadel, Claudia; Massardo, Loreto; González, Alfonso; Soza, Andrea

    2009-01-01

    Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity. PMID:19276072

  20. Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells.

    PubMed

    Yi, Jason; Wu, Xufeng S; Crites, Travis; Hammer, John A

    2012-03-01

    Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin-a novel reporter for F-actin-we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function-associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse.

  1. Altered expression of miR-125a-5p in thymoma-associated myasthenia gravis and its down-regulation of foxp3 expression in Jurkat cells.

    PubMed

    Li, Jinpin; Qiu, Di; Chen, Zezhi; Du, Weiwei; Liu, Jingli; Mo, Xuean

    2016-04-01

    Myasthenia gravis is an autoantibody-mediated and T cell-dependent autoimmune disease of neuromuscular junctions. Thymomas may play a crucial role in the pathogenesis of thymoma-associated myasthenia gravis (TAMG), but the thymic pathogenesis of TAMG is unknown. MicroRNAs (miRNAs) are non-coding RNA molecules 21-24 nt in length that regulate the expression of their target genes in a post-transcriptional manner. In this study, we used a miRNA microarray chip to identify, for the first time, 137 miRNAs in normal tissue adjacent to the thymoma from TAMG patients that were significantly dysregulated compared with normal thymus controls. We confirmed the differential expression of miR-125a-5p in larger samples using quantitative real-time polymerase chain reaction (qRT-PCR). Using bioinformatics analysis, we identified the foxp3 3' untranslated region (UTR) as a target of miR-125a-5p. Importantly, miR-125a-5p expression exhibited a negative correlation with foxp3 expression in normal tissue adjacent to the thymoma from TAMG patients. Furthermore, we demonstrated that the expression of the foxp3 gene was modulated by miR-125a-5p in Jurkat cells. Taken together, our results suggest that the abnormal expression of miR-125a-5p and its effect on foxp3 expression are likely involved in the pathogenesis of TAMG.

  2. A20 inhibits tumor necrosis factor (TNF) alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in Jurkat T cells.

    PubMed

    He, Kai-Li; Ting, Adrian T

    2002-09-01

    Tumor necrosis factor receptor 1 (TNFR1) can trigger distinct signaling pathways leading to either the activation of NF-kappaB transcription factors or apoptosis. NF-kappaB activation results in the expression of antiapoptotic genes that inhibit the apoptosis pathway that is activated in parallel. However, the molecular mechanism of this inhibition remains poorly characterized. We have isolated a Jurkat T-cell mutant that exhibits enhanced sensitivity to TNF-induced apoptosis as a result of a deficiency in I-kappaB kinase gamma (IKKgamma)/NEMO, an essential component of the IKK complex and NF-kappaB pathway. We show here that the zinc finger protein A20 is an NF-kappaB-inducible gene that can protect the IKKgamma-deficient cells from TNF-induced apoptosis by disrupting the recruitment of the death domain signaling molecules TRADD and RIP to the receptor signaling complex. Our study, together with reports on the role of other antiapoptotic proteins such as c-FLIP and c-IAP, suggests that, in order to ensure an effective shutdown of the apoptotic pathway, TNF induces multiple NF-kappaB-dependent genes that inhibit successive steps in the TNFR1 death signaling pathway.

  3. Integrated mRNA and micro RNA profiling reveals epigenetic mechanism of differential sensitivity of Jurkat T cells to AgNPs and Ag ions.

    PubMed

    Eom, Hyun-Jeong; Chatterjee, Nivedita; Lee, Jeongsoo; Choi, Jinhee

    2014-08-17

    In our previous in vitro study of the toxicity on silver nanoparticles (AgNPs), we observed a dramatically higher sensitivity of Jurkat T cells to AgNPs than to Ag ions, and DNA damage and apoptosis were found to be involved in that toxicity. In this study, to understand underlying mechanism of different sensitivity of Jurket T cells to AgNPs and Ag ions, mRNA microarray and micro RNA microarray were concomitantly conducted on AgNPs and Ag ions exposed Jurkat T cells. Surprisingly only a small number of genes were differentially expressed by exposure to each of the silver (15 altered mRNA by AgNPs exposure, whereas 4 altered mRNA by Ag ions exposure, as determined 1.5-fold change as the cut-off value). miRNA microarray revealed that the expression of 63 miRNAs was altered by AgNPs exposure, whereas that of 32 miRNAs was altered by Ag ions exposure. An integrated analysis of mRNA and miRNA expression revealed that the expression of hsa-miR-219-5p, was negatively correlated with the expression of metallothionein 1F (MT1F) and tribbles homolog 3 (TRIB3), in cells exposed to AgNPs; whereas, the expression of hsa-miR-654-3p was negatively correlated with the expression of mRNA, endonuclease G-like 1 (EDGL1) in cells exposed to Ag ions. Network analysis were further conducted on mRNA-miRNA pairs, which revealed that miR-219-5p-MT1F and -TRIB3 pairs by AgNPs are being involved in various cellular processes, such as, oxidative stress, cell cycle and apoptosis, whereas, miR-654-3p and ENDOGL1 pair by Ag ions generated a much simpler network. The putative target genes of AgNPs-induced miR-504, miR-33 and miR-302 identified by Tarbase 6.0 are also found to be involved in DNA damage and apoptosis. These results collectively suggest that distinct epigenetic regulation may be an underlying mechanism of different sensitivity of Jurkat T cells to AgNPs and Ag ion. Further identification of putative target genes of DE miRNA by AgNPs and Ag ions may provide additional clues for the

  4. Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells.

    PubMed Central

    Hanekom, C; Nel, A; Gittinger, C; Rheeder, A; Landreth, G

    1989-01-01

    Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells. Images Fig. 1. Fig. 7. Fig. 8. PMID:2552997

  5. Wolfberry Water Soluble Phytochemicals Down-Regulate ER Stress Biomarkers and Modulate Multiple Signaling Pathways Leading To Inhibition of Proliferation and Induction of Apoptosis in Jurkat Cells

    PubMed Central

    Jiang, Yu; Zhang, Yunong; Wark, Logan; Ortiz, Edlin; Lim, Soyoung; He, Hui; Wang, Weiqun; Medeiros, Denis; Lin, Dingbo

    2012-01-01

    Phytochemicals have received much recent attention in cancer prevention through simultaneous targeting multiple pathways in the disease progression. Here we determined that wolfberry phytochemicals was chemopreventive on the leukemic Jurkat cell. The water soluble wolfberry fractions (i.e., wolfberry phytochemicals) were enriched in carbohydrates (73.4 ± 4.5 % (w/w)), polyphenolics (1555 ± 112 mg quercetin equivalent/100 g freeze dry powder, including 213 mg rutin/100 g freeze dry powder), and had enhanced antioxidant activity (7771 ± 207 μM Trolox equivalent/100 g freeze dry powder). Wolfberry phytochemicals, but not purified wolfberry polysaccharide fractions, inhibited Jurkat cell proliferation, induced cycle arrest at the G2/M phase in a dose dependent manner starting at 1 mg/ml for 48 h. Wolfberry phytochemicals eliminated cellular reactive oxygen species, declined expression of endoplasmic reticulum (ER) stress biomarkers, including glucose regulated protein 78, inositol-requiring protein 1(IRE1), activating transcription factor 6 (ATF6), protein kinase RNA-like ER kinase (PERK), and c/EBP-homologous protein, and induced activation of AMP activated protein kinase, stabilization of β-catenin, and inhibition of NFκB, and AKT activity. Simultaneous siRNA knockdown of ATF6, IRE1 and PERK caused inhibition of cell proliferation and induction of apoptosis. Data suggested that ER stress and multiple survival/apoptosis signaling pathways were modulated by wolfberry phytochemicals during the apoptotic progression. Consumption of wolfberry could be an efficacious dietary strategy for preventing leukemia. PMID:22685690

  6. Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor.

    PubMed Central

    Rouquette-Jazdanian, Alexandre K; Pelassy, Claudette; Breittmayer, Jean-Philippe; Cousin, Jean-Louis; Aussel, Claude

    2002-01-01

    Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts. PMID:11964165

  7. Mollugin induces apoptosis in human Jurkat T cells through endoplasmic reticulum stress-mediated activation of JNK and caspase-12 and subsequent activation of mitochondria-dependent caspase cascade regulated by Bcl-xL

    SciTech Connect

    Kim, Sun Mi; Park, Hae Sun; Jun, Do Youn; Woo, Hyun Ju; Woo, Mi Hee; Yang, Chae Ha; Kim, Young Ho

    2009-12-01

    Exposure of Jurkat T cells to mollugin (15-30 muM), purified from the roots of Rubia cordifolia L., caused cytotoxicity and apoptotic DNA fragmentation along with mitochondrial membrane potential disruption, mitochondrial cytochrome c release, phosphorylation of c-Jun N-terminal kinase (JNK), activation of caspase-12, -9, -7, -3, and -8, cleavage of FLIP and Bid, and PARP degradation, without accompanying necrosis. While these mollugin-induced cytotoxicity and apoptotic events including activation of caspase-8 and mitochondria-dependent activation of caspase cascade were completely prevented by overexpression of Bcl-xL, the activation of JNK and caspase-12 was prevented to much lesser extent. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk), the caspase-9 inhibitor (z-LEHD-fmk), the caspase-3 inhibitor (z-DEVD-fmk) or the caspase-12 inhibitor (z-ATAD-fmk) at the minimal concentration to prevent mollugin-induced apoptosis appeared to completely block the activation of caspase-7 and -8, and PARP degradation, but failed to block the activation of caspase-9 and -3 with allowing a slight enhancement in the level of JNK phosphorylation. Both FADD-positive wild-type Jurkat clone A3 and FADD-deficient Jurkat clone I2.1 exhibited a similar susceptibility to the cytotoxicity of mollugin, excluding involvement of Fas/FasL system in triggering mollugin-induced apoptosis. Normal peripheral T cells were more refractory to the cytotoxicity of mollugin than were Jurkat T cells. These results demonstrated that mollugin-induced cytotoxicity in Jurkat T cells was mainly attributable to apoptosis provoked via endoplasmic reticulum (ER) stress-mediated activation of JNK and caspase-12, and subsequent mitochondria-dependent activation of caspase-9 and -3, leading to activation of caspase-7 and -8, which could be regulated by Bcl-xL.

  8. JS-K, a nitric oxide-releasing prodrug, modulates ß-catenin/TCF signaling in leukemic Jurkat cells: evidence of an S-nitrosylated mechanism.

    PubMed

    Nath, Niharika; Chattopadhyay, Mitali; Pospishil, Liliya; Cieciura, Lucyna Z; Goswami, Satindra; Kodela, Ravinder; Saavedra, Joseph E; Keefer, Larry K; Kashfi, Khosrow

    2010-12-01

    β-Catenin is a central player of the Wnt signaling pathway that regulates cell-cell adhesion and may promote leukemia cell proliferation. We examined whether JS-K, an NO-donating prodrug, modulates the Wnt/β-catenin/TCF-4 signaling pathway in Jurkat T-Acute Lymphoblastic Leukemia cells. JS-K inhibited Jurkat T cell growth in a concentration and time-dependent manner. The IC(50)s for cell growth inhibition were 14±0.7 and 9±1.2μM at 24 and 48h, respectively. Treatment of the cells with JS-K for 24h, caused a dose-dependent increase in apoptosis from 16±3.3% at 10μM to 74.8±2% at 100μM and a decrease in proliferation. This growth inhibition was also due, in part, to alterations in the different phases of the cell cycle. JS-K exhibited a dose-dependent cytotoxicity as measured by LDH release at 24h. However, between 2 and 8h, LDH release was less than 20% for any indicated JS-K concentration. The β-catenin/TCF-4 transcriptional inhibitory activity was reduced by 32±8, 63±5, and 93±2% at 2, 10, and 25μM JS-K, respectively, based on luciferase reporter assays. JS-K reduced nuclear β-catenin and cyclin D1 protein levels, but cytosolic β-catenin expression did not change. Based on a time-course assay of S-nitrosylation of proteins by a biotin switch assay, S-nitrsolyation of nuclear β-catenin was determined to precede its degradation. A comparison of the S-nitrosylated nuclear β-catenin to the total nuclear β-catenin showed that β-catenin protein levels were degraded at 24h, while S-nitrosylation of β-catenin occurred earlier at 0-6h. The NO scavenger PTIO abrogated the JS-K mediated degradation of β-catenin demonstrating the need for NO.

  9. Selective role of intracellular chloride in the regulation of the intrinsic but not extrinsic pathway of apoptosis in Jurkat T-cells.

    PubMed

    Heimlich, Gerd; Cidlowski, John A

    2006-01-27

    Apoptosis is a genetic program for the removal of unwanted cells from an organism, which is distinct from necrosis by its characteristic volume loss or apoptotic volume decrease. This cell shrinkage is the result of ion redistribution that is crucial for both the activation and execution of apoptosis. Here we report that UV-C but not Fas ligand treatment results in a significant decrease in intracellular chloride that can be abolished by modulation of chloride flux using either the chloride channel inhibitor SITS or medium with a reduced chloride concentration. Accordingly, downstream events are diminished during UV-C-induced apoptosis following chloride flux modulation, whereas Fas ligand-induced apoptotic characteristics are not affected. Moreover, the activation of the mitogen-activated protein kinase signal transduction pathway early in the apoptotic signaling cascade was affected by chloride flux in Jurkat T-cells. Thus, an alteration of intracellular chloride plays an important role in the activation of signaling molecules upstream of the mitochondria, specifically impairing the intrinsic but not extrinsic apoptotic pathway.

  10. CD50 (intercellular adhesion molecule 3) stimulation induces calcium mobilization and tyrosine phosphorylation through p59fyn and p56lck in Jurkat T cell line

    PubMed Central

    1994-01-01

    The leukocyte differentiation antigen, CD50, has been recently identified as the intercellular adhesion molecule 3 (ICAM-3), the third counter-receptor of leukocyte function-associated antigen 1 (LFA-1). This molecule seems to be specially involved in the adhesion events of the initial phases of the immune response. To characterize the role of CD50 in leukocyte interactions, the different molecular events induced after cross-linking of CD50 on T cell-derived Jurkat cell line have been analyzed. When cells were incubated with anti-CD50 mAbs and cross- linked with polyclonal goat anti-mouse immunoglobulins, a rise in intracellular calcium concentration ([Ca2+]i) was observed. This increase in [Ca2+]i was mainly due to the uptake of extracellular Ca2+. This Ca2+ flux involved tyrosine phosphorylations and was further increased by CD3 costimulation. These data, together with those obtained by phosphotyrosine (P-Tyr) immunoprecipitation and in vitro kinase assays, suggested the involvement of protein-tyrosine kinases (PTK) in CD50 transduction pathways. By using specific antisera, the presence of p56lck and p59fyn protein tyrosine kinases (PTK) was clearly demonstrated in the CD50 immunoprecipitates. These findings suggest that the interaction of CD50 with its natural ligand (LFA-1) may result in T lymphocyte activation events, in which CD50 could play a very active role after antigen triggering. PMID:7515097

  11. Activation of endogenous c-fos proto-oncogene expression by human T-cell leukemia virus type I-encoded p40 sup tax protein in the human T-cell line, Jurkat

    SciTech Connect

    Nagata, Kinya; Ohtani, Kiyoshi; Nakamura, Masataka; Sugamura, Kazuo )

    1989-08-01

    The authors examined the ability of the trans-acting factor p40{sup tax} of human T-cell leukemia virus type I (HTLV-I), which is thought to be a crucial molecule in T-cell transformation by HTLV-I, to activate expression of a set of endogenous cellular genes related to T-cell proliferation. For this purpose, they established a subclone (JPX-9) of Jurkat cells that was stably transfected with an expression plasmid containing the p40{sup tax} gene, whose expression is definitively dependent on heavy-metal ions. Expression of the interleukin-2 receptor {alpha} chain in JPX-9 cells was induced in response to the induction of p40{sup tax} expression, as has been demonstrated by others in transient transfection experiments with Jurkat cells. In addition, they found that significant enhancement of expression of the nuclear proto-oncogene c-fos was closely associated with expression of p40{sup tax}. Continuous enhancement in the level of c-fos mRNA was observed in the presence of p40{sup tax}. These results suggest that (i) in addition to the interleukin-2-interleukin-2 receptor system, cellular genes such as c-fos, which regulate normal T-cell growth, are also activated directly or indirectly by p40{sup tax} and (ii) p40{sup tax}-induced modulation of gene expression plays a crucial role in T-cell transformation by HTLV-I.

  12. A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.

    PubMed

    Chen, Lihong; Meng, Qingli; Jing, Xian; Xu, Pingxiang; Luo, Dali

    2011-02-01

    Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux.

  13. Effect of vibrational stress and spaceflight on regulation of heat shock proteins hsp70 and hsp27 in human lymphocytes (Jurkat)

    NASA Technical Reports Server (NTRS)

    Cubano, L. A.; Lewis, M. L.

    2001-01-01

    Heat shock protein levels are increased in cells as a result of exposure to stress. To determine whether heat shock protein regulation could be used to evaluate stress in cells during spaceflight, the response of Jurkat cells to spaceflight and simulated space shuttle launch vibration was investigated by evaluating hsp70 and hsp27 gene expression. Gene expression was assessed by reverse transcription-polymerase chain reaction using mRNA extracted from vibrated, nonvibrated, space-flown, and ground control cells. Results indicate that mechanical stresses of vibration and low gravity do not up-regulate the mRNA for hsp70, although the gene encoding hsp27 is up-regulated by spaceflight but not by vibration. In ground controls, the mRNA for hsp70 and hsp27 increased with time in culture. We conclude that hsp70 gene expression is a useful indicator of stress related to culture density but is not an indicator of the stresses of launch vibration or microgravity. Up-regulation of hsp27 gene expression in microgravity is a new finding.

  14. Transcriptional regulation of the human IL5 gene by ionizing radiation in Jurkat T cells: evidence for repression by an NF-AT-like element.

    PubMed

    Lu-Hesselmann, J; Messer, G; van Beuningen, D; Kind, P; Peter, R U

    1997-12-01

    Eosinophilia is often observed in patients with parasitic infections and atopic diseases like allergic asthma and atopic dermatitis. Additionally, it is a typical feature of the inflammatory reaction after therapeutic and accidental exposure to ionizing radiation. This uniquely specific phenomenon regulated by the cytokine interleukin 5 (IL-5) suggests specific control for IL5 gene expression. In this study, we generated promoter-CAT constructs containing different human IL-5 promoter regions spanning from positions -507 to +43. Transfection experiments in Jurkat T cells revealed that the promoter sequence from -57 to +43 was required for constitutive and inducible IL-5 promoter activity. Low baseline CAT activity could be enhanced by treatment with phenylmercuric acetate (PMA) or the combination of PMA and calcium ionophore. The promoter region between positions -97 and +43 showed responsiveness to low-dose X rays. Electrophoretic mobility shift assays demonstrated that the region from -117 to -97 was responsive to irradiation. Transcription factors specifically bound to this sequence showed a dose-dependent response to single doses of X rays between 1 and 8 Gy. Competition analysis indicated that the protein-DNA complexes at this region were related to the nuclear factor of activated T cells (NF-AT). Further confirmation was obtained by the addition of specific antibodies into protein-DNA reactions. For the first time, we have demonstrated that specific DNA binding of NF-ATp at the promoter region from -117 to -97 is involved in transcriptional regulation of the human IL5 gene in response to ionizing radiation.

  15. Inhibition of phosphatidylserine synthesis during Jurkat T cell activation. The phosphatase inhibitor, sodium ortho-vanadate bypasses the CD3/T cell receptor-induced second messenger signaling pathway.

    PubMed

    Pelassy, C; Breittmayer, J P; Aussel, C

    2000-02-01

    Sodium ortho-vanadate (Na3VO4), an inhibitor of protein tyrosine phosphatase, induces a rapid (15 min) and strong inhibition of phosphatidylserine synthesis with an IC50 = 100 microM. The mode of action of Na3VO4 was compared to that of CD3 mAbs. It was found that Na3VO4 bypasses the major CD3-induced T cell activation signals including protein tyrosine phosphorylation, p56lck activation and the generation of second messengers including inositol phosphates and its subsequent Ca2+ mobilization as well as diacylglycerol production. These facts were confirmed by using a panel of Jurkat clones that differs by the expression of either tyrosine kinases involved in the CD3-induced T cell activation pathway such as p56lck, p72syk and ZAP-70 or some cell surface receptors such as the CD3/TCR complex or the CD45 phosphatase.

  16. Prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim reduces the association of Bcl-2 with Bak or Bim, provoking Bak activation and mitochondrial apoptosis in nocodazole-treated Jurkat T cells.

    PubMed

    Han, Cho Rong; Jun, Do Youn; Lee, Ji Young; Kim, Young Ho

    2014-01-01

    Treatment of Jurkat T cells with the microtubule-depolymerizing agent nocodazole (NOC) caused prometaphase arrest and apoptosis. NOC-induced mitochondrial apoptotic events including Bak activation, Δψm loss, cytochrome c release, and caspase cascade activation were blocked by Bcl-2 overexpression. However, mitotic arrest, Cdc25C activation, upregulation of cyclin B1 levels, Cdk1 activation, Bcl-2 phosphorylation at Thr-56 and Ser-70, and Bim phosphorylation were retained. The treatment of Jurkat T cells concomitantly with NOC and the G1/S-blocking agent hydroxyurea resulted in G1/S arrest and complete abrogation of all apoptotic events. The association of Bcl-2 with Bim or Bak declined after the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, whereas the association of Bcl-2 with Bax remained relatively constant. Although Bax was redistributed from the cytosol to the mitochondria, resulting in an increase in the mitochondrial level of Bax following NOC treatment, the subcellular localization of Bcl-2, Bim, Bak and apoptosis-inducing factor was confined to the mitochondrial fraction irrespective of NOC treatment. Experiments using selective caspase inhibitors showed that mitochondria-dependent activation of caspase-9 and -3 was crucial for NOC-induced apoptosis. NOC-induced phosphorylation of Bcl-2 and Bim, Δψm loss, and mitochondria-dependent apoptotic events were significantly suppressed by a Cdk1 inhibitor roscovitine, but not by the JNK inhibitor SP600125 or the p38 MAPK inhibitor SB203580. These results show that the prometaphase arrest-dependent phosphorylation of Bcl-2 and Bim, which was mediated by Cdk1, could reduce the association of Bcl-2 with Bak or Bim to allow Bak activation and mitochondrial apoptotic events in Jurkat T cells exposed to NOC.

  17. Effects of teicoplanin on cell number of cultured cell lines

    PubMed Central

    Kashkolinejad-Koohi, Tahere; Saadat, Iraj

    2015-01-01

    Teicoplanin is a glycopeptide antibiotic with a wide variation in human serum half-life. It is also a valuable alternative of vancomycin. There is however no study on its effect on cultured cells. The aim of the present study was to test the effect of teicoplanin on cultured cell lines CHO, Jurkat E6.1 and MCF-7. The cultured cells were exposed to teicoplanin at final concentrations of 0–11000 μg/ml for 24 hours. To determine cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed. At low concentrations of teicoplanin the numbers of cultured cells (due to cell proliferation) were increased in the three cell lines examined. The maximum cell proliferation rates were observed at concentrations of 1000, 400, and 200 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. Cell toxicity was observed at final concentrations over 2000, 6000, and 400 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. A dose-dependent manner of cell toxicity was observed. Our present findings indicated that teicoplanin at clinically used concentrations induced cell proliferation. It should therefore be used cautiously, particularly in children, pregnant women and patients with cancer. PMID:27486356

  18. Quantitative Analysis of Human T-Lymphotropic Virus Type 1 (HTLV-1) Infection Using Co-Culture with Jurkat LTR-Luciferase or Jurkat LTR-GFP Reporter Cells.

    PubMed

    Alais, Sandrine; Dutartre, Hélène; Mahieux, Renaud

    2017-01-01

    Unlike HIV-1, HTLV-1 viral transmission requires cell-to-cell contacts, while cell-free virions are poorly infectious and almost absent from body fluids. Though the virus uses three nonexclusive mechanisms to infect new target cells: (1) MTOC polarization followed by formation of a virological synapse and viral transfer into a synaptic cleft, (2) genesis of a viral biofilm and its transfer of embedded viruses, or (3) HTLV-1 transmission using conduits. The Tax transactivator and the p8 viral proteins are involved in virological synapse and nanotube formation respectively.HTLV-1 transcription from the viral promoter (i.e., LTR) requires the Tax protein that is absent from the viral particle and is expressed after productive infection. The present chapter focuses on a series of protocols used to quantify HTLV-1 de novo infection of target cells. These techniques do not discriminate between the different modes of transmission, but allow an accurate measure of productive infection. We used cell lines that are stably transfected with LTR-GFP or LTR-luciferase plasmids and quantified Green Fluorescent Protein expression or luciferase activity, since both of them reflect Tax expression.

  19. Fas/APO-1 protein is increased in spaceflown lymphocytes (Jurkat)

    NASA Technical Reports Server (NTRS)

    Cubano, L. A.; Lewis, M. L.

    2000-01-01

    Human lymphocytes flown on the Space Shuttle respond poorly to mitogen stimulation and populations of the lymphoblastoid T cell line, Jurkat, manifest growth arrest, increase in apoptosis and time- and microgravity-dependent increases in the soluble form of the cell death factor, Fas/APO-1 (sFas). The potential role of apoptosis in population dynamics of space-flown lymphocytes has not been investigated previously. We flew Jurkat cells on Space Transportation System (STS)-80 and STS-95 to determine whether apoptosis and the apparent microgravity-related release of sFas are characteristic of lymphocytes in microgravity. The effects of spaceflight and ground-based tests simulating spaceflight experimental conditions, including high cell density and low serum concentration, were assessed. Immunofluorescence microscopy showed increased cell associated Fas in flown cells. Results of STS-80 and STS-95 confirmed increase in apoptosis during spaceflight and the release of sFas as a repeatable, time-dependent and microgravity-related response. Ground-based tests showed that holding cells at 1.5 million/ml in medium containing 2% serum before launch did not increase sFas. Reports of increased Fas in cells of the elderly and the increases in spaceflown cells suggest possible similarities between aging and spaceflight effects on lymphocytes.

  20. Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant.

    PubMed Central

    Erdogan, S; Houslay, M D

    1997-01-01

    cells. Forskolin treatment led to a marked decrease of this novel PDE4A species and allowed the detection of a strong signal for an approximately 67 kDa PDE4D species, suggested to be PDE4D1, but did not induce PDE4B and PDE4C isoforms. Elevation of intracellular cAMP concentrations in Jurkat T-cells thus exerts a highly selective effect on the transcriptional activity of the genes encoding the various PDE4 isoforms. This leads to the down-regulation of a novel PDE4A splice variant and the induction of PDE4D1 and PDE4D2 splice variants, leading to a net increase in the total PDE4 activity of Jurkat T-cells. PMID:9003416

  1. Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

    SciTech Connect

    Jun, Do Youn; Kim, Jun Seok; Park, Hae Sun; Song, Woo Sun; Bae, Young Seuk; Kim, Young Ho . E-mail: ykim@knu.ac.kr

    2007-07-15

    To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 {mu}M) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins.

  2. The inhibition of voltage-gated H(+) channel (HVCN1) induces acidification of leukemic Jurkat T cells promoting cell death by apoptosis.

    PubMed

    Asuaje, Agustín; Smaldini, Paola; Martín, Pedro; Enrique, Nicolás; Orlowski, Alejandro; Aiello, Ernesto A; Gonzalez León, Carlos; Docena, Guillermo; Milesi, Verónica

    2017-02-01

    Cellular energetic deregulation is widely known to produce an overproduction of acidic species in cancer cells. This acid overload must be counterbalanced with a high rate of H(+) extrusion to maintain cell viability. In this sense, many H(+) transporters have been reported to be crucial for cell survival and proposed as antineoplastic target. By the way, voltage-gated proton channels (Hv1) mediate highly selective H(+) outward currents, capable to compensate acid burden in brief periods of time. This structure is canonically described acting as NADPH oxidase counterbalance in reactive oxygen species production. In this work, we show, for the first time in a oncohematologic cell line, that inhibition of Hv1 channels by Zn(2+) and the more selective blocker 2-(6-chloro-1H-benzimidazol-2-yl)guanidine (ClGBI) progressively decreases intracellular pH in resting conditions. This acidification is evident minutes after blockade and progresses under prolonged exposure (2, 17, and 48 h), and we firstly demonstrate that this is followed by cell death through apoptosis (annexin V binding). Altogether, these results contribute strong evidence that this channel might be a new therapeutic target in cancer.

  3. The Genomic Sequences Bound to Special AT-rich Sequence-binding Protein 1 (SATB1) In Vivo in Jurkat T Cells Are Tightly Associated with the Nuclear Matrix at the Bases of the Chromatin Loops

    PubMed Central

    de Belle, Ian; Cai, Shutao; Kohwi-Shigematsu, Terumi

    1998-01-01

    Special AT-rich sequence-binding protein 1 (SATB1), a DNA-binding protein expressed predominantly in thymocytes, recognizes an ATC sequence context that consists of a cluster of sequence stretches with well-mixed A's, T's, and C's without G's on one strand. Such regions confer a high propensity for stable base unpairing. Using an in vivo cross-linking strategy, specialized genomic sequences (0.1–1.1 kbp) that bind to SATB1 in human lymphoblastic cell line Jurkat cells were individually isolated and characterized. All in vivo SATB1-binding sequences examined contained typical ATC sequence contexts, with some exhibiting homology to autonomously replicating sequences from the yeast Saccharomyces cerevisiae that function as replication origins in yeast cells. In addition, LINE 1 elements, satellite 2 sequences, and CpG island–containing DNA were identified. To examine the higher-order packaging of these in vivo SATB1-binding sequences, high-resolution in situ fluorescence hybridization was performed with both nuclear “halos” with distended loops and the nuclear matrix after the majority of DNA had been removed by nuclease digestion. In vivo SATB1-binding sequences hybridized to genomic DNA as single spots within the residual nucleus circumscribed by the halo of DNA and remained as single spots in the nuclear matrix, indicating that these sequences are localized at the base of chromatin loops. In human breast cancer SK-BR-3 cells that do not express SATB1, at least one such sequence was found not anchored onto the nuclear matrix. These findings provide the first evidence that a cell type–specific factor such as SATB1 binds to the base of chromatin loops in vivo and suggests that a specific chromatin loop domain structure is involved in T cell–specific gene regulation. PMID:9548713

  4. Resveratrol-3-O-glucuronide and resveratrol-4’-O-glucuronide reduce DNA strand breakage but not apoptosis in Jurkat T cells treated with camptothecin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resveratrol has been reported to inhibit or induce DNA damage depending upon the type of cell and experimental conditions. Dietary resveratrol is present in the body mostly as metabolites and little is known about the activities of these metabolic products. We evaluated physiologically obtainable ...

  5. Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    PubMed Central

    Jin, Hongping; Li, Dongsheng; Sivakumaran, Haran; Lor, Mary; Rustanti, Lina; Cloonan, Nicole; Wani, Shivangi

    2016-01-01

    ABSTRACT Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells. PMID:27381288

  6. Analysis of CD2 and TCR-{beta} gene expression in jurkat cell mutants suggests a cis regulation of gene transcription

    SciTech Connect

    Kamoun, M.; Woods, J.S.; Sano, N.

    1995-10-15

    Thirty CD2{sup -} J32 stable clones, derived by mutagenesis and subsequent immunoselection with anti-CD2 Ab, were used to study the regulation of CD2 and TCR gene expression. Analysis of RNA expression revealed that the loss of surface expression of CD2 was due to a lack of expression of CD2 mRNA and was associated with a lack of expression of VDJ TCR-{beta} transcripts in 12 of these mutants, sparing the expression of DJ TCR-{beta}, TCR-{alpha}, CD3{gamma}, {delta}, {epsilon}, and {zeta} RNA. The expression of other differentiation molecules was unaffected, except for CD1, CD4, and CD5, which were either decreased or absent in most of these mutants. A gain in the expression of TCR-{gamma} transcripts was observed in each of these mutants, while, as expected, no TCR-{gamma} transcripts were detected in wild-type J32 cells. Several mutants were able to use the human CD2 enhancer and the murine TCR-{beta} enhancer and promoter to activate transcription from reporter genes in the context of heterologous promoters, indicating that the mutation(s) does not affect transcription pathways. Consistent with this finding is the adequate expression in these mutants of several lineage-specific transcription factors. The expression of CD2 in several of these mutants was rescued by gene transfer using a genomic 28.5-kb CD2 fragment, suggesting that the enchancer function of this gene may be dependent on the enhancer site. These observations suggest that the coordinate expressions of CD2 and TCR-{beta} genes share common regulatory mechanisms involving factors regulating chromatin structure and accessibility. 51 refs., 6 figs., 3 tabs.

  7. Spaceflight alters microtubules and increases apoptosis in human lymphocytes (Jurkat)

    NASA Technical Reports Server (NTRS)

    Lewis, M. L.; Reynolds, J. L.; Cubano, L. A.; Hatton, J. P.; Lawless, B. D.; Piepmeier, E. H.

    1998-01-01

    Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.

  8. Neuro-Immune Interactions for Cell-based Model under the Conditions of Irradiation

    NASA Astrophysics Data System (ADS)

    Zhu, Huiyang; Deng, Yulin; Lei, Runhong; Ma, Hong; Zhuang, Fengyuan

    AIM: To explore the proliferation effect of the SH-SY5Y cells which is damaged by the radiation on the Jurkat cells and THP-1 cells. METHODS: The first, SH-SY5Y cells was on the dose of 7 Gy γ-ray radiation, then the cells were cultured at 37℃ until 24 hours later, and the culture medium was changed. The second, culture of SH-SY5Y cells was removed, and it was used for Jurkat cells and THP-1 cells. The third, Jurkat cells and THP-1 cells were cultured until 24 hours later. At this moment the cell number was counted by the MTS. RESULTS: On the MTS results, the number of Jurkat cells in the experimental group was significantly lower than the control group, but the number of THP-1 cells in the experimental group was much higher than the control group. CONCLUSION: The 24 hours medium culture from SH-SY5Y cells which was damaged by radiation would Inhibition of the Jurkat cell growth, but it would Promote the growth of the THP-1 cell.

  9. Increased sensitivity of early apoptotic cells to complement-mediated lysis.

    PubMed

    Attali, Gitit; Gancz, Dana; Fishelson, Zvi

    2004-11-01

    Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

  10. Inhibition of proteasome activity by various fruits and vegetables is associated with cancer cell death.

    PubMed

    Chen, Marina S; Chen, Di; Dou, Q Ping

    2004-01-01

    There is a large amount of scientific evidence showing that fruits and vegetables lower the risk of cancer. However, the responsible molecular mechanisms remain poorly understood. Our previous studies have demonstrated that inhibition of proteasomal chymotrypsin-like activity is associated with cancer cell apoptosis, which may also be the major mechanism responsible for the anticancer effects of green tea polyphenols. In the current study, we tested the hypothesis that some fruits and vegetables inhibit tumor cell proteasome activity and that this inhibition contributes to their cancer-preventative activities. We report that the extracts of apple and grape are more potent than onion, tomato and celery in: (i) inhibiting the proteasomal chymotrypsin-like activity in leukemia Jurkat T cell extract; (ii) accumulating the polyubiquitinated proteins in intact Jurkat T cells; (iii) inducing activation of caspase-3/-7 and cleavage of poly(ADP-ribose) polymerase in intact Jurkat T cells; and (iv) inducing the appearance of spherical cells preferentially in prostate cancer PC-3 over the normal NIH 3T3 cell line. We also found that strawberry extract had some effect on Jurkat T cell extract and the prostate PC-3 cell line but not on intact Jurkat T cells. Our findings suggest that the proteasome is a cancer-related molecular target for, at least, the extracts of apple, grape and onion, and that the inhibition of proteasome activity by these fruits or vegetable may contribute to their cancer-preventative effects, although other molecular mechanisms may also be involved.

  11. Antitumor activity of 6-(cyclohexylamino)-1, 3-dimethyl-5(2-pyridyl)furo[2,3-d]pyrimidine-2,4(1H,3H)-dione and Its Ti(IV), Zn(II), Fe(III), and Pd(II) complexes on K562 and Jurkat cell lines.

    PubMed

    Shabani, Fahmideh; Ghammamy, Shahriar; Mehrani, Khayroallah; Teimouri, Mohammad Bagher; Soleimani, Masoud; Kaviani, Saeid

    2008-01-01

    (6-(cyclohexylamino)-1,3-dimethyl-5(2-pyridyl)furo[2,3-d]pyrimidine-2,4(1H,3H)-dione) abbreviated as CDP was synthesized and characterized. Ti(IV), Zn(II), Fe(III), and Pd(II) metal complexes of this ligand are prepared by the reaction of salts of Ti(IV), Zn(II), Fe(III), and Pd(II) with CDP in acetonitrile. Characterization of the ligand and its complexes was made by microanalyses, FT-IR, (1)H NMR, (13)C NMR, and UV-Visible spectroscopy. All complexes were characterized by several techniques using elemental analysis (C, H, N), FT-IR, electronic spectra, and molar conductance measurements. The elemental analysis data suggest the stoichiometry to be 1:1 [M:L] ratio formation. The molar conductance measurements reveal the presence of 1:1 electrolytic nature complexes. These new complexes showed excellent antitumor activity against two kinds of cancer cells that are K562 (human chronic myeloid leukemia) cells and Jurkat (human T lymphocyte carcinoma) cells.

  12. In Vitro Cytotoxic Potential of Essential Oils of Eucalyptus benthamii and Its Related Terpenes on Tumor Cell Lines

    PubMed Central

    Döll-Boscardin, Patrícia Mathias; Sartoratto, Adilson; Sales Maia, Beatriz Helena Lameiro de Noronha; Padilha de Paula, Josiane; Nakashima, Tomoe; Farago, Paulo Vitor; Kanunfre, Carla Cristine

    2012-01-01

    Eucalyptus L. is traditionally used for many medicinal purposes. In particular, some Eucalyptus species have currently shown cytotoxic properties. Local Brazilian communities have used leaves of E. benthamii as a herbal remedy for various diseases, including cancer. Considering the lack of available data for supporting this cytotoxic effect, the goal of this paper was to study the in vitro cytotoxic potential of the essential oils from young and adult leaves of E. benthamii and some related terpenes (α-pinene, terpinen-4-ol, and γ-terpinene) on Jurkat, J774A.1 and HeLa cells lines. Regarding the cytotoxic activity based on MTT assay, the essential oils showed improved results than α-pinene and γ-terpinene, particularly for Jurkat and HeLa cell lines. Terpinen-4-ol revealed a cytotoxic effect against Jurkat cells similar to that observed for volatile oils. The results of LDH activity indicated that cytotoxic activity of samples against Jurkat cells probably involved cell death by apoptosis. The decrease of cell DNA content was demonstrated due to inhibition of Jurkat cells proliferation by samples as a result of cytotoxicity. In general, the essential oils from young and adult leaves of E. benthamii presented cytotoxicity against the investigated tumor cell lines which confirms their antitumor potential. PMID:22645627

  13. Effect of selected flavones on cancer and endothelial cells.

    PubMed

    Pilátová, Martina; Stupáková, Viktória; Varinská, Lenka; Sarisský, Marek; Mirossay, Ladislav; Mirossay, Andrej; Gál, Peter; Kraus, Vladimír; Dianisková, Katarína; Mojzis, Ján

    2010-06-01

    In our study we used quercetin (3,3 ,4 ,5,7-pentahydroxyflavone) as the reference standard to compare antiproliferative and antiangiogenic effects of chrysin (5,7-dihydroxyflavone) and 3-hydroxyflavone. Our data indicates that chrysin and 3-hydroxyflavone showed significantly higher cytotoxic effect than reference standard quercetin. These tested agents significantly decreased cell survival with the efficacy of 65-85% at the concentration 100 micromol/l for HUVEC, lung carcinoma and leukemic cells being the most sensitive. Cell cycle analysis indicates that quercetin and 3-hydroxyflavone might affect the cell cycle of Jurkat cells by a similar or the same mechanism of action which lead to G2/M arrest as well as to an increase in sub-G0/G1 fraction. Treatment of Jurkat cells with chrysin resulted only increase in the fraction of cells with sub-G0/G1 DNA content, which is considered to be a marker of apoptotic cell death. Apoptosis was confirmed by DNA fragmentation and by staining with annexin V. All three tested flavones inhibited endothelial cell migration after 24 h of incubation at a concentration 100 micromol/l. At a lower concentration (10 micromol/l) only quercetin significantly inhibited migration of endothelial cells. Furthermore, in our experiments decreased secretion of matrix metalloproteinases (MMP-2 and MMP-9) was observed after a 72 h treatment with quercetin. No decrease in secretion of MMP-2 and MMP-9 was seen after chrysin and 3-hydroxyflavone treatment. On the other hand, our results showed that none of three flavonoids blocked microcapillary tube formation. Further studies are necessary to investigate the mechanism of action and to find out the relationship between the structure, character and position of substituents of natural substances and their biological activities.

  14. Cytotoxic Effects of Dimorfolido- N-Trichloroacetylphosphorylamide and Dimorfolido- N-Benzoylphosphorylamide in Combination with C60 Fullerene on Leukemic Cells and Docking Study of Their Interaction with DNA

    NASA Astrophysics Data System (ADS)

    Prylutska, S.; Grynyuk, I.; Grebinyk, A.; Hurmach, V.; Shatrava, Iu.; Sliva, T.; Amirkhanov, V.; Prylutskyy, Yu.; Matyshevska, O.; Slobodyanik, M.; Frohme, M.; Ritter, U.

    2017-02-01

    Dimorfolido- N-trichloroacetylphosphorylamide (HL1) and dimorfolido- N-benzoylphosphorylamide (HL2) as representatives of carbacylamidophosphates were synthesized and identified by the methods of IR, 1H, and 31P NMR spectroscopy. In vitro HL1 and HL2 at 1 mM concentration caused cell specific and time-dependent decrease of leukemic cell viability. Compounds caused the similar gradual decrease of Jurkat cells viability at 72 h (by 35%). HL1 had earlier and more profound toxic effect as compared to HL2 regardless on leukemic cell line. Viability of Molt-16 and CCRF-CEM cells under the action of HL1 was decreased at 24 h (by 32 and 45%, respectively) with no substantial further reducing up to 72 h. Toxic effect of HL2 was detected only at 72 h of incubation of Jurkat and Molt-16 cells (cell viability was decreased by 40 and 45%, respectively).

  15. Immune Suppressive Effect of Cinnamaldehyde Due to Inhibition of Proliferation and Induction of Apoptosis in Immune Cells: Implications in Cancer

    PubMed Central

    Gomez-Casado, Cristina; Diaz-Perales, Araceli; Oida, Kumiko; Singer, Josef; Kinaciyan, Tamar; Fuchs, Heidemarie C.; Jensen-Jarolim, Erika

    2014-01-01

    Background Besides its anti-inflammatory effects, cinnamaldehyde has been reported to have anti-carcinogenic activity. Here, we investigated its impact on immune cells. Methods Activation of nuclear factor-κB by cinnamaldehyde (0–10 µg/ml) alone or in combination with lipopolysaccharide was assessed in THP1XBlue human monocytic cell line and in human peripheral blood mononuclear cells (PBMCs). Proliferation and secretion of cytokines (IL10 and TNFα) was determined in primary immune cells and the human cell lines (THP1, Jurkat E6-1 and Raji cell lines) stimulated with cinnamaldehyde alone or in conjunction with lipopolysaccharide. Nitric oxide was determined in mouse RAW264.7 cells. Moreover, different treated PBMCs were stained for CD3, CD20 and AnnexinV. Results Low concentrations (up to 1 µg/ml) of cinnamaldehyde resulted in a slight increase in nuclar factor-kB activation, whereas higher concentrations led to a dose-dependent decrease of nuclear factor-kB activation (up to 50%) in lipopolysachharide-stimulated THP1 cells and PBMCs. Accordingly, nitric oxide, interleukin 10 secretion as well as cell proliferation were reduced in lipopolysachharide-stimulated RAW264.7 cells, PBMCs and THP1, Raji and Jurkat-E6 immune cells in the presence of cinnamaldehyde in a concentration-dependent manner. Flow cytometric analysis of PBMCs revealed that CD3+ were more affected than CD20+ cells to apopotosis by cinnamaldehyde. Conclusion We attribute the anti-inflammatory properties of cinnamaldehyde to its ability to block nuclear factor-κB activation in immune cells. Treatment with cinnamaldehyde led to inhibition of cell viability, proliferation and induced apoptosis in a dose-dependent manner in primary and immortalized immune cells. Therefore, despite its described anti-carcinogenic property, treatment with cinnamaldehyde in cancer patients might be contraindicated due to its ability to inhibit immune cell activation. PMID:25271635

  16. Specific inhibition of Wee1 kinase and Rad51 recombinase: A strategy to enhance the sensitivity of leukemic T-cells to ionizing radiation-induced DNA double-strand breaks

    SciTech Connect

    Havelek, Radim; Cmielova, Jana; Kralovec, Karel; Bruckova, Lenka; Bilkova, Zuzana; Fousova, Ivana; Sinkorova, Zuzana; Vavrova, Jirina; Rezacova, Martina

    2014-10-24

    Highlights: • Pre-treatment with the inhibitors increased the sensitivity of Jurkat cells to irradiation. • Combining both inhibitors together resulted in a G2 cell cycle arrest abrogation in Jurkat. • Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24 h upon irradiation. • Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction in MOLT-4 cells. • When dosed together, the combination decreased MOLT-4 cell survival. - Abstract: Present-day oncology sees at least two-thirds of cancer patients receiving radiation therapy as a part of their anticancer treatment. The objectives of the current study were to investigate the effects of the small molecule inhibitors of Wee1 kinase II (681641) and Rad51 (RI-1) on cell cycle progression, DNA double-strand breaks repair and apoptosis following ionizing radiation exposure in human leukemic T-cells Jurkat and MOLT-4. Pre-treatment with the Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to irradiation, however combining both inhibitors together resulted in a further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for γH2AX foci 24 h upon irradiation. MOLT-4 cells were less affected by inhibitors application prior to ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had no effect on apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4 cells.

  17. The cathepsin B inhibitor, z-FA-CMK is toxic and readily induced cell death in human T lymphocytes

    SciTech Connect

    Liow, K.Y.; Chow, S.C.

    2013-11-01

    The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induced cell death in the human T cell line, Jurkat, whereas two other analogs benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose–response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. - Highlights: • z-FA-CMK is toxic and induce cell death in the human T cells. • z-FA-CMK toxicity requires the CMK group, alanine and the benzyloxycarbonyl group. • z-FA-CMK induced apoptosis at low concentration and necrosis at high concentration.

  18. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  19. TU-100 (Daikenchuto) and ginger ameliorate anti-CD3 antibody induced T cell-mediated murine enteritis: microbe-independent effects involving Akt and NF-κB suppression.

    PubMed

    Ueno, Nobuhiro; Hasebe, Takumu; Kaneko, Atsushi; Yamamoto, Masahiro; Fujiya, Mikihiro; Kohgo, Yutaka; Kono, Toru; Wang, Chong-Zhi; Yuan, Chun-Su; Bissonnette, Marc; Chang, Eugene B; Musch, Mark W

    2014-01-01

    The Japanese traditional medicine daikenchuto (TU-100) has anti-inflammatory activities, but the mechanisms remain incompletely understood. TU-100 includes ginger, ginseng, and Japanese pepper, each component possessing bioactive properties. The effects of TU-100 and individual components were investigated in a model of intestinal T lymphocyte activation using anti-CD3 antibody. To determine contribution of intestinal bacteria, specific pathogen free (SPF) and germ free (GF) mice were used. TU-100 or its components were delivered by diet or by gavage. Anti-CD3 antibody increased jejunal accumulation of fluid, increased TNFα, and induced intestinal epithelial apoptosis in both SPF and GF mice, which was blocked by either TU-100 or ginger, but not by ginseng or Japanese pepper. TU-100 and ginger also blocked anti-CD3-stimulated Akt and NF-κB activation. A co-culture system of colonic Caco2BBE and Jurkat-1 cells was used to examine T-lymphocyte/epithelial cells interactions. Jurkat-1 cells were stimulated with anti-CD3 to produce TNFα that activates epithelial cell NF-κB. TU-100 and ginger blocked anti-CD3 antibody activation of Akt in Jurkat cells, decreasing their TNFα production. Additionally, TU-100 and ginger alone blocked direct TNFα stimulation of Caco2BBE cells and decreased activation of caspase-3 and polyADP ribose. The present studies demonstrate a new anti-inflammatory action of TU-100 that is microbe-independent and due to its ginger component.

  20. The effect of strong static magnetic field on lymphocytes.

    PubMed

    Aldinucci, Carlo; Garcia, Julian Blanco; Palmi, Mitri; Sgaragli, Gianpietro; Benocci, Alberto; Meini, Antonella; Pessina, Federica; Rossi, Claudio; Bonechi, Claudia; Pessina, Gian Paolo

    2003-02-01

    We investigated whether static electromagnetic fields (EMFs) at a flux density of 4.75 T, generated by an NMR apparatus (NMRF), could promote movements of Ca2+, cell proliferation, and the eventual production of proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC) as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 mg/ml phytohaemagglutinin (PHA). Our results clearly demonstrate that static NMRF exposure has neither proliferative, nor activating, nor proinflammatory effects on both normal and PHA activated PBMC. Moreover, the concentration of interleukin-1beta, interleukin-2, interleukin-6, interferon, and tumour necrosis factor alpha (TNFalpha) remained unvaried in exposed cells. Exposure of Jurkat cells statistically decreased the proliferation and the proliferation indexes, which 24 and 48 h after exposure were 0.7 +/- 0.29 and 0.87 +/- 0.12, respectively. Moreover, in Jurkat cells the [Ca2+]i was higher than in PBMC and was reduced significantly to about one half after exposure. This is consistent with the decrease of proliferation and with the low levels of IL-2 measured. On the whole, our data suggest that NMRF exposure failed to affect the physiologic behaviour of normal lymphomonocytes. Instead in Jurkat cells, by changing the properties of cell membranes, NMRF can influence Ca2+ transport processes, and hence Ca2+ homeostasis with improvement of proliferation.

  1. The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes.

    PubMed Central

    Chuang, T H; Hahn, K M; Lee, J D; Danley, D E; Bokoch, G M

    1997-01-01

    Apoptosis plays an important role in regulating development and homeostasis of the immune system, yet the elements of the signaling pathways that control cell death have not been well defined. When expressed in Jurkat T cells, an activated form of the small GTPase Cdc42 induces cell death exhibiting the characteristics of apoptosis. The death response induced by Cdc42 is mediated by activation of a protein kinase cascade leading to stimulation of c-Jun amino terminal kinase (JNK). Apoptosis initiated by Cdc42 is inhibited by dominant negative components of the JNK cascade and by reagents that block activity of the ICE protease (caspase) family, suggesting that stimulation of the JNK kinase cascade can lead to caspase activation. The sequence of morphological events observed typically in apoptotic cells is modified in the presence of activated Cdc42, suggesting that this GTPase may account for some aspects of cytoskeletal regulation during the apoptotic program. These data suggest a means through which the biochemical and morphological events occurring during apoptosis may be coordinately regulated. Images PMID:9307966

  2. Effect of sodium butyrate on pro-matrix metalloproteinase-9 and -2 differential secretion in pediatric tumors and cell lines.

    PubMed

    Rodríguez-Salvador, J; Armas-Pineda, C; Perezpeña-Diazconti, M; Chico-Ponce de León, F; Sosa-Sáinz, G; Lezama, P; Recillas-Targa, F; Arenas-Huertero, F

    2005-09-01

    Matrix metalloproteinases (MMPs) are enzymes responsible for extracellular matrix degradation and contribute to local and distant cell invasion during cancer progression or metastasis. The effects of chromatin structure on gene expression and the use of histone deacetylase inhibitors such as sodium butyrate (NaBu) may directly influence pro-MMPs secretion. In the present study, we evaluated the effect of NaBu on pro-MMP-9 and pro-MMP-2 secretion in human Jurkat and HT1080 cells, and in 36 pediatric solid tumors. Cell lines and samples were exposed to 8 mM of NaBu and proteinase activity was evaluated in the supernatant by gelatin zymograms. Our results showed, for Jurkat cells treated with NaBu, increases of 2-fold and 1.5-fold in pro-MMP-9 and pro-MMP-2 secretion, respectively. A 50% decrease in pro-MMP-9 secretion due to NaBu was observed in HT1080 cells. NaBu induced a 0.62 reduction in levels of pro-MMP-9 secretion in untreated tumors. For cell lines and some NaBu-treated tumors we found histone H4 hyperacetylation. We conclude that pro-MMPs gene expression and their secretion can be epigenetically mis-regulated in tumoral processes.

  3. Cellular taurine release triggered by stimulation of the Fas(CD95) receptor in Jurkat lymphocytes.

    PubMed

    Lang, F; Madlung, J; Uhlemann, A C; Risler, T; Gulbins, E

    1998-08-01

    One of the hallmarks of apoptosis is cell shrinkage which appears to be important for cell death. The mechanisms mediating cell volume decrease have, however, not been addressed. Mechanisms employed by swollen cells to decrease their cell volume include activation of ion transport pathways, such as ion channels and KCl cotransport, and release of cellular osmolytes, such as taurine, sorbitol, betaine and inositol. The present study has been performed to test for release of taurine. To this end Jurkat human T-lymphocytes were loaded with [3H]taurine and apoptotic cell death induced by triggering the Fas(CD95) receptor with monoclonal crosslinking antibody. Triggering the Fas(CD95) receptor led to a release of 60+/-5% of cellular taurine within 90 min. The release did not occur prior to 45 min. The release coincided with cell shrinkage as evidenced from forward scatter in FACS analysis and preceeded DNA fragmentation according to propidium iodide staining. The delay of taurine release was not influenced by exchange of medium and thus was not due to extracellular accumulation of a stimulator. The Fas(CD95)-induced taurine release, cell shrinkage and DNA fragmentation were blunted by lowering of ambient temperature to 23 degreesC. Following pretreatment of cells with Fas(CD95) antibody at 23 degreesC rewarming led to rapid taurine release, cell shrinkage and DNA fragmentation, indicating that the temperature-sensitive step is distal to the mechanisms accounting for the delay. Osmotic cell swelling led to an immediate release of taurine. In conclusion, Fas(CD95) triggering leads to delayed taurine release through a temperature-sensitive mechanism.

  4. Characterization of the Metabolic Flux and Apoptotic Effects of O-Hydroxyl- and N-Acyl-Modified N-Acetylmannosamine Analogs in Jurkat Cells*

    DTIC Science & Technology

    2004-04-30

    min, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. The primers used for the detection of human sialidase 1 were 5-CAC TGC CAC AGG GGT...18345 and sialidases were monitored to test whether the link between the enzymes responsible for the surface expression of sialic acid and apoptosis...RNA was isolated, and RT-PCR was performed using specific primers for two sialyl- transferases and a sialidase to confirm the alteration of gene

  5. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: Involvement of interleukin-2 system

    SciTech Connect

    Cervia, Davide; Catalani, Elisabetta; Belardinelli, Maria Cristina; Perrotta, Cristiana; Picchietti, Simona; Alimenti, Claudio; Casini, Giovanni; Fausto, Anna Maria; Vallesi, Adriana

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ► Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ► Er-1 increases the T-cell production of specific cytokines. ► Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ► The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.

  6. Low toxic and high soluble camptothecin derivative 2-47 effectively induces apoptosis of tumor cells in vitro.

    PubMed

    Zhou, Yao; Zhao, Hong-Ye; Jiang, Du; Wang, Lu-Yao; Xiang, Cen; Wen, Shao-Peng; Fan, Zhen-Chuan; Zhang, Yong-Min; Guo, Na; Teng, Yu-Ou; Yu, Peng

    2016-04-08

    The cytotoxic activity of camptothecin derivatives is so high that these compounds need to be further modified before their successful application as anti-cancer agents clinically. In this study, we reported the synthesis and biological evaluation of a novel camptothecin derivative called compound 2-47. The changes in structure did not reduce its activity to inhibit DNA topoisomerase I. Compound 2-47 induced apoptosis of many tumor cells including leukemia cells K562, Jurkat, HL-60, breast cancer cell BT-549, colon cancer cell HT-29 and liver cancer cell HepG2 with a half maximal inhibitory concentration (IC50) of 2- to 3-fold lower than HCPT as a control. In particular, 2-47 inhibited the proliferation of Jurkat cells with an IC50 of as low as 40 nM. By making use of Jurkat cell as a model, following treatment of Jurkat cells, compound 2-47 activated caspase-3 and PARP, resulting in a decreased Bcl-2/Bax ratio. These data showed that compound 2-47 induces Jurkat cell death through the mitochondrial apoptotic pathway. In addition, compound 2-47 showed a decreased cytotoxic activity against normal cells and an improved solubility in low-polar solvent. For example, compound 2-47 solutes in CHCl3 130-fold higher than HCPT. Taken together, our data demonstrated that camptothecin derivative 2-47 notably inhibits the tumor cell proliferation through mitochondrial-mediated apoptosis in vitro.

  7. 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.

  8. Fullerene C60 Penetration into Leukemic Cells and Its Photoinduced Cytotoxic Effects

    NASA Astrophysics Data System (ADS)

    Franskevych, D.; Palyvoda, K.; Petukhov, D.; Prylutska, S.; Grynyuk, I.; Schuetze, C.; Drobot, L.; Matyshevska, O.; Ritter, U.

    2017-01-01

    Fullerene C60 as a representative of carbon nanocompounds is suggested to be promising agent for application in photodynamic therapy due to its unique physicochemical properties. The goal of this study was to estimate the accumulation of fullerene C60 in leukemic cells and to investigate its phototoxic effect on parental and resistant to cisplatin leukemic cells. Stable homogeneous water colloid solution of pristine C60 with average 50-nm diameter of nanoparticles was used in experiments. Fluorescent labeled C60 was synthesized by covalent conjugation of C60 with rhodamine B isothiocyanate. The results of confocal microscopy showed that leukemic Jurkat cells could effectively uptake fullerene C60 from the medium. Light-emitting diode lamp (100 mW cm-2, λ = 420-700 nm) was used for excitation of accumulated C60. A time-dependent decrease of viability was detected when leukemic Jurkat cells were exposed to combined treatment with C60 and visible light. The cytotoxic effect of photoexcited C60 was comparable with that induced by H2O2, as both agents caused 50% decrease of cell viability at 24 h at concentrations about 50 μM. Using immunoblot analysis, protein phosphotyrosine levels in cells were estimated. Combined action of C60 and visible light was followed by decrease of cellular proteins phosphorylation on tyrosine residues though less intensive as compared with that induced by H2O2 or protein tyrosine kinase inhibitor staurosporine. All tested agents reduced phosphorylation of 55, 70, and 90 kDa proteins while total suppression of 26 kDa protein phosphorylation was specific only for photoexcited C60.

  9. Differential effects of Helenalin, an anti-inflammatory sesquiterpene lactone, on the proteome, metabolome and the oxidative stress response in several immune cell types.

    PubMed

    Zwicker, Paula; Schultze, Nadin; Niehs, Sarah; Albrecht, Dirk; Methling, Karen; Wurster, Martina; Wachlin, Gerhild; Lalk, Michael; Lindequist, Ulrike; Haertel, Beate

    2016-12-18

    Extracts of Arnica spp. are traditionally used due to their anti-inflammatory effects for the topical treatment of e.g. haematoma or muscle distortions. One of the main active compounds is Helenalin, a sesquiterpene lactone that can be found in various Asteraceae. However, immunotoxic effects of the compound are only poorly analysed. In this study, a 2D gel electrophoresis based proteomic approach together with a membrane based proteomic assay, metabolomics and the detection of intracellular reactive oxygen species (iROS) were used to investigate potential immunotoxic properties of Helenalin on the human immune cell lines Jurkat and THP-1 and on human peripheral blood mononuclear cells (PBMC). The study revealed a dose-dependent cytotoxicity towards both tested cell lines and the PBMC. However, the cell lines were less sensitive to the Helenalin treatment than the PBMC. The proteomic assays showed strong effects on the carbohydrate metabolism and the protein folding in THP-1 cells but only weak impact on Jurkat cells. Metabolomic studies as well as iROS detection in THP-1 cells verified the results of the proteomic analysis. In summary, the approaches used in this study were able to identify target pathways of Helenalin especially in THP-1 monocytes and thus enable a risk assessment of the substance.

  10. The apoptosis-inducing effect of ginsenoside F4 from steamed notoginseng on human lymphocytoma JK cells.

    PubMed

    Chen, Bin; Shen, Yu-Ping; Zhang, Dong-Feng; Cheng, Jie; Jia, Xiao-Bin

    2013-01-01

    In this study, the inhibitory and apoptosis-inducing effect of ginsenoside F4 (GF4) from steamed notoginseng was investigated by using human lymphocytoma Jurkat (JK) cell. Cell Counting Kit-8 method was then used to assess the anti-proliferative effect of GF4, and Western blotting was run to detect the expression level of two apoptosis-related proteins including Bax and the Bcl-2. The results suggested that GF4 can effectively inhibit the proliferation of the cells, and Bax expression increased gradually, but Bcl-2 expression reduced with the increase of GF4 concentration. In conclusion, GF4 has inhibitory effect on human lymphocytoma JK cell by inducing its apoptosis. The mechanism of action could be related to the mitochondrial dysfunction and the increase of Bax expression and decrease of Bcl-2 expression by GF4.

  11. Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 tax oncoproteins modulate cell cycle progression and apoptosis.

    PubMed

    Sieburg, Michelle; Tripp, Adam; Ma, Jung-Woo; Feuer, Gerold

    2004-10-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. HTLV-2, although highly related to HTLV-1 at the molecular level, has not been conclusively linked to development of lymphoproliferative disorders. Differences between the biological activities of the respective tax gene products (Tax1 and Tax2) may be one factor which accounts for the differential pathogenicities associated with infection. To develop an in vitro model to investigate and compare the effects of constitutive expression of Tax1 and Tax2, Jurkat T-cell lines were infected with lentivirus vectors encoding Tax1 and Tax2 in conjunction with green fluorescent protein, and stably transduced clonal cell lines were generated by serial dilution in the absence of drug selection. Jurkat cells that constitutively express Tax1 and Tax2 (Tax1/Jurkat and Tax2/Jurkat, respectively) showed notably reduced kinetics of cellular replication, and Tax1 inhibited cellular replication to a higher degree in comparison to Tax2. Tax1 markedly activated transcription from the cdk inhibitor p21(cip1/waf1) promoter in comparison to Tax2, suggesting that upregulation of p21(cip1/waf1) may account for the differential inhibition of cellular replication kinetics displayed by Tax1/Jurkat and Tax2/Jurkat cells. The presence of binucleated and multinucleated cells, reminiscent of large lymphocytes with cleaved or cerebriform nuclei often seen in HTLV-1- and -2-seropositive patients, was noted in cultures expressing Tax1 and Tax2. Although Tax1 and Tax2 expression mediated elevated resistance to apoptosis in Jurkat cells after serum deprivation, Tax1 was unique in protection from apoptosis after exposure to camptothecin and etoposide, inhibitors of topoisomerase I and II, respectively. Characterization of the unique phenotypes displayed by Tax1 and Tax2 in vitro will provide information as to the relative roles of

  12. Synergistic Apoptosis-Inducing Antileukemic Effects of Arsenic Trioxide and Mucuna macrocarpa Stem Extract in Human Leukemic Cells via a Reactive Oxygen Species-Dependent Mechanism

    PubMed Central

    Lu, Kuan-Hung; Lee, Hui-Ju; Huang, Min-Li; Lai, Shang-Chih; Ho, Yu-Ling; Chang, Yuan-Shiun; Chi, Chin-Wen

    2012-01-01

    The objective of this study was to examine the potential of enhancing the antileukemic activity of arsenic trioxide (ATO) by combining it with a folk remedy, crude methanolic extract of Mucuna macrocarpa (CMEMM). Human leukemia cells HL-60, Jurkat, and Molt-3 were treated with various doses of ATO, CMEMM, and combinations thereof for 24 and 48 h. Results indicated that the combination of 2.5 μM ATO and 50 μg/mL CMEMM synergistically inhibited cell proliferation in HL-60 and Jurkat cell lines. Apoptosis triggered by ATO/CMEMM treatment was confirmed by accumulation of cells in the sub-G1 phase in cell cycle analyses, characteristic apoptotic nuclear fragmentation, and increased percentage of annexin V-positive apoptotic cells. Such combination treatments also led to elevation of reactive oxygen species (ROS). The antioxidants N-acetyl cysteine (NAC), butylated hydroxytoluene, and α-tocopherol prevented cells from ATO/CMEMM-induced apoptosis. The ATO/CMEMM-induced activation of caspase-3 and caspase-9 can be blocked by NAC. In summary, these results suggest that ATO/CMEMM combination treatment exerts synergistic apoptosis-inducing effects in human leukemic cells through a ROS-dependent mechanism and may provide a promising antileukemic approach in the future. PMID:21826188

  13. Modifications in host cell cytoskeleton structure and function mediated by intracellular HIV-1 Tat protein are greatly dependent on the second coding exon

    PubMed Central

    López-Huertas, M. R.; Callejas, S.; Abia, D.; Mateos, E.; Dopazo, A.; Alcamí, J.; Coiras, M.

    2010-01-01

    The human immunodeficiency virus type 1 (HIV-1) regulator Tat is essential for viral replication because it achieves complete elongation of viral transcripts. Tat can be released to the extracellular space and taken up by adjacent cells, exerting profound cytoskeleton rearrangements that lead to apoptosis. In contrast, intracellular Tat has been described as protector from apoptosis. Tat gene is composed by two coding exons that yield a protein of 101 amino acids (aa). First exon (1–72aa) is sufficient for viral transcript elongation and second exon (73–101 aa) appears to contribute to non-transcriptional functions. We observed that Jurkat cells stably expressing intracellular Tat101 showed gene expression deregulation 4-fold higher than cells expressing Tat72. Functional experiments were performed to evaluate the effect of this deregulation. First, NF-κB-, NF-AT- and Sp1-dependent transcriptional activities were greatly enhanced in Jurkat-Tat101, whereas Tat72 induced milder but efficient activation. Second, cytoskeleton-related functions as cell morphology, proliferation, chemotaxis, polarization and actin polymerization were deeply altered in Jurkat-Tat101, but not in Jurkat-Tat72. Finally, expression of several cell surface receptors was dramatically impaired by intracellular Tat101 but not by Tat72. Consequently, these modifications were greatly dependent on Tat second exon and they could be related to the anergy observed in HIV-1-infected T cells. PMID:20139419

  14. Probing nanoparticle interactions in cell culture media.

    PubMed

    Sabuncu, Ahmet C; Grubbs, Janna; Qian, Shizhi; Abdel-Fattah, Tarek M; Stacey, Michael W; Beskok, Ali

    2012-06-15

    Nanoparticle research is often performed in vitro with little emphasis on the potential role of cell culture medium. In this study, gold nanoparticle interactions with cell culture medium and two cancer cell lines (human T-cell leukemia Jurkat and human pancreatic carcinoma PANC1) were investigated. Gold nanoparticles of 10, 25, 50, and 100 nm in diameter at fixed mass concentration were tested. Size distributions and zeta potentials of gold nanoparticles suspended in deionized (DI) water and Dulbecco's Modified Eagle's Media (DMEM) supplemented with fetal calf serum (FCS) were measured using dynamic light scattering (DLS) technique. In DI water, particle size distributions exhibited peaks around their nominal diameters. However, the gold nanoparticles suspended in DMEM supplemented with FCS formed complexes around 100 nm, regardless of their nominal sizes. The DLS and UV-vis spectroscopy results indicate gold nanoparticle agglomeration in DMEM that is not supplemented by FCS. The zeta potential results indicate that protein rich FCS increases the dispersion quality of gold nanoparticle suspensions through steric effects. Cellular uptake of 25 and 50 nm gold nanoparticles by Jurkat and PANC1 cell lines were investigated using inductively coupled plasma-mass spectroscopy. The intracellular gold level of PANC1 cells was higher than that of Jurkat cells, where 50 nm particles enter cells at faster rates than the 25 nm particles.

  15. Articulatin-D induces apoptosis via activation of caspase-8 in acute T-cell leukemia cell line.

    PubMed

    Mishra, Ruchi; Das, Mrinal K; Singh, Savita; Sharma, Radhey Shyam; Sharma, Sadhna; Mishra, Vandana

    2017-02-01

    Leukemia is among the most aggressive and prevalent human malignant carcinoma. Chemotherapy is the preferred therapeutic strategy; however, recurrence of cancer and non-selective cytotoxicity are the major concerns. Unlike synthetic chemotherapeutic agents, mistletoe ribosome-inactivating protein (RIP) displays anti-tumor function in various types of cancers. However, its effect on leukemia cells is little explored. In this study, we assessed the impact of Viscum articulatum RIP (Articulatin-D) on the survival of acute T-cell leukemia cells and the involved molecular and cellular mechanisms. Cell proliferation assay showed that Articulatin-D suppressed the viability of leukemia cells selectively. We further confirmed that the elevation of mitochondrial membrane potential and exposure of phosphatidylserine are the early events of apoptosis induction in Articulatin-D-treated Jurkat cells. Subsequently, we found that Articulatin-D treatment induces apoptosis in Jurkat cells in a time- and concentration-dependent manner. In conclusion, we provided evidence that Articulatin-D efficiently activates caspase-8 involved in extrinsic pathway of apoptosis induction, which ultimately results in caspase-3-dependent DNA fragmentation of Jurkat cells. Further evaluation of Articulatin-D in cell culture and animal models may provide novel information on selective cytotoxicity to acute T-cell leukemia and its involvement in targeting tumor cell survival pathways.

  16. Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

    PubMed

    Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

    2017-02-01

    We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

  17. Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines

    PubMed Central

    Gabrani, Reema; Jain, Ramya; Sharma, Anjali; Sarethy, Indira P.; Dang, Shweta; Gupta, S.

    2012-01-01

    Solanum nigrum is used in various traditional medical systems for antiproliferative, antiinflammatory, antiseizure and hepatoprotective activities. We have evaluated organic solvent and aqueous extracts obtained from berries of Solanum nigrum for antiproliferative activity on leukemic cell lines, Jurkat and HL-60 (Human promyelocytic leukemia cells). The cell viability after the treatment with Solanum nigrum extract was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Results indicated increased cytotoxicity with increasing extract concentrations. Comparative analysis indicated that 50% inhibitory concentration value of methanol extract is the lowest on both cell lines. PMID:23716874

  18. Specific Anti-Leukemic Activity of the Peptide Warnericin RK and Analogues and Visualization of Their Effect on Cancer Cells by Chemical Raman Imaging

    PubMed Central

    Loiseau, Clémence; Augenstreich, Jacques; Marchand, Adrienne; Harté, Etienne; Garcia, Martine; Verdon, Julien; Mesnil, Marc; Lecomte, Sophie; Berjeaud, Jean-Marc

    2016-01-01

    Antimicrobial peptides can be used as therapeutic agents against cancer cells. Warnericin RK and derivatives (WarnG20D and WarnF14V) were tested on various, solid tumor or leukemia, cancer cells. These peptides appeared to be cytotoxic on all the cell types tested, cancerous as well healthy, but very interestingly displayed no deleterious effect on healthy mononuclear cells. The mode of action of the peptide was proposed to be membranolytic, using chemical Raman imaging. Addition of peptide induced a large disorganization of the membrane leading to the loss of the content of inner compartments of Jurkat cell, whereas no effect was observed on the healthy mononuclear cells. The less hemolytic peptides WarnG20D and WarnF14V could be good candidates for the leukemia treatment. PMID:27598770

  19. Non-POU Domain-Containing Octamer-Binding Protein Negatively Regulates HIV-1 Infection in CD4(+) T Cells.

    PubMed

    St Gelais, Corine; Roger, Jonathan; Wu, Li

    2015-08-01

    HIV-1 interacts with numerous cellular proteins during viral replication. Identifying such host proteins and characterizing their roles in HIV-1 infection can deepen our understanding of the dynamic interplay between host and pathogen. We previously identified non-POU domain-containing octamer-binding protein (NonO or p54nrb) as one of host factors associated with catalytically active preintegration complexes (PIC) of HIV-1 in infected CD4(+) T cells. NonO is involved in nuclear processes including transcriptional regulation and RNA splicing. Although NonO has been identified as an HIV-1 interactant in several recent studies, its role in HIV-1 replication has not been characterized. We investigated the effect of NonO on the HIV-1 life cycle in CD4(+) T cell lines and primary CD4(+) T cells using single-cycle and replication-competent HIV-1 infection assays. We observed that short hairpin RNA (shRNA)-mediated stable NonO knockdown in a CD4(+) Jurkat T cell line and primary CD4(+) T cells did not affect cell viability or proliferation, but enhanced HIV-1 infection. The enhancement of HIV-1 infection in Jurkat T cells correlated with increased viral reverse transcription and gene expression. Knockdown of NonO expression in Jurkat T cells modestly enhanced HIV-1 gag mRNA expression and Gag protein synthesis, suggesting that viral gene expression and RNA regulation are the predominantly affected events causing enhanced HIV-1 replication in NonO knockdown (KD) cells. Furthermore, overexpression of NonO in Jurkat T cells reduced HIV-1 single-cycle infection by 41% compared to control cells. Our data suggest that NonO negatively regulates HIV-1 infection in CD4(+) T cells, albeit it has modest effects on early and late stages of the viral life cycle, highlighting the importance of host proteins associated with HIV-1 PIC in regulating viral replication.

  20. Surface active stabilizer Tyloxapol in colloidal dispersions exerts cytostatic effects and apoptotic dismissal of cells

    SciTech Connect

    Kristl, Julijana; Teskac, Karmen; Milek, Miha; Mlinaric-Rascan, Irena

    2008-10-15

    Solid lipid nanoparticles (SLN) have been praised for their advantageous drug delivery properties such as biocompatibility, controlled release and passive drug targeting. However, the cytotoxicity of SLN and their ingredients, especially over a longer time period, has not been investigated in detail. We examined the critical issues regarding the use of a surface active stabilizer Tyloxapol (Tyl) for the preparation of solid lipid particles (SLP) and their effects on cellular functions and viability. SLP composed of behenate, phospholipids and a stabilizer, Tyloxapol or Lutrol (Lut), were prepared by the lipid melt method, labeled with a fluorescent dye and tested on Jurkat or HEK293 cells. The nano-sized particles were rapidly internalized and exhibited cytoplasmic localization. Incubation of cells with SLP-Tyl resulted in a dose- and time-dependent cytostatic effect, and also caused moderate and delayed cytotoxicity. Tyloxapol solution or SLP-Tyl dispersion caused the detachment of HEK293 cells, a decrease in cell proliferation and alterations in cellular morphology. Cell cycle analysis revealed that, while the unfavourable effects of SLP-Tyl and Tyloxapol solution are similar initially, longer incubation results in partial recovery of cells incubated with the dispersion of SLP-Tyl, whereas the presence of Tyloxapol solution induces apoptotic cell death. These findings indicate that Tyloxapol is an unfavourable stabilizer of SLP used for intracellular delivery and reinforce the role of stabilizers in a design of SLP with minimal cytotoxic properties.

  1. Protective Role of Hsp27 Protein Against Gamma Radiation-Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

    SciTech Connect

    Aloy, Marie-Therese Hadchity, Elie; Bionda, Clara; Diaz-Latoud, Chantal; Claude, Line; Rousson, Robert; Arrigo, Andre-Patrick; Rodriguez-Lafrasse, Claire

    2008-02-01

    Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. Methods and Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively or in response to {gamma}-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. Results: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Conclusion: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.

  2. Caspase-10 Is the Key Initiator Caspase Involved in Tributyltin-Mediated Apoptosis in Human Immune Cells

    PubMed Central

    Krug, Harald F.

    2012-01-01

    Tributyltin (TBT) is one of the most toxic compounds produced by man and distributed in the environment. A multitude of toxic activities have been described, for example, immunotoxic, neurotoxic, and endocrine disruptive effects. Moreover, it has been shown for many cell types that they undergo apoptosis after treatment with TBT and the cell death of immune cells could be the molecular background of its immunotoxic effect. As low as 200 nM up to 1 μM of TBT induces all signs of apoptosis in Jurkat T cells within 1 to 24 hrs of treatment. When compared to Fas-ligand control stimulation, the same sequence of events occurs: membrane blebbing, phosphatidylserine externalisation, the activation of the “death-inducing signalling complex,” and the following sequence of cleavage processes. In genetically modified caspase-8-deficient Jurkat cells, the apoptotic effects are only slightly reduced, whereas, in FADD-negative Jurkat cells, the TBT effect is significantly diminished. We could show that caspase-10 is recruited by the TRAIL-R2 receptor and apoptosis is totally prevented when caspase-10 is specifically inhibited in all three cell lines. PMID:22287961

  3. Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells.

    PubMed

    Miyajima, Chiharu; Itoh, Yuka; Inoue, Yasumichi; Hayashi, Hidetoshi

    2015-01-01

    Tribbles 1 (TRB1), a member of the Tribbles family, is a pseudokinase that is conserved among species and implicated in various human diseases including leukemia, cardiovascular diseases, and metabolic disorders. However, the role of TRB1 in the immune response is not understood. To evaluate this role, we examined regulation of TRB1 expression and the function of TRB1 in interleukin-2 (IL-2) induction in Jurkat cells, a human acute T cell leukemia cell line. We found that TRB1 was strongly induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin in these cells. IL-2 expression was induced in Jurkat cells activated by PMA and ionomycin; however, knockdown of TRB1 resulted in decreased induction of IL-2. TRB1 null Jurkat cells established using the CRISPR/Cas9 system also showed reduction of IL-2 expression on PMA/ionomycin stimulation. TRB1 knockdown also markedly inhibited IL-2 promoter activation. To determine the mechanism of the stimulatory effect on IL-2 induction, we focused on histone deacetylases (HDACs), and found that HDAC1 preferentially interacts with TRB1. TRB1 suppressed the interaction of HDAC1 with nuclear factor of activated T cells 2 (NFAT2), which is a crucial transcription factor for IL-2 induction. These results indicate that TRB1 is a positive regulator of IL-2 induction in activated T cells.

  4. Synthesis of N-methylarylnitrones derived from alkyloxybenzaldehydes and antineoplastic effect on human cancer cell lines.

    PubMed

    Costa, Débora S S; Martino, Thiago; Magalhães, Fernanda C; Justo, Graça; Coelho, Marsen G P; Barcellos, Julio C F; Moura, Victor B; Costa, Paulo R R; Sabino, Kátia C C; Dias, Ayres G

    2015-05-01

    New O-isoprenylated-N-methylarylnitrones derived from isomeric o, m and p-hydroxybenzaldehydes have been prepared and the antineoplastic effects on human cancer cell lines were evaluated. The O-geranylated nitrone LQB-278 (1b) and its isomers 2b and 3b inhibited the NO production, but the anti-leukemic activity was drastically dependent on nitrone isomer, with the 1b being the most effective one (IC₅₀ of 6.7 μM) on Jurkat leukemia cell, by MTT assay. In addition, 1b up-regulated p21CIP1/WAF1/Sdi1 protein expression (flow cytometry), a cell cycle inhibitor, reduced cell growth, and induced DNA fragmentation (increased sub-G1 phase cells) and phosphatidylserine externalization in plasmatic membrane (increased annexin V positive cells). Finally, the 1b up-regulation of p21 expression and apoptosis induction seem to be the mechanisms by which it promotes its anti-leukemic effects, making this new molecular architecture a promising prototype for leukemia intervention.

  5. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: involvement of interleukin-2 system.

    PubMed

    Cervia, Davide; Catalani, Elisabetta; Belardinelli, Maria Cristina; Perrotta, Cristiana; Picchietti, Simona; Alimenti, Claudio; Casini, Giovanni; Fausto, Anna Maria; Vallesi, Adriana

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell-cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the β and γ subunits of IL-2 receptor (IL-2R), while the mRNA levels of the α subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2Rα subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth.

  6. Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

    PubMed

    Horn, S; Endl, E; Fehse, B; Weck, M M; Mayr, G W; Jücker, M

    2004-11-01

    The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

  7. Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells

    PubMed Central

    2011-01-01

    Background Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization. Results We first investigated the subcellular localization of ASP by transfecting Jurkat T cells with vectors expressing ASP tagged with the Flag epitope to its N-terminus. Using immunofluorescence microscopy, we found that ASP localized to the plasma membrane in transfected Jurkat T cells, but with different staining patterns. In addition to an entire distribution to the plasma membrane, ASP showed an asymmetric localization and could also be detected in membrane connections between two cells. We then infected Jurkat T cells with NL4.3 virus coding for ASP tagged with the Flag epitope at its C-terminal end. By this approach, we were capable of showing that ASP is effectively expressed from the HIV-1 3' LTR in infected T cells, with an asymmetric localization of the viral protein at the plasma membrane. Conclusion These results demonstrate for the first time that ASP can be detected when expressed from full-length HIV-1 proviral DNA and that its localization is consistent with Jurkat T cells overexpressing ASP. PMID:21929758

  8. Cytotoxicity of recombinant tamapin and related toxin-like peptides on model cell lines.

    PubMed

    Ramírez-Cordero, Belén; Toledano, Yanis; Cano-Sánchez, Patricia; Hernández-López, Rogelio; Flores-Solis, David; Saucedo-Yáñez, Alma L; Chávez-Uribe, Isabel; Brieba, Luis G; del Río-Portilla, Federico

    2014-06-16

    The scorpion toxin tamapin displays the most potent and selective blockage against KCa2.2 channels known to date. In this work, we report the biosynthesis, three-dimensional structure, and cytotoxicity on cancer cell lines (Jurkat E6-1 and human mammary breast cancer MDA-MB-231) of recombinant tamapin and five related peptides bearing mutations on residues (R6A,R7A, R13A, R6A-R7A, and GS-tamapin) that were previously suggested to be important for tamapin's activity. The indicated cell lines were used as they constitutively express KCa2.2 channels. The studied toxin-like peptides displayed lethal responses on Jurkat T cells and breast cancer cells; their effect is dose- and time-dependent with IC50 values in the nanomolar range. The order of potency is r-tamapin>GS-tamapin>R6A>R13A>R6A-R7A>R7A for Jurkat T cells and r-tamapin>R7A for MDA-MB-231 breast cancer cells. Our structural determination by NMR demonstrated that r-tamapin preserves the folding of the αKTx5 subfamily and that neither single nor double alanine mutations affect the three-dimensional structure of the wild-type peptide. In contrast, our activity assays show that changes in cytotoxicity are related to the chemical nature of certain residues. Our results suggest that the toxic activity of r-tamapin on Jurkat and breast cancer cells could be mediated by the interaction of charged residues in tamapin with KCa2.2 channels via the apoptotic cell death pathway.

  9. Polyalkoxybenzenes from plants. 5. Parsley seed extract in synthesis of azapodophyllotoxins featuring strong tubulin destabilizing activity in the sea urchin embryo and cell culture assays.

    PubMed

    Semenova, Marina N; Kiselyov, Alex S; Tsyganov, Dmitry V; Konyushkin, Leonid D; Firgang, Sergei I; Semenov, Roman V; Malyshev, Oleg R; Raihstat, Mikhail M; Fuchs, Fabian; Stielow, Anne; Lantow, Margareta; Philchenkov, Alex A; Zavelevich, Michael P; Zefirov, Nikolay S; Kuznetsov, Sergei A; Semenov, Victor V

    2011-10-27

    A series of 4-azapodophyllotoxin derivatives with modified rings B and E have been synthesized using allylpolyalkoxybenzenes from parsley seed oil. The targeted molecules were evaluated in vivo in a phenotypic sea urchin embryo assay for antimitotic and tubulin destabilizing activity. The most active compounds identified by the in vivo sea urchin embryo assay featured myristicin-derived ring E. These molecules were determined to be more potent than podophyllotoxin. Cytotoxic effects of selected molecules were further confirmed and evaluated by conventional assays with A549 and Jurkat human leukemic T-cell lines including cell growth inhibition, cell cycle arrest, cellular microtubule disruption, and induction of apoptosis. The ring B modification yielded 6-OMe substituted molecule as the most active compound. Finally, in Jurkat cells, compound induced caspase-dependent apoptosis mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic pathway.

  10. Targeted Lymphoma Cell Death by Novel Signal Transduction Modifications

    DTIC Science & Technology

    2009-07-01

    effects in human NHL xenografts. We also have the c apacity to use small animal immuno-positron emission tomography (iPET). IP ET is a new, sop...CLL, mantle cell:Karpas 519, figure 6. CD22 Binding Peptide 41 Treatment of Differnt Lymphoma cell Lines (Figure 6) 0 20 40 60 80 100 120 Jurkat...the anti- CD22 mAb HB22.7 to B cells. Thus we used a flow cytometry-based assay to examine the effects of peptide 41 on HB22.7 binding to B cells

  11. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

    NASA Astrophysics Data System (ADS)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

    1986-08-01

    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  12. The effect of exposure to high flux density static and pulsed magnetic fields on lymphocyte function.

    PubMed

    Aldinucci, Carlo; Garcia, Julian Blanco; Palmi, Mitri; Sgaragli, Gianpietro; Benocci, Alberto; Meini, Antonella; Pessina, Federica; Rossi, Claudio; Bonechi, Claudia; Pessina, Gian Paolo

    2003-09-01

    We investigated whether a combination of static electromagnetic field (EMF) at a flux density of 4.75 T together with pulsed EMF at a flux density of 0.7 mT generated by an NMR apparatus (NMRF), could promote movements of Ca(2+), cell proliferation, and the eventual production of proinflammatory cytokines in human lymphocytes as well as in Jurkat cells, after exposure to the field for 1 h. The same study was also performed after activation of cells with 5 micro g/ml phytohaemagglutinin (PHA) immediately before the exposure period. Our results clearly demonstrate that NMRF exposure increases the [Ca(2+)](i), without any proliferative, or activating, or proinflammatory effect on both normal and PHA stimulated lymphocytes. Accordingly, the levels of interferon gamma, tumor necrosis factor alpha, interleukin-1beta, interleukin-2, and interleukin-6 remained unvaried after exposure. Exposure of Jurkat cells statistically decreased the [Ca(2+)](i) and the proliferation. This is consistent with the low levels of IL-2 measured in supernatants of these cells after exposure. On the whole our data suggest that static and pulsed NMRF exposure contribute synergistically in the increase of the [Ca(2+)](i) without any activating or proinflammatory effect either in normal or in PHA challenged lymphocytes. In Jurkat cells, by changing the properties of cell membranes, NMRF exposure can influence Ca(2+) transport processes and hence Ca(2+) homeostasis, causing a marked decrease of proliferation.

  13. A novel 2,6-diisopropylphenyl-docosahexaenoamide conjugate induces apoptosis in T cell acute lymphoblastic leukemia cell lines

    SciTech Connect

    Altenburg, Jeffrey D.; Harvey, Kevin A.; McCray, Sharon; Xu, Zhidong; Siddiqui, Rafat A.

    2011-07-29

    Highlights: {yields} 2,6-Diisopropylphenyl-docosahexaenoamide conjugates (DIP-DHA) inhibits the proliferation of T-cell leukemic cell lines. {yields} DIP-DHA resulted in increased activation of caspase-3, and caspase-7. {yields} DIP-DHA significantly downregulated CXCR4 surface expression. -- Abstract: We have previously characterized the effects of 2,6-diisopropylphenyl-docosahexaenoamide (DIP-DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP-DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP-DHA resulted in significantly greater inhibition of proliferation and induction of apoptosis than that of parent compounds, 2,6-diisopropylphenol (DIP) or docosahexaenoate (DHA). Treatment of the cells with DIP-DHA resulted in increased activation of caspase-3, and caspase-7. Furthermore, induction of apoptosis in both cell lines was reversed in the presence of a caspase family inhibitor. Treatment with DIP-DHA reduced mitochondrial membrane potential. These observations suggest that the effects are driven by intrinsic apoptotic pathways. DIP-DHA treatment also downregulated surface CXCR4 expression, an important chemokine receptor involved in cancer metastasis that is highly expressed in both CEM and Jurkat cells. In conclusion, our data suggest that the DIP-DHA conjugate exhibits significantly more potent effects on CEM and Jurkat cells than that of DIP or DHA alone. These conjugates have potential use for treatment of patients with T cell acute lymphoblastic leukemia.

  14. Laccase purified from Cerrena unicolor exerts antitumor activity against leukemic cells

    PubMed Central

    MATUSZEWSKA, ANNA; KARP, MARTA; JASZEK, MAGDALENA; JANUSZ, GRZEGORZ; OSIŃSKA-JAROSZUK, MONIKA; SULEJ, JUSTYNA; STEFANIUK, DAWID; TOMCZAK, WALDEMAR; GIANNOPOULOS, KRZYSZTOF

    2016-01-01

    Chronic lymphocytic leukemia (CLL) is the most commonly observed adult hematological malignancy in Western countries. Despite the fact that recent improvements in CLL treatment have led to an increased percentage of complete remissions, CLL remains an incurable disease. Cerrena unicolor is a novel fungal source of highly active extracellular laccase (ex-LAC) that is currently used in industry. However, to the best of our knowledge, no reports regarding its anti-leukemic activity have been published thus far. In the present study, it was hypothesized that C. unicolor ex-LAC may possess cytotoxic activity against leukemic cell lines and CLL primary cells. C. unicolor ex-LAC was separated using anion exchange chromatography on diethylaminoethyl cellulose-Sepharose and Sephadex G-50 columns. The cytotoxic effects of ex-LAC upon 24- and 48-h treatment on HL-60, Jurkat, RPMI 8226 and K562 cell lines, as well as CLL primary cells of nine patients with CLL, were evaluated using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Annexin V/propidium iodide staining of Jurkat cells treated with ex-LAC was used to investigate apoptosis via flow cytometry. Ex-LAC induced changes in Jurkat and RPMI 8226 cells, as visualized by fluorescence and scanning electron microscopy (SEM). The XTT assay revealed high cytotoxic rates following treatment with various concentrations of ex-LAC on all the cell lines and CLL primary cells analyzed, with a half maximal inhibitory concentration ranging from 0.4 to 1.1 µg/ml. Fluorescence microscopy and SEM observations additionally revealed apoptotic changes in Jurkat and RPMI 8226 cells treated with ex-LAC, compared with control cells. These results were in agreement with the apoptosis analysis of Jurkat cells on flow cytometry. In conclusion, C. unicolor ex-LAC was able to significantly induce cell apoptosis, and may represent a novel therapeutic agent for the treatment of various hematological neoplasms. PMID

  15. Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells

    NASA Astrophysics Data System (ADS)

    Schatten, Heide; Lewis, Marian L.; Chakrabarti, Amitabha

    2001-08-01

    The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis.

  16. Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells

    NASA Technical Reports Server (NTRS)

    Schatten, H.; Lewis, M. L.; Chakrabarti, A.

    2001-01-01

    The cytoskeleton is a complex network of fibers that is sensitive to environmental factors including microgravity and altered gravitational forces. Cellular functions such as transport of cell organelles depend on cytoskeletal integrity; regulation of cytoskeletal activity plays a role in cell maintenance, cell division, and apoptosis. Here we report cytoskeletal and mitochondria alterations in cultured human lymphocyte (Jurkat) cells after exposure to spaceflight and in insect cells of Drosophila melanogaster (Schneider S-1) after exposure to conditions created by clinostat rotation. Jurkat cells were flown on the space shuttle in Biorack cassettes while Schneider S-1 cells were exposed to altered gravity forces as produced by clinostat rotation. The effects of both treatments were similar in the different cell types. Fifty percent of cells displayed effects on the microtubule network in both cell lines. Under these experimental conditions mitochondria clustering and morphological alterations of mitochondrial cristae was observed to various degrees after 4 and 48 hours of culture. Jurkat cells underwent cell divisions during exposure to spaceflight but a large number of apoptotic cells was also observed. Similar results were obtained in Schneider S-1 cells cultured under clinostat rotation. Both cell lines displayed mitochondria abnormalities and mitochondria clustering toward one side of the cells which is interpreted to be the result of microtubule disruption and failure of mitochondria transport along microtubules. The number of mitochondria was increased in cells exposed to altered gravity while cristae morphology was severely affected indicating altered mitochondria function. These results show that spaceflight as well as altered gravity produced by clinostat rotation affects microtubule and mitochondria organization and results in increases in apoptosis. Grant numbers: NAG 10-0224, NAG2-985. c 2001. Elsevier Science Ltd. All rights reserved.

  17. Cell death caused by selenium deficiency and protective effect of antioxidants.

    PubMed

    Saito, Yoshiro; Yoshida, Yasukazu; Akazawa, Takashi; Takahashi, Kazuhiko; Niki, Etsuo

    2003-10-10

    Selenium is an essential trace element and it is well known that selenium is necessary for cell culture. However, the mechanism underlying the role of selenium in cellular proliferation and survival is still unknown. The present study using Jurkat cells showed that selenium deficiency in a serum-free medium decreased the selenium-dependent enzyme activity (glutathione peroxidases and thioredoxin reductase) within cells and cell viability. To understand the mechanism of this effect of selenium, we examined the effect of other antioxidants, which act by different mechanisms. Vitamin E, a lipid-soluble radical-scavenging antioxidant, completely blocked selenium deficiency-induced cell death, although alpha-tocopherol (biologically the most active form of vitamin E) could not preserve selenium-dependent enzyme activity. Other antioxidants, such as different isoforms and derivatives of vitamin E, BO-653 and deferoxamine mesylate, also exerted an inhibitory effect. However, the water-soluble antioxidants, such as ascorbic acid, N-acetyl cysteine, and glutathione, displayed no such effect. Dichlorodihydrofluorescein (DCF) assay revealed that cellular reactive oxygen species (ROS) increased before cell death, and sodium selenite and alpha-tocopherol inhibited ROS increase in a dose-dependent manner. The generation of lipid hydroperoxides was observed by fluorescence probe diphenyl-1-pyrenylphosphine (DPPP) and HPLC chemiluminescence only in selenium-deficient cells. These results suggest that the ROS, especially lipid hydroperoxides, are involved in the cell death caused by selenium deficiency and that selenium and vitamin E cooperate in the defense against oxidative stress upon cells by detoxifying and inhibiting the formation of lipid hydroperoxides.

  18. Role of ascorbate in the activation of NF-kappaB by tumour necrosis factor-alpha in T-cells.

    PubMed

    Muñoz, E; Blázquez, M V; Ortiz, C; Gomez-Díaz, C; Navas, P

    1997-07-01

    The first product of ascorbate oxidation, the ascorbate free radical (AFR), acts in biological systems mainly as an oxidant, and through its role in the plasma membrane redox system exerts different effects on the cell. We have investigated the role of ascorbate, AFR and dehydroascorbate (DHA) in the activation of the NF-kappaB transcription factor in Jurkat T-cells stimulated by tumour necrosis factor-alpha (TNF-alpha). Here we show, by electrophoretic mobility shift assays, that ascorbate increases the binding of NF-kappaB to DNA in TNF-alpha-stimulated Jurkat cells. The ability of ascorbate to enhance cytoplasmic inhibitory IkBalpha protein degradation correlates completely with its capacity to induce NF-kappaB binding to DNA and to potentiate NF-kappaB-mediated transactivation of the HIV-1 long terminal repeat promoter in TNF-alpha-stimulated Jurkat cells but not in cells stimulated with PMA plus ionomycin. AFR behaves like ascorbate, while DHA and ascorbate phosphate do not affect TNF-alpha-mediated NF-kappaB activation. These results provide new evidence for a possible relationship between the activation of the electron-transport system at the plasma membrane by ascorbate or its free radical and redox-dependent gene transcription in T-cells.

  19. Chemical sporulation and germination: cytoprotective nanocoating of individual mammalian cells with a degradable tannic acid-FeIII complex.

    PubMed

    Lee, Juno; Cho, Hyeoncheol; Choi, Jinsu; Kim, Doyeon; Hong, Daewha; Park, Ji Hun; Yang, Sung Ho; Choi, Insung S

    2015-12-07

    Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-Fe(III) nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-Fe(III) nanocoat, mimicking the sporulation and germination processes found in nature.

  20. Killing of human myelomonocytic leukemia and lymphocytic cell lines by Actinobacillus actinomycetemcomitans leukotoxin.

    PubMed Central

    Simpson, D L; Berthold, P; Taichman, N S

    1988-01-01

    The purified leukotoxin of Actinobacillus actinomycetemcomitans kills human leukemic cell lines (e.g., HL-60, U937, and KG-1) and human T- and B-cell lines (e.g., JURKAT, MOLT-4, Daudi, and Raji) in a dose- and time-dependent manner. The 50% effective doses for these cell lines are similar to those established for human polymorphonuclear leukocytes and monocytes. In contrast, other human and nonhuman tumor cell lines are not susceptible to the leukotoxin. These human leukemia and lymphoid cell lines will serve as useful model systems with which to study the molecular specificity and mechanism(s) of action of the actinobacillus leukotoxin. Images PMID:3258584

  1. 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.

  2. High expression of IMPACT protein promotes resistance to indoleamine 2,3-dioxygenase-induced cell death.

    PubMed

    Habibi, Darya; Jalili, Reza B; Forouzandeh, Farshad; Ong, Christopher J; Ghahary, Aziz

    2010-10-01

    Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO-induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non-derepressible-2(GCN2) kinase stress-responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO-induced low tryptophan environment. IDO-expressing human fibroblasts were co-cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO-expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO-induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co-cultured with IDO-expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT-expressing vector, was rescued significantly in tryptophan-deficient but not IDO-expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO-mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO-generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non-immune cells such as skin cells acts as a protective mechanism against IDO-induced GCN2 activation, therefore, makes them resistant to the amino acid-deprived environment caused by IDO.

  3. The effect of hypogravity and hypergravity on cells of the immune system

    NASA Technical Reports Server (NTRS)

    Cogoli, A.

    1993-01-01

    This article reviews the gravity effects discovered in T lymphocytes and other cells of the immune system. The strong depression of mitogenic activation first observed in an experiment conducted in Spacelab 1 in 1983 triggered several other investigations in space and on the ground in the clinostat and in the centrifuge in the past 10 years. During this period, great progress was made in our knowledge of the complex mechanism of T cell activation as well as the technology to analyze the lymphokines produced during stimulation. Nevertheless, several aspects of the steps leading to activation are not yet clear. Studies in hypogravity and hypergravity may contribute to answering some of the questions. A recent investigation in the U.S. Spacelab SLS-1, based on a new technology in which leukocytes are attached to microcarrier beads, showed that the strong inhibition of activation in microgravity is due to a malfunction of monocytes acting as accessory cells. In fact, interleukin-1 production is nearly nil in resuspended monocytes, whereas T cell activation is doubled in attached cells. In hypergravity, but not at 1g, concanavalin A bound to erythrocytes activates B lymphocytes in addition to T cells. The activation of Jurkat cells is also severely impaired in space. These recent results have raised new questions that have to be answered in experiments to be conducted in space and on Earth in this decade. The experimental system, based on the mitogenic activation of T lymphocytes and accessory cells attached to microcarriers, offers an optimum model for studying basic biological mechanisms of the cell to assess the immunological fitness of humans in space and to test the feasibility of bioprocesses in space as well as on Earth.

  4. The effect of hypogravity and hypergravity on cells of the immune system.

    PubMed

    Cogoli, A

    1993-09-01

    This article reviews the gravity effects discovered in T lymphocytes and other cells of the immune system. The strong depression of mitogenic activation first observed in an experiment conducted in Spacelab 1 in 1983 triggered several other investigations in space and on the ground in the clinostat and in the centrifuge in the past 10 years. During this period, great progress was made in our knowledge of the complex mechanism of T cell activation as well as the technology to analyze the lymphokines produced during stimulation. Nevertheless, several aspects of the steps leading to activation are not yet clear. Studies in hypogravity and hypergravity may contribute to answering some of the questions. A recent investigation in the U.S. Spacelab SLS-1, based on a new technology in which leukocytes are attached to microcarrier beads, showed that the strong inhibition of activation in microgravity is due to a malfunction of monocytes acting as accessory cells. In fact, interleukin-1 production is nearly nil in resuspended monocytes, whereas T cell activation is doubled in attached cells. In hypergravity, but not at 1g, concanavalin A bound to erythrocytes activates B lymphocytes in addition to T cells. The activation of Jurkat cells is also severely impaired in space. These recent results have raised new questions that have to be answered in experiments to be conducted in space and on Earth in this decade. The experimental system, based on the mitogenic activation of T lymphocytes and accessory cells attached to microcarriers, offers an optimum model for studying basic biological mechanisms of the cell to assess the immunological fitness of humans in space and to test the feasibility of bioprocesses in space as well as on Earth.

  5. Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

    PubMed

    Budagian, Vadim; Bulanova, Elena; Brovko, Luba; Orinska, Zane; Fayad, Raja; Paus, Ralf; Bulfone-Paus, Silvia

    2003-01-17

    ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate

  6. Up-regulated A20 promotes proliferation, regulates cell cycle progression and induces chemotherapy resistance of acute lymphoblastic leukemia cells.

    PubMed

    Chen, Shuying; Xing, Haiyan; Li, Shouyun; Yu, Jing; Li, Huan; Liu, Shuang; Tian, Zheng; Tang, Kejing; Rao, Qing; Wang, Min; Wang, Jianxiang

    2015-09-01

    A20, also known as tumor necrosis factor-α (TNFα)-induced protein 3 (TNFAIP3), has been identified as a key regulator of cell survival in many solid tumors. However, little is known about the protein expression level and function of A20 in acute lymphoblastic leukemia (ALL). In this study, we found that A20 is up-regulated in ALL patients and several cell lines. Knockdown of A20 in Jurkat, Nalm-6, and Reh cells resulted in reduced cell proliferation, which was associated with cell cycle arrest. Phospho-ERK (p-ERK) was also down-regulated, while p53 and p21 were up-regulated in A20 knockdown cells. In addition, A20 knockdown induced apoptosis in Jurkat and Reh cells and enhanced the sensitivity of these cell lines to chemotherapeutic drugs. These results indicate that A20 may stimulate cell proliferation by regulating cell cycle progression. A20 inhibited apoptosis in some types of ALL cells, thereby enhancing their resistance to chemotherapy. This effect was abolished through A20 silencing. These findings suggest that A20 may contribute to the pathogenesis of ALL and that it may be used as a new therapeutic target for ALL treatment.

  7. Effect of prolactin on carcinoembryonic antigen-specific cytotoxic T lymphocyte response induced by dendritic cells.

    PubMed

    Matera, L; Beltramo, E; Martinuzzi, E; Buttiglieri, S

    2004-08-01

    The cytokine hormone prolactin (PRL) has been shown previously to modulate native cellular responses and maturation of antigen-presenting cells. Here we have addressed its effect on the antigen-specific response of cytotoxic T lymphocytes (CTL). CTL were generated from HLA-A2 lymphocytes after three rounds of stimulation with autologous dendritic cells loaded with HLA-A2-restricted carcinoembrionic antigen (CEA) Cap-1 (YLSGANLNL) peptide. Selected cultures were expanded on cytokine-supplemented feeder-layers, enriched for CD8+ lymphocytes and analysed for PRL-receptor (PRL-R) expression and PRL responsiveness. Resting CD8+ lymphocytes were negative for PRL-R, whereas antigen-activated CD8+ lymphocytes derived from long-term cultures were highly positive. Results of a 51Cr release assay showed CTL killing of CEA-loaded, but not unloaded, T2 cell line and the CEA-positive gastric carcinoma cell line KATO, but not of the CEA-negative T leukaemia cell line Jurkat. Interferon (IFN)-gamma release, evaluated in an ELISPOT assay against CEA-loaded T2, was enhanced (P < 0.05) by concentrations of PRL (12-25 ng/ml) very close to the physiological levels (6-20 ng/ml), but was decreased (P < 0.05) by high concentrations (200 ng/ml). Pre-incubation of the stimulators with the anti-MHC class I MoAb W6.32 induced a 40-60% decrease of the PRL-boosted IFN-gamma release, thus proving the MHC restriction of the lymphocyte response. Cytotoxicity against CEA-loaded T2 and KATO cell lines was also increased by 12-25 ng (P < 0.05) and decreased (P < 0.05) by 200 ng PRL. Pre-incubation of CTL with an antibody specific for the PRL-R almost completely abrogated this effect.

  8. Delivery of anticancer drugs and antibodies into cells using ultrasound

    NASA Astrophysics Data System (ADS)

    Wu, Junru; Pepe, Jason; Rincon, Mercedes

    2005-04-01

    It has been shown experimentally in cell suspensions that pulsed ultrasound (2.0 MHz) could be used to deliver an anti-cancer drug (Adriamycin hydrochloride) into Jurkat lymphocytes and antibodies (goat anti rabbit IgG and anti mouse IgD) into human peripheral blood mononuclear (PBMC) cells and Jurkat lymphocytes assisted by encapsulated microbubbles (Optison). When Adriamycin hydrochloride (ADR) was delivered, the delivery efficiency reached 4.80% and control baseline (no ultrasound and no ADR) was 0.17%. When anti-rabbit IgD was delivered, the efficiencies were 34.90% (control baseline was 1.33%) and 32.50% (control baseline was 1.66%) respectively for Jurkat cells and PBMC. When goat anti rabbit IgG was delivered, the efficiencies were 78.60% (control baseline was 1.60%) and 57.50% (control baseline was 11.30%) respectively for Jurkat cells and PBMC.

  9. Targeting of T/Tn Antigens with a Plant Lectin to Kill Human Leukemia Cells by Photochemotherapy

    PubMed Central

    Poiroux, Guillaume; Lafont, Elodie; Ségui, Bruno; Van Damme, Els J. M.; Peumans, Willy J.; Bernadou, Jean; Levade, Thierry; Rougé, Pierre; Barre, Annick; Benoist, Hervé

    2011-01-01

    Photochemotherapy is used both for solid tumors and in extracorporeal treatment of various hematologic disorders. Nevertheless, its development in oncology remains limited, because of the low selectivity of photosensitizers (PS) towards human tumor cells. To enhance PS efficiency, we recently covalently linked a porphyrin (TrMPyP) to a plant lectin (Morniga G), known to recognize with high affinity tumor-associated T and Tn antigens. The conjugation allowed a quick uptake of PS by Tn-positive Jurkat leukemia cells and efficient PS-induced phototoxicity. The present study was performed: (i) to evaluate the targeting potential of the conjugate towards tumor and normal cells and its phototoxicity on various leukemia cells, (ii) to investigate the mechanism of conjugate-mediated cell death. The conjugate: (i) strongly increased (×1000) the PS phototoxicity towards leukemic Jurkat T cells through an O-glycan-dependent process; (ii) specifically purged tumor cells from a 1∶1 mixture of Jurkat leukemia (Tn-positive) and healthy (Tn-negative) lymphocytes, preserving the activation potential of healthy lymphocytes; (iii) was effective against various leukemic cell lines with distinct phenotypes, as well as fresh human primary acute and chronic lymphoid leukemia cells; (iv) induced mostly a caspase-independent cell death, which might be an advantage as tumor cells often resist caspase-dependent cell death. Altogether, the present observations suggest that conjugation with plant lectins can allow targeting of photosensitizers towards aberrant glycosylation of tumor cells, e.g. to purge leukemia cells from blood and to preserve the normal leukocytes in extracorporeal photochemotherapy. PMID:21858067

  10. Bioorthogonal Click Chemistry-Based Synthetic Cell Glue.

    PubMed

    Koo, Heebeom; Choi, Myunghwan; Kim, Eunha; Hahn, Sei Kwang; Weissleder, Ralph; Yun, Seok Hyun

    2015-12-22

    Artificial methods of cell adhesion can be effective in building functional cell complexes in vitro, but methods for in vivo use are currently lacking. Here, a chemical cell glue based on bioorthogonal click chemistry with high stability and robustness is introduced. Tetrazine (Tz) and trans-cyclooctene (TCO) conjugated to the cell surface form covalent bonds between cells within 10 min in aqueous conditions. Glued, homogeneous, or heterogeneous cell pairs remain viable and stably attached in a microfluidic flow channel at a shear stress of 20 dyn cm(-2) . Upon intravenous injection of assembled Jurkat T cells into live mice, fluorescence microscopy shows the trafficking of cell pairs in circulation and their infiltration into lung tissues. These results demonstrate the promising potential of chemically glued cell pairs for various applications ranging from delivering therapeutic cells to studying cell-cell interactions in vivo.

  11. Wogonin, a plant flavone, potentiates etoposide-induced apoptosis in cancer cells.

    PubMed

    Lee, Eibai; Enomoto, Riyo; Suzuki, Chie; Ohno, Masataka; Ohashi, Toshinori; Miyauchi, Azusa; Tanimoto, Eriko; Maeda, Kaori; Hirano, Hiroyuki; Yokoi, Toshio; Sugahara, Chiyoko

    2007-01-01

    Etoposide, a podophylotoxin anticancer agent, induces apoptotic cell death in normal and cancer cells. Etoposide-induced apoptosis plays a role in not only anticancer effect but also adverse reaction, such as myelosuppression. Since we have found that wogonin, a flavone found in Scutellaria baicalensis Georgi, prevents thymocyte apoptosis induced by various compounds including etoposide, we examined the effect of this flavone on etoposide-induced apoptosis in cancer cells. Although 100 muM wogonin itself significantly increased DNA fragmentation in HL-60 cells, this change was not observed in Jurkat cells. On the other hand, this flavone significantly potentiated etoposide-induced apoptosis in Jurkat and HL-60 cells. Similarly, wogonin accelerated etoposide-induced cell death in lung cancer cells. Since wogonin had no effect on the action of other anticancer agents, such as 5-FU and cisplatin, this flavone seems to accelerate only etoposide-induced apoptotic cell death in cancer cells. These results suggest that the modification of etoposide-induced apoptosis by wogonin may be available to reduce the adverse reaction of this agent.

  12. Aplotaxene blocks T cell activation by modulation of protein kinase C-θ-dependent pathway.

    PubMed

    Na, Bo-Ra; Kim, Hye-Ran; Kwon, Min-Sung; Lee, Hyun-Su; Piragyte, Indre; Choi, Eun-Ju; Choi, Hyun-Kyu; Han, Weon-Cheol; Lee, Seung-Ho; Jun, Chang-Duk

    2013-12-01

    Aplotaxene, (8Z, 11Z, 14Z)-heptadeca-1, 8, 11, 14-tetraene, is one of the major components of essential oil obtained from Inula helenium root, which is used in Oriental medicine. However, the effects of aplotaxene on immunity have not been investigated. Here, we show that aplotaxene inhibits T cell activation in terms of IL-2 and CD69 expression. Aplotaxene, at a concentration that optimally inhibits IL-2 production, has little effect on apoptotic or necrotic cell death, suggesting that apoptosis is not a mechanism for aplotaxene-mediated inhibition of T cell activation. Aplotaxene affects neither superantigeninduced conjugate formation between Jurkat T cells and Raji B cells nor clustering of CD3 and LFA-1 at the immunological synapse. Aplotaxene significantly inhibits PKC-θ phosphorylation and translocation to the immunological synapse, and blocks PMA-induced T-cell receptor internalization. Furthermore, aplotaxene leads to inhibition of mitogen-activated protein kinases (JNK, ERK and p38) phosphorylation and NF-κB, NF-AT, and AP-1 promoter activities in Jurkat T cells. Taken together, our findings provide evidence for the immunosuppressive effect of aplotaxene on activated T cells through the modulation of the PKC-θ and MAPK pathways, suggesting that aplotaxene may be a novel immunotherapeutic agent for immunological diseases related to the overactivation of T cells.

  13. Chemical sporulation and germination: cytoprotective nanocoating of individual mammalian cells with a degradable tannic acid-FeIII complex

    NASA Astrophysics Data System (ADS)

    Lee, Juno; Cho, Hyeoncheol; Choi, Jinsu; Kim, Doyeon; Hong, Daewha; Park, Ji Hun; Yang, Sung Ho; Choi, Insung S.

    2015-11-01

    Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-FeIII nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-FeIII nanocoat, mimicking the sporulation and germination processes found in nature.Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-FeIII nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-FeIII nanocoat, mimicking the sporulation and germination processes found in nature. Electronic supplementary information (ESI) available: Experimental details, LSCM images, and SEM and TEM images. See DOI: 10.1039/c5nr05573c

  14. Cell cycle effects of drugs

    SciTech Connect

    Dethlefsen, L.A.

    1986-01-01

    This book contains 11 chapters. Some of the chapter titles are: Cell Growth and Division Cycle; Cell Cycle Effects of Alkylating Agents; Biological Effects of Folic Acid Antagonists with Antineoplastic Activity; and Bleomycin-Mode of Action with Particular Reference to the Cell Cycle.

  15. Methylenedioxy flavonoids: assessment of cytotoxic and anti-cancer potential in human leukemia cells.

    PubMed

    Orlikova, Barbora; Menezes, José C J M D S; Ji, Seungwon; Kamat, Shrivallabh P; Cavaleiro, José A S; Diederich, Marc

    2014-09-12

    A new series of chalcones, flavanones and flavones with methylenedioxy group at the 3',4' position in chalcone, 7,8 position in flavanones and flavones with mono-, di- and trimethoxy groups in the benzaldehyde ring have been assessed for their effect on proliferation, cytotoxic potential and apoptosis in human leukemia cells. Among the tested compounds, the chalcone series showed the best activity and chalcone 3 (mono methoxy group at the ortho position in A-ring) showed a significant effect on down-regulation of cancer cell proliferation and viability in three different leukemia cell lines (K562, Jurkat, U937). The executioner caspase cleavage analyses indicated that the cytotoxic effect mediated by chalcone 3 is due to induction of apoptotic cell death. Interestingly, the cytotoxic effect was cell type-specific and targeted preferentially cancer cells as peripheral blood mononuclear cells (PBMCs) from healthy donors were less affected by the treatment compared to K562, Jurkat and U937 leukemia cells. Altogether our results indicate a potential drug candidate with interesting differential toxicity obeying Lipinski's rule of five.

  16. γ-Tocotrienol induces apoptosis in human T cell lymphoma through activation of both intrinsic and extrinsic pathways.

    PubMed

    Wilankar, Chandan; Khan, Nazir M; Checker, Rahul; Sharma, Deepak; Patwardhan, Raghavendra; Gota, Vikram; Sandur, Santosh Kumar; Devasagayam, T P A

    2011-01-01

    Tocotrienols are members of vitamin E family and possess broad biological activities including antioxidant, anti-inflammatory and antitumor effects. In the present study, we examine the potential of α-tocotrienol (AT) and γ-tocotrienol (GT) in inhibiting the proliferation of human T cell lymphoma Jurkat cells and elucidate the pathways involved in anti tumor effects of GT. GT but not AT inhibited proliferation and induced apoptosis in Jurkat cells in a dose dependent manner. GT treatment resulted in elevated mitochondrial ROS production, activation of JNK and suppression of ERK and p38 MAPK. GT also induced calcium release, loss of mitochondrial membrane potential and cytochrome c release from the mitochondria. These changes were accompanied by increase in Bax expression with a concomitant decrease in Bcl-xl expression suggesting activation of mitochondrial apoptotic pathway. GT induced increase in mitochondrial ROS was abrogated by catalase. Besides, GT also up-regulated surface expression of Fas and FasL on Jurkat cells. Further, caspase activation and PARP degradation were also seen in cells treated with GT. Inhibitors of caspase-8 and caspase-9 significantly abrogated GT mediated apoptosis. In contrast GT was not toxic to normal human peripheral blood mononuclear cells suggesting differential cytotoxicity towards normal lymphocytes and transformed lymphoma cells. Cellular uptake studies with tocotrienols showed higher intracellular accumulation of GT as compared to AT which may be responsible for its better antitumor activity. Our results show antitumor effects of GT in human lymphoma cells via increased mitochondrial ROS generation and activation of both intrinsic and extrinsic apoptotic pathways.

  17. The Novel Organic Arsenical Darinaparsin Induces MAPK-Mediated and SHP1-Dependent Cell Death in T-cell Lymphoma and Hodgkin’s Lymphoma Cells and Human Xenograft Models

    PubMed Central

    Ravi, Dashnamoorthy; Bhalla, Savita; Gartenhaus, Ronald B; Crombie, Jennifer; Kandela, Irawati; Sharma, Jaya; Mazar, Andrew; Evens, Andrew M.

    2014-01-01

    Purpose Darinaparsin (Zio-101) is a novel organic arsenical compound with encouraging clinical activity in relapsed/refractory T-cell lymphoma (TCL) and Hodgkin lymphoma (HL), however little is known regarding its mechanism of action. Experimental Design TCL cell lines (Jurkat, Hut78, and HH) and HL cell lines (L428, L540, and L1236) were examined for in vitro cell death by MTT assay and Annexin-V based flow cytometry. Jurkat and L540-derived xenografts in SCID mice were examined for in vivo tumor inhibition and survival. Biological effects of darinaparsin on MAPK pathway were investigated using pharmacological inhibitors, RNA interference (RNAi) and transient transfection for overexpression for SHP1 and MEK. Results Darinaparsin treatment resulted in dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines. Additionally, darinaparsin had more rapid, higher, and sustained intracellular arsenic levels compared with arsenic trioxide via mass spectrometry. In vivo experiments with Jurkat (TCL) and L540 (HL)-derived lymphoma xenografts showed significant inhibition of tumor growth and improved survival in darinaparsin-treated SCID mice. Biologically, darinaparsin caused phosphorylation of ERK (and relevant downstream substrates) primarily by decreasing the inhibitory SHP1 phosphatase and co-immunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, while co-treatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) as well as MEK constitutive activation decreased darinaparsin-related cell death. Conclusions Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms. PMID:25316819

  18. Investigation of a direct effect of nanosecond pulse electric fields on mitochondria

    NASA Astrophysics Data System (ADS)

    Estlack, Larry E.; Roth, Caleb C.; Cerna, Cesario Z.; Wilmink, Gerald J.; Ibey, Bennett L.

    2014-03-01

    The unique cellular response to nanosecond pulsed electric field (nsPEF) exposure, as compared to longer pulse exposure, has been theorized to be due to permeabilization of intracellular organelles including the mitochondria. In this investigation, we utilized a high-throughput oxygen and pH sensing system (Seahorse® XF24 extracellular flux analyzer) to assess the mitochondrial activity of Jurkat and U937 cells after nsPEF. The XF Analyzer uses a transient micro-chamber of only a few μL in specialized cell culture micro-plates to enable oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to be monitored in real-time. We found that for nsPEF exposures of 10 pulses at 10-ns pulse width and at 50 kV/cm e-field, we were able to cause an increase in OCR in both U937 and Jurkat cells. We also found that high pulse numbers (>100) caused a significant decrease in OCR. Higher amplitude 150 kV/cm exposures had no effect on U937 cells and yet they had a deleterious effect on Jurkat cells, matching previously published 24 hour survival data. These results suggest that the exposures were modulating metabolic activity in cells possibly due to direct effects on the mitochondria themselves. To validate this hypothesis, we isolated mitochondria from U937 cells and exposed them similarly and found no significant change in metabolic activity for any pulse number. In a final experiment, we removed calcium from the buffer solution that the cells were exposed in and found that no significant enhancement in metabolic activity was observed. These results suggest that direct permeabilization of the mitochondria is unlikely a primary effect of nsPEF exposure and calcium-mediated intracellular pathway activation is likely responsible for observed pulse-induced mitochondrial effects.

  19. Continuous high throughput molecular adhesion based cell sorting using ridged microchannels

    NASA Astrophysics Data System (ADS)

    Tasadduq, Bushra; Wang, Gonghao; Alexeev, Alexander; Sarioglu, Ali Fatih; Sulchek, Todd

    2016-11-01

    Cell molecular interactions govern important physiological processes such as stem cell homing, inflammation and cancer metastasis. But due to a lack of effective separation technologies selective to these interactions it is challenging to specifically sort cells. Other label free separation techniques based on size, stiffness and shape do not provide enough specificity to cell type, and correlation to clinical condition. We propose a novel microfluidic device capable of high throughput molecule dependent separation of cells by flowing them through a microchannel decorated with molecule specific coated ridges. The unique aspect of this sorting design is the use of optimized gap size which is small enough to lightly squeeze the cells while flowing under the ridged part of the channel to increase the surface area for interaction between the ligand on cell surface and coated receptor molecule but large enough so that biomechanical markers, stiffness and viscoelasticity, do not dominate the cell separation mechanism. We are able to separate Jurkat cells based on its expression of PSGL-1ligand using ridged channel coated with P selectin at a flow rate of 0.045ml/min and achieve 2-fold and 5-fold enrichment of PSGL-1 positive and negative Jurkat cells respectively.

  20. All-trans-retinoic acid induces integrin-independent B-cell adhesion to ADAM disintegrin domains.

    PubMed

    Bridges, Lance C; Lingo, Joshuah D; Grandon, Rachel A; Kelley, Melissa D

    2008-04-15

    Cell adhesion is an integral aspect of immunity facilitating extravasation of immune cells during homing and activation. All -trans-Retinoic acid ( t-RA) regulates leukocyte differentiation, proliferation, and transmigration. However, the role of t-RA in immune cell adhesion is poorly defined. In this study, we evaluated the impact of t-RA and its metabolism on B and T cell adhesion. Specifically, we address the impact of t-RA on the adhesive properties of the human mature B and T cell lines RPMI 8866, Daudi and Jurkats. The effect of t-RA exposure on cell adhesion to vascular cell adhesion molecule-1 (VCAM-1), a well-established integrin counter receptor involved in immunity, and to nonconventional ADAM integrin ligands was assessed. We show for the first time that t-RA potently induces B cell adhesion in an integrin-independent manner to both VCAM-1 and select ADAM disintegrin domains. Using retinoid extraction and reverse-phase HPLC analysis, we identify the retinoid that is functionally responsible for this augmented adhesion. We also provide evidence that this novel t-RA adhesive response is not prototypical of lymphocytes since both Daudi and Jurkats do not alter their adhesive properties upon t-RA treatment. Further, the t-RA metabolic profiles between these lineages is distinct with 9- cis-retinoic acid being exclusively detected in Jurkat media. This study is the first to demonstrate that t-RA directly induces B cell adhesion in an integrin-independent manner and is not contingent upon t-RA metabolism.

  1. Microfluidic immunomagnetic cell separation using integrated permanent micromagnets

    PubMed Central

    Osman, O.; Toru, S.; Dumas-Bouchiat, F.; Dempsey, N. M.; Haddour, N.; Zanini, L.-F.; Buret, F.; Reyne, G.; Frénéa-Robin, M.

    2013-01-01

    In this paper, we demonstrate the possibility to trap and sort labeled cells under flow conditions using a microfluidic device with an integrated flat micro-patterned hard magnetic film. The proposed technique is illustrated using a cell suspension containing a mixture of Jurkat cells and HEK (Human Embryonic Kidney) 293 cells. Prior to sorting experiments, the Jurkat cells were specifically labeled with immunomagnetic nanoparticles, while the HEK 293 cells were unlabeled. Droplet-based experiments demonstrated that the Jurkat cells were attracted to regions of maximum stray field flux density while the HEK 293 cells settled in random positions. When the mixture was passed through a polydimethylsiloxane (PDMS) microfluidic channel containing integrated micromagnets, the labeled Jurkat cells were selectively trapped under fluid flow, while the HEK cells were eluted towards the device outlet. Increasing the flow rate produced a second eluate much enriched in Jurkat cells, as revealed by flow cytometry. The separation efficiency of this biocompatible, compact micro-fluidic separation chamber was compared with that obtained using two commercial magnetic cell separation kits. PMID:24396526

  2. Microfluidic immunomagnetic cell separation using integrated permanent micromagnets.

    PubMed

    Osman, O; Toru, S; Dumas-Bouchiat, F; Dempsey, N M; Haddour, N; Zanini, L-F; Buret, F; Reyne, G; Frénéa-Robin, M

    2013-01-01

    In this paper, we demonstrate the possibility to trap and sort labeled cells under flow conditions using a microfluidic device with an integrated flat micro-patterned hard magnetic film. The proposed technique is illustrated using a cell suspension containing a mixture of Jurkat cells and HEK (Human Embryonic Kidney) 293 cells. Prior to sorting experiments, the Jurkat cells were specifically labeled with immunomagnetic nanoparticles, while the HEK 293 cells were unlabeled. Droplet-based experiments demonstrated that the Jurkat cells were attracted to regions of maximum stray field flux density while the HEK 293 cells settled in random positions. When the mixture was passed through a polydimethylsiloxane (PDMS) microfluidic channel containing integrated micromagnets, the labeled Jurkat cells were selectively trapped under fluid flow, while the HEK cells were eluted towards the device outlet. Increasing the flow rate produced a second eluate much enriched in Jurkat cells, as revealed by flow cytometry. The separation efficiency of this biocompatible, compact micro-fluidic separation chamber was compared with that obtained using two commercial magnetic cell separation kits.

  3. Synthetic Coprisin analog peptide, D-CopA3 has antimicrobial activity and pro-apoptotic effects in human leukemia cells.

    PubMed

    Kim, Soon-ja; Kim, In-Woo; Kwon, Yong-Nam; Yun, Eun-Young; Hwang, Jae-Sam

    2012-02-01

    Recently, we reported that the synthetic Coprisin analog peptide 9-mer dimer CopA3 (consisted of all-L amino acid sequence) was designed based on a defensin-like peptide, Coprisin isolated from Copris tripartitus. The 9-mer dimer CopA3 (L-CopA3) had antibacterial activity and induced apoptosis in human leukemia cells via a caspase-independent pathway. In this study, all of amino acid sequences of L-CopA3 were modified to all D-form amino acids (D-CopA3) to develop a more effective antimicrobial peptide. We investigated whether D-CopA3 had antimicrobial activities against pathogenic microorganisms and proapoptotic effects in human leukemia cells (U937, Jurkat, and AML-2). The synthetic peptide D-CopA3 had antimicrobial activities against various pathogenic bacteria and yeast fungus with MIC values in the 4~64 microM range. Moreover, D-CopA3 caused cell growth inhibition, and increased the chromosomal DNA fragmentation and the expression of inflammatory cytokines, TNF-alpha and IL1-beta, transcripts in human leukemia cells. The all-D amino acid peptide D-CopA3 proved as effective as the L-CopA3 reported previously. These results provide the basis for developing D-CopA3 as a new antibiotic peptide.

  4. Mechanisms of rapid induction of interleukin-22 in activated T cells and its modulation by cyclosporin a.

    PubMed

    Rudloff, Ina; Bachmann, Malte; Pfeilschifter, Josef; Mühl, Heiko

    2012-02-10

    IL-22 is an immunoregulatory cytokine displaying pathological functions in models of autoimmunity like experimental psoriasis. Understanding molecular mechanisms driving IL-22, together with knowledge on the capacity of current immunosuppressive drugs to target this process, may open an avenue to novel therapeutic options. Here, we sought to characterize regulation of human IL22 gene expression with focus on the established model of Jurkat T cells. Moreover, effects of the prototypic immunosuppressant cyclosporin A (CsA) were investigated. We report that IL-22 induction by TPA/A23187 (T/A) or αCD3 is inhibited by CsA or related FK506. Similar data were obtained with peripheral blood mononuclear cells or purified CD3(+) T cells. IL22 promoter analysis (-1074 to +156 bp) revealed a role of an NF-AT (-95/-91 nt) and a CREB (-194/-190 nt) binding site for gene induction. Indeed, binding of CREB and NF-ATc2, but not c-Rel, under the influence of T/A to those elements could be proven by ChIP. Because CsA has the capability to impair IκB kinase (IKK) complex activation, the IKKα/β inhibitor IKKVII was evaluated. IKKVII likewise reduced IL-22 induction in Jurkat cells and peripheral blood mononuclear cells. Interestingly, transfection of Jurkat cells with siRNA directed against IKKα impaired IL22 gene expression. Data presented suggest that NF-AT, CREB, and IKKα contribute to rapid IL22 gene induction. In particular the crucial role of NF-AT detected herein may form the basis of direct action of CsA on IL-22 expression by T cells, which may contribute to therapeutic efficacy of the drug in autoimmunity.

  5. Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

    DTIC Science & Technology

    2011-01-01

    staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat...staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat...membrane, we chose to monitor the externalization of phosphatidylserine (PS) molecules on the outer leaflet of the plasma membrane by utilizing FITC

  6. Radiation effects on bacterial cells

    NASA Technical Reports Server (NTRS)

    Powers, E. L.

    1968-01-01

    Study reveals the physicochemical and biochemical mechanisms which alter or modify the effects of high-energy radiation on living cells. An in-depth discussion is presented emphasizing the importance of optimizing bacterial treatment with glycerol.

  7. Synthetic ligands of death receptor 5 display a cell-selective agonistic effect at different oligomerization levels

    PubMed Central

    Beyrath, Julien; Chekkat, Neila; Smulski, Cristian R.; Lombardo, Caterina M.; Lechner, Marie-Charlotte; Seguin, Cendrine; Decossas, Marion; Spanedda, Maria Vittoria; Frisch, Benoît; Guichard, Gilles; Fournel, Sylvie

    2016-01-01

    DR4 (Death Receptor 4) and DR5 (Death Receptor 5) are two potential targets for cancer therapy due to their ability to trigger apoptosis of cancer cells, but not normal ones, when activated by their cognate ligand TRAIL (TNF related apoptosis-inducing ligand). Therapies based on soluble recombinant TRAIL or agonist antibodies directed against one of the receptors are currently under clinical trials. However, TRAIL-R positive tumor cells are frequently resistant to TRAIL induced apoptosis. The precise mechanisms of this resistance are still not entirely understood. We have previously reported on synthetic peptides that bind to DR5 (TRAILmim/DR5) and induce tumor cell apoptosis in vitro and in vivo. Here, we showed that while hexameric soluble TRAIL is able to efficiently kill the DR5 positive lymphoma Jurkat or the carcinoma HCT116, these cells are resistant to apoptosis induced by the divalent form of TRAILmim/DR5 and are poorly sensitive to apoptosis induced by an anti-DR5 agonist monoclonal antibody. This resistance can be restored by the cross-linking of anti-DR5 agonist antibody but not by the cross-linking of the divalent form of TRAILmim/DR5. Interestingly, the divalent form of TRAILmim/DR5 that induced apoptosis of DR5 positive BJAB cells, acts as an inhibitor of TRAIL-induced apoptosis on Jurkat and HCT116 cells. The rapid internalization of DR5 observed when treated with divalent form of TRAILmim/DR5 could explain the antagonist activity of the ligand on Jurkat and HCT116 cells but also highlights the independence of the mechanisms responsible for internalization and activation when triggering the DR5 apoptotic cascade. PMID:27409341

  8. Leptin Reverts Pro-Apoptotic and Antiproliferative Effects of α-Linolenic Acids in BCR-ABL Positive Leukemic Cells: Involvement of PI3K Pathway

    PubMed Central

    Beaulieu, Aurore; Poncin, Géraldine; Belaid-Choucair, Zakia; Humblet, Chantal; Bogdanovic, Gordana; Lognay, Georges; Boniver, Jacques; Defresne, Marie-Paule

    2011-01-01

    It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced. PMID:21991326

  9. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

    SciTech Connect

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-08-30

    Highlights: •Nanosecond pulsed electric field (nsPEF) is a new and unique means for life sciences. •Apoptosis was induced by nsPEF exposure in Jurkat cells. •No signs of apoptosis were detected in HeLa S3 cells exposed to nsPEFs. •Formation of poly(ADP-ribose) was induced in nsPEF-exposed HeLa S3 cells. •Two distinct modes of cell death were activated by nsPEF in a cell-dependent manner. -- Abstract: Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.

  10. Pumping of mammalian cells with a nozzle-diffuser micropump.

    PubMed

    Yamahata, Christophe; Vandevyver, Caroline; Lacharme, Frédéric; Izewska, Paulina; Vogel, Horst; Freitag, Ruth; Gijs, Martin A M

    2005-10-01

    We discuss the successful transport of jurkat cells and 5D10 hybridoma cells using a reciprocating micropump with nozzle-diffuser elements. The effect of the pumping action on cell viability and proliferation, as well as on the damaging of cellular membranes is quantified using four types of well-established biological tests: a trypan blue solution, the tetrazolium salt WST-1 reagent, the LDH cytotoxicity assay and the calcium imaging ATP test. The high viability levels obtained after pumping, even for the most sensitive cells (5D10), indicate that a micropump with nozzle-diffuser elements can be very appropriate for handling living cells in cell-on-a-chip applications.

  11. The mitochondrial effects of novel apoptogenic molecules generated by psoralen photolysis as a crucial mechanism in PUVA therapy.

    PubMed

    Caffieri, Sergio; Di Lisa, Fabio; Bolesani, Federico; Facco, Monica; Semenzato, Gianpietro; Dall'Acqua, Francesco; Canton, Marcella

    2007-06-01

    The generation of photoproducts of psoralen (POPs) might be relevant in cell death induced by psoralen plus UVA, namely PUVA, which is a recognized effective treatment for cutaneous T-cell lymphoma, chronic graft-versus-host disease, and psoriasis. We investigated the occurrence of POP-induced cell death and the underlying mechanisms. POPs were produced by irradiating a psoralen solution with UVA. Jurkat cells treated in the dark with these mixtures died mainly through an apoptotic mechanism. POPs were separated by high-performance liquid chromatography (HPLC), and cells were added with each of these fractions. A total of 2 dimers of psoralen and 6-formyl-7-hydroxycoumarin (FHC) were identified in the apoptogenic fractions. Apoptosis was preceded by mitochondrial dysfunction caused by the opening of the mitochondrial permeability transition pore (PTP). In fact, both mitochondrial depolarization and cell death were prevented by the PTP inhibitor cyclosporin A (CsA). PTP opening was also documented in isolated mitochondria added with POP, suggesting that apoptosis is caused by a direct effect of POP on mitochondria. In fact, FHC alone induced PTP opening and CsA-inhibitable cell death of Jurkat cells, whereas nontransformed T lymphocytes were resistant. Along with identifying novel apoptogenic molecules, the present results indicate that POP generation directs transformed cells to apoptosis.

  12. Small-sized, stable lipid nanoparticle for the efficient delivery of siRNA to human immune cell lines

    PubMed Central

    Nakamura, Takashi; Kuroi, Moeka; Fujiwara, Yuki; Warashina, Shota; Sato, Yusuke; Harashima, Hideyoshi

    2016-01-01

    Gene silencing by small interfering RNA (siRNA) is useful for analyzing the functions of human immune cells. However, the transfection of siRNA to human immune cells is difficult. Here, we used a multifunctional envelope-type nanodevice (MEND) containing YSK12-C4 (YSK12-MEND) to efficiently introduce siRNA to human immune cell lines, Jurkat, THP-1, KG-1 and NK92. The YSK12-MEND was transfected to human immune cell lines at a siRNA dose range of 1–30 nM, resulting that maximum gene silencing efficiencies at the mRNA level in Jurkat, THP-1, KG-1 and NK92 were 96%, 96%, 91% and 75%, respectively. The corresponding values for Lipofectamine RNAiMAX (RNAiMAX) were 37%, 56%, 43% and 19%, respectively. The process associated with cellular uptake played a role in effective gene silencing effect of the YSK12-MEND. The small size and high non-aggregability of the YSK12-MEND were advantageous for the cellular internalization of siRNA to immune cell lines. In the case of RNAiMAX, a drastic increase in particles size was observed in the medium used, which inhibited cellular uptake. The YSK12-MEND reported in herein appears to be appropriate for delivering siRNA to human immune cells, and the small particle size and non-aggregability are essential properties. PMID:27892533

  13. PKCθ and HIV-1 Transcriptional Regulator Tat Co-exist at the LTR Promoter in CD4+ T Cells

    PubMed Central

    López-Huertas, María Rosa; Li, Jasmine; Zafar, Anjum; Rodríguez-Mora, Sara; García-Domínguez, Carlota; Mateos, Elena; Alcamí, José; Rao, Sudha; Coiras, Mayte

    2016-01-01

    PKCθ is essential for the activation of CD4+ T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4+ T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T538 residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4+ T cells and could be used as a

  14. TNF induced cleavage of HSP90 by cathepsin D potentiates apoptotic cell death

    PubMed Central

    Fritsch, Jürgen; Fickers, Ricarda; Klawitter, Jan; Särchen, Vinzenz; Zingler, Philipp; Adam, Dieter; Janssen, Ottmar; Krause, Eberhard; Schütze, Stefan

    2016-01-01

    During apoptosis induction by TNF, the extrinsic and intrinsic apoptosis pathways converge at the lysosomal-mitochondrial interface. Earlier studies showed that the lysosomal aspartic protease Cathepsin D (CtsD) cleaves Bid to tBid, resulting in the amplification of the initial apoptotic cascade via mitochondrial outer membrane permeabilization (MOMP). The goal of this study was to identify further targets for CtsD that might be involved in activation upon death receptor ligation. Using a proteomics screen, we identified the heat shock protein 90 (HSP90) to be cleaved by CtsD after stimulation of U937 or other cell lines with TNF, FasL and TRAIL. HSP90 cleavage corresponded to apoptosis sensitivity of the cell lines to the different stimuli. After mutation of the cleavage site, HSP90 partially prevented apoptosis induction in U937 and Jurkat cells. Overexpression of the cleavage fragments in U937 and Jurkat cells showed no effect on apoptosis, excluding a direct pro-apoptotic function of these fragments. Pharmacological inhibition of HSP90 with 17AAG boosted ligand mediated apoptosis by enhancing Bid cleavage and caspase-9 activation. Together, we demonstrated that HSP90 plays an anti-apoptotic role in death receptor signalling and that CtsD-mediated cleavage of HSP90 sensitizes cells for apoptosis. These findings identify HSP90 as a potential target for cancer therapy in combination with death ligands (e.g. TNF or TRAIL). PMID:27716614

  15. miRNA-149* promotes cell proliferation and suppresses apoptosis by mediating JunB in T-cell acute lymphoblastic leukemia.

    PubMed

    Fan, Sheng-Jin; Li, Hui-Bo; Cui, Gang; Kong, Xiao-Lin; Sun, Li-Li; Zhao, Yan-Qiu; Li, Ying-Hua; Zhou, Jin

    2016-02-01

    MicroRNA-149* (miRNA-149*) functions as an oncogenic regulator in human melanoma. However, the effect of miRNA-149* on T-cell acute lymphoblastic leukemia (T-ALL) is unclear. Here we aimed to analyze the effects of miRNA-149* on in vitro T-ALL cells and to uncover the target for miRNA-149* in these cells. The miRNA-149* level was determined in multiple cell lines and bone marrow cells derived from patients with T-ALL, B acute lymphoblastic leukemia (B-ALL), acute myelocytic leukemia (AML), and healthy donors. We found that miRNA-149* was highly expressed in T-ALL cell lines and T-ALL patients' bone marrow samples. JunB was identified as a direct target of miR-149*. miRNA-149* mimics downregulated JunB levels in Molt-4 and Jurkat cells, while miRNA-149* inhibitors dramatically upregulated JunB expression in these cells. miRNA-149* mimics promoted proliferation, decreased the proportion of cells in G1 phase, and reduced cell apoptosis in T-ALL cells, while miRNA-149* inhibitors prevented these effects. miRNA-149* mimics downregulated p21 and upregulated cyclinD1, 4EBP1, and p70s6k in Molt-4 and Jurkat cells. Again, inhibitors prevented these effects. Our findings demonstrate that miRNA-149* may serve as an oncogenic regulator in T-ALL by negatively regulating JunB.

  16. Comprehensive analysis of human cells motion under an irrotational AC electric field in an electro-microfluidic chip.

    PubMed

    Vaillier, Clarisse; Honegger, Thibault; Kermarrec, Frédérique; Gidrol, Xavier; Peyrade, David

    2014-01-01

    AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines.

  17. Comprehensive Analysis of Human Cells Motion under an Irrotational AC Electric Field in an Electro-Microfluidic Chip

    PubMed Central

    Kermarrec, Frédérique; Gidrol, Xavier; Peyrade, David

    2014-01-01

    AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines. PMID:24736275

  18. Induction of Apoptosis in Human Leukemia Cells by Grape Seed Extract Occurs via Activation of JNK

    PubMed Central

    Gao, Ning; Budhraja, Amit; Cheng, Senping; Yao, Hua; Zhang, Zhuo; Shi, Xianglin

    2009-01-01

    Purpose To characterize the functional role of JNK and other apoptotic pathways in grape seed extract (GSE)-induced apoptosis in human leukemia cells by using pharmacologic and genetic approaches. Experimental Design Jurkat cells were treated with various concentrations of GSE for 12 h and 24 h, or with 50 μg/ml of GSE for various time intervals, after which apoptosis, caspase activation, and cell signaling pathways were evaluated. Parallel studies were performed in U937 and HL-60 human leukemia cells. Results Exposure of Jurkat cells to GSE resulted in dose- and time-dependent increase in apoptosis and caspase activation, events associated with the pronounced increase in Cip1/p21 protein level. Furthermore, treatment of Jurkat cells with GSE resulted in marked increase in levels of phospho-JNK. Conversely, interruption of the JNK pathway by pharmacological inhibitor (e.g. SP600125) or genetic (e.g. siRNA) approaches displayed significant protection against GSE mediated lethality in Jurkat cells. Conclusions The result of the present study showed that GSE induces apoptosis in Jurkat cells through a process that involves sustained JNK activation and Cip1/p21 up-regulation, culminating in caspase activation. PMID:19118041

  19. Dispersion state and toxicity of mwCNTs in cell culture medium with different T80 concentrations.

    PubMed

    Sabuncu, Ahmet C; Kalluri, Bhargava S; Qian, Shizhi; Stacey, Michael W; Beskok, Ali

    2010-06-15

    In this study, size distribution, zeta potential, shape, and toxicity of multi-walled carbon nanotubes (mwCNTs), and effect of non-ionic detergent Tween 80 (T80) concentrations (0%, 0.2%, and 1%) on the dispersion quality and cell viability are investigated. Nanotubes are suspended in biological solutions (DMEM, RPMI) with three different concentrations (10, 50, and 100 microg/ml) and toxicological investigations are carried on human T-cell leukemia (Jurkat) and human pancreatic carcinoma (PANC1) cell lines. According to light and transmission electron microscopy results, mwCNTs form well-defined and regular bundles in the presence of 1% T80 surfactant; whereas more irregular structures are present in absence of T80. Dispersion quality is represented in terms of the size distribution from dynamic light scattering (DLS) experiments and its second moment. Dispersion quality of the mwCNTs decreases with decreasing T80 concentration, while the constituents of RPMI and DMEM increase the dispersion quality. No significant differences between the dispersive effects of RPMI and DMEM suspensions are observed. Zeta potential of the mwCNTs is measured using electrophoretic light scattering. Variations in the nanotube and T80 concentrations do not change the zeta potential significantly. T80 concentrations above 0.2% are found to be toxic for Jurkat andPANC1 cells.

  20. 4-Chlorobenzoyl berbamine induces apoptosis and G2/M cell cycle arrest through the PI3K/Akt and NF-kappaB signal pathway in lymphoma cells.

    PubMed

    Du, Hua-Ping; Shen, Jia-Kun; Yang, Min; Wang, Yun-Qui; Yuan, Xiang-Qui; Ma, Qiu-Ling; Jin, Jie

    2010-03-01

    Berbamine is an herbal compound derived from Berberis amurensis, which is used in Chinese traditional medicine. However, few studies have investigated this anti-tumor effect or the underlying mechanisms of berbamine on lymphoma cells. We investigate the effect, as well as the mechanism of action, of 4-chlorobenzoyl berbamine (BBD9) on Raji, L428, Namalwa and Jurkat lymphoma cells lines. Our findings show that BBD9 inhibits cell proliferation and induces cell apoptosis in lymphoma cell lines as well as G2/M cell cycle arrest through PI3K/Akt and NF-kappaB signaling pathways in a caspase-dependent manner. These results may provide new insights into the treatment of lymphoma.

  1. Immunomodulatory effects of ethanol extract of germinated ice plant (Mesembryanthemum crystallinum)

    PubMed Central

    Choi, Joo-Hee; Jo, Sung-Gang; Jung, Seoung-Ki; Park, Woo-Tae; Kim, Keun-Young; Park, Yong-Wook

    2017-01-01

    The purpose of this study was to investigate the immunomodulatory activity of ice plant (Mesembryanthemum crystallinum) extract (IPE) in vitro and in vivo. Raji (a human B cell line) and Jurkat (a human T cell line) cells were treated with various doses of IPE and cell proliferation was measured by WST assay. Results showed that IPE promoted the proliferation of both Raji and Jurkat cells in a dose-dependent manner. IPE also enhanced IL-6 and TNF-α production in macrophages in the presence of lipopolysaccharide (LPS), although IPE alone did not induce cytokine production. Moreover, IPE treatment upregulated iNOS gene expression in macrophages in a time- and dose-dependent manner and led to the production of nitric oxide in macrophages in the presence of IFNγ. In vivo studies revealed that oral administration of IPE for 2 weeks increased the differentiation of CD4+, CD8+, and CD19+ cells in splenocytes. These findings suggested that IPE has immunomodulatory effects and could be developed as an immunomodulatory supplement.

  2. Estimating the number of plasmids taken up by a eukaryotic cell during transfection and evidence that antisense RNA abolishes gene expression in Physarum polycephalum.

    PubMed

    Materna, Stefan C; Marwan, Wolfgang

    2005-02-01

    We have estimated the statistical distribution of the number of plasmids taken up by individual Jurkat lymphoma cells during electroporation in the presence of two plasmids, one encoding for yellow (EYFP) the other for cyan (ECFP) fluorescent protein. The plasmid concentration at which most of the cells take up only one plasmid or several molecules was determined by statistical analysis. We found that cells behaved slightly heterogeneous in plasmid uptake and describe how the homogeneity of a cell population can be quantified by Poisson statistics in order to identify experimental conditions that yield homogeneously transfection-competent cell populations. The experimental procedure worked out with Jurkat cells was applied to assay the effectiveness of antisense RNA in knocking down gene expression in Physarum polycephalum. Double transfection of flagellates with vectors encoding EYFP and antisense-EYFP revealed for the first time that gene expression can be suppressed by co-expression of antisense RNA in Physarum. Quantitative analysis revealed that one copy of antisense expressing gene per EYFP gene was sufficient to completely suppress formation of the EYFP protein in Physarum.

  3. Effect of p40tax trans-activator of human T cell lymphotropic virus type I on expression of autoantigens.

    PubMed

    Banki, K; Ablonczy, E; Nakamura, M; Perl, A

    1994-03-01

    The possibility of a retroviral etiology has long been raised in a number of autoimmune disorders. More recently, Sjögren's syndrome and rheumatoid arthritis were noted in transgenic mice carrying the tax gene of human T cell leukemia virus type I (HTLV-I). To evaluate the involvement of HTLV-I Tax in autoimmunity, its effect on expression of autoantigens was investigated. A metallothionein promoter-driven p40tax expression plasmid, pMAXRHneo-1, was stably transfected into Molt4 and Jurkat cells and the p40tax protein was induced with CdCl2. trans-Activation or trans-repression of autoantigens by HTLV-I Tax was studied by Western blot analysis utilizing autoantigen-specific murine monoclonal and rabbit polyvalent antibodies as well as sera from 161 autoimmune patients. Induction of p40tax of HTLV-I had no significant effect on levels of expression of common autoantigens U1 snRNP, Sm, Ro, La, HSP-70, topoisomerase I/Scl70, PCNA, and HRES-1. Expression of two potentially novel autoantigens, 44 and 46 kDa, was induced by p40tax as detected by sera of progressive systemic sclerosis patients, BAK and VAR. By contrast, expression of 24- and 34-kDa proteins was suppressed in response to induction of p40tax as detected by sera of systemic lupus erythematosus patients PUS and HOR. Because none of these patients were infected by HTLV-I, a protein functionally similar to p40tax may be involved in eliciting autoantigen expression and a subsequent autoantibody response in a minority of patients with PSS and SLE. Sera of autoimmune patients may also be utilized to detect novel proteins trans-activated or trans-repressed by p40tax of HTLV-I.

  4. Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia

    PubMed Central

    Chen, Yu-Jen; Fang, Li-Wen; Su, Wen-Chi; Hsu, Wen-Yi; Yang, Kai-Chien; Huang, Huey-Lan

    2016-01-01

    Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her) superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL) cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA) partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of macrophagic differentiation in AML U937 cells by lapatinib. We also noted the synergistic effects of the use of lapatinib and cytotoxic drugs in U937 leukemia cells. These results indicate that lapatinib may have potential for development as a novel antileukemia agent. PMID:27499639

  5. Control of type I interferon-induced cell death by Orai1-mediated calcium entry in T cells.

    PubMed

    Yue, Chanyu; Soboloff, Jonathan; Gamero, Ana M

    2012-01-27

    Store-operated Ca(2+) entry (SOCE) is an essential process in T cell activation. SOCE is controlled by the Ca(2+) release-activated Ca(2+) (CRAC) channel encoded by the gene Orai1 that is expressed on the plasma membrane and activated by STIM1 when ER Ca(2+) stores are depleted. Our earlier work showed that a somatic T-cell line Jurkat mutant H123 bearing a defect in Ca(2+) signaling was susceptible to the apoptotic effects of type I interferons (IFN-α/β). The nature of the mutation and whether this mutation was linked to IFN-α/β apoptotic susceptibility was unknown. Here we show that H123 cells lacked Orai1 and exhibit reduced STIM1 protein. Reconstitution of both Orai1 and STIM1 in H123 cells rescued SOCE in response to thapsigargin and ionomycin and abrogated IFN-α/β-induced apoptosis. Reciprocally, overexpression of the dominant negative Orai1-E106A in either parental Jurkat cells or an unrelated human T cell line (CEM391) inhibited SOCE and led to sensitization to IFN-α/β-induced apoptosis. Furthermore, we showed that the Ca(2+) response pathway antagonized the IFN-α/β -induced transcriptional responses; in the absence of SOCE, this negative regulatory effect was lost. However, the inhibitory effect of Ca(2+) on type I IFN-induced gene transcription was diminished by pharmacological inhibition of NF-κB in cells with intact SOCE. Our findings reveal an unexpected and novel regulatory crosstalk mechanism between type I IFNs and store-operated Ca(2+) signaling pathways mediated at least in part by NF-κB activity with significant clinical implications to both viral and tumor immunology.

  6. Cimetidine down-regulates stability of Foxp3 protein via Stub1 in Treg cells.

    PubMed

    Zhang, Yizhi; Chen, Zhoujia; Luo, Xuerui; Wu, Bin; Li, Bin; Wang, Bin

    2016-10-02

    Foxp3-expressing Treg cells have been well documented to provide immune regulation by promoting immune tolerance and suppressing immune over-reaction. Cimetidine (CIM), used to inhibit stomach acid secretion, has been reported to promote immune responses and suppress Treg cell function in several studies. However, the underlying mechanism is unknown. To investigate CIM effects on the suppressive function of Treg and Foxp3, here we used CIM to stimulate human CD4(+)CD25(+) Treg cells and Jurkat T cells and evaluated changes of Foxp3 expression and stability. Our data showed that CIM leads to a reduction of Foxp3 via E3 ligase Stub1-mediated proteosomal degradation, which is dependent on an activated PI3K-AKT-mTOR pathway. Thus, CIM affects the suppressive function of Treg cells by destabilizing their Foxp3 expression.

  7. Knockdown of ERM family member moesin in host cells increases HIV type 1 replication.

    PubMed

    Capalbo, Gianni; Mueller-Kuller, Thea; Markovic, Sandra; Klein, Stefan A; Dietrich, Ursula; Hoelzer, Dieter; Ottmann, Oliver G; Scheuring, Urban J

    2011-12-01

    Moesin is a member of the ERM (ezrin, radixin, moesin) family of cytoskeleton/membrane structure organizing and signal transduction proteins. Previously, we found an increased expression of moesin during HIV-1 infection. Moesin was also reported to be incorporated into HIV-1 virions. To analyze whether moesin is a host factor affecting the replication cycle of human immunodeficiency virus type 1 (HIV-1), we used small interfering RNAs (siRNAs) to evaluate the effect of moesin knockdown on HIV-1 replication in P4-CCR5 cells. Moesin's knockdown did not affect the cell viability or cell phenotype. Interestingly, we observed a marked increase in viral replication, as demonstrated by enhanced HIV-1 RNA, p24 antigen, and ß-galactosidase reporter expression. Moesin-dependent enhancement of HIV-1 replication was confirmed in lymphocytic host cells (Jurkat). These results suggest an overall rather restrictive role of moesin for HIV-1 replication in host cells in vitro.

  8. Echinacea purpurea (L.) Moench modulates human T-cell cytokine response☆

    PubMed Central

    Fonseca, Fabiana N.; Papanicolaou, Genovefa; Lin, Hong; Lau, Clara B.S.; Kennelly, Edward J.; Cassileth, Barrie R.; Cunningham-Rundles, Susanna

    2014-01-01

    The study objective was to evaluate the composition of a neutral and weakly acidic water-soluble extract from Echinacea purpurea (L.) Moench (EchNWA) previously shown to modify murine influenza infection, and to assess immunomodulatory effects on human T-cells. EchNWA extract from fresh aerial parts was extracted with water, ethanolic precipitation, and size-exclusion chromatography. The chemical profile of EchNWA was characterized by chromatography (size-exclusion, HPLC, GC–MS), and small molecule finger-print analysis performed by HPLC–PDA. Jurkat T-cells at high and low cell density were pretreated or not with doses of EchNWA, followed by activation with phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). Interleukin-2 (IL-2) and interferon gamma (IFNg) cytokine secretions were measured by multi-cytokine luminex technology. Results showed that EchNWA contains 80% polysaccharides, predominantly a 10 kDa entity; phenolic compounds, cynarin, cichoric and caftaric acids, but no detectable alkylamides. Cytokine production required stimulation and was lower after PMA+I activation in high-density compared to low-density conditions. EchNWA mediated a strong dose-dependent enhancement of high-density T-cell production of IL-2 and IFNg response to PMA+I. EchNWA alone did not stimulate T-cells. EchNWA enhanced mean fluorescence intensity of IL-2 in Jurkat T-cells activated by PMA+1 or ionomycin alone. Conversely EchNWA mediated modest but significant suppression of IFNg response and reduced the percentage of CD25+ T-cells under low-density conditions. Conclusions are that EchNWA polysaccharides, but not phenolic compounds have dose-related adjuvant effects on human T-cell cytokine responses characterized by enhancing and suppressive effects that are regulated by T-cell density. PMID:24434371

  9. Echinacea purpurea (L.) Moench modulates human T-cell cytokine response.

    PubMed

    Fonseca, Fabiana N; Papanicolaou, Genovefa; Lin, Hong; Lau, Clara B S; Kennelly, Edward J; Cassileth, Barrie R; Cunningham-Rundles, Susanna

    2014-03-01

    The study objective was to evaluate the composition of a neutral and weakly acidic water-soluble extract from Echinacea purpurea (L.) Moench (EchNWA) previously shown to modify murine influenza infection, and to assess immunomodulatory effects on human T-cells. EchNWA extract from fresh aerial parts was extracted with water, ethanolic precipitation, and size-exclusion chromatography. The chemical profile of EchNWA was characterized by chromatography (size-exclusion, HPLC, GC-MS), and small molecule fingerprint analysis performed by HPLC-PDA. Jurkat T-cells at high and low cell density were pretreated or not with doses of EchNWA, followed by activation with phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). Interleukin-2 (IL-2) and interferon gamma (IFNg) cytokine secretions were measured by multi-cytokine luminex technology. Results showed that EchNWA contains 80% polysaccharides, predominantly a 10kDa entity; phenolic compounds, cynarin, cichoric and caftaric acids, but no detectable alkylamides. Cytokine production required stimulation and was lower after PMA+I activation in high-density compared to low-density conditions. EchNWA mediated a strong dose-dependent enhancement of high-density T-cell production of IL-2 and IFNg response to PMA+I. EchNWA alone did not stimulate T-cells. EchNWA enhanced mean fluorescence intensity of IL-2 in Jurkat T-cells activated by PMA+1 or ionomycin alone. Conversely EchNWA mediated modest but significant suppression of IFNg response and reduced the percentage of CD25+ T-cells under low-density conditions. Conclusions are that EchNWA polysaccharides, but not phenolic compounds have dose-related adjuvant effects on human T-cell cytokine responses characterized by enhancing and suppressive effects that are regulated by T-cell density.

  10. Involvement of T-cell immunoregulation by ochnaflavone in therapeutic effect on fungal arthritis due to Candida albicans.

    PubMed

    Lee, Jue-Hee

    2011-07-01

    Arthritis due to pathogenic fungi is a serious disease causing rapid destruction of the joint. In the pathogenesis of arthritis, T lymphocytes are considered to be one of the major immune cells. In present study, we examined the T cell immunoregulatory effect by ochnaflavone (Och), a biflavonoid, on arthritis caused by Candida albicans that is the most commonly associated with fungal arthritis. To examine the effects of ochnaflavonon Candida albicans-caused septic arthritis, an emulsified mixture of C. albicans cell wall and complete Freund's adjuvant (CACW/CFA) was injected into BALB/c mice via hind footpad route on days -3, -2, and -1. On Day 0, Och at 1 or 2 mg/dose/time was intratraperitoneally given to mice with the swollen footpad every other day for 3 times. The footpad-edema was measured for 20 days. Results revealed that Och reduced the edema at all dose levels and furthermore, there was app. 45% reduction of the edema in animals given 2 mg-dose at the peak of septic arthritis (p < 0.05). This anti-arthritic effect was accompanied by the diminishing of the DTH (delayed type hypersensitivity) activity against the CACW and by the provoking of the dominant T helper 2 (Th2) type cytokines production (IL-4 and Il-10), which appeared to result in a suppression of T helper 1 cytokines (IFN-γ and IL-2). Besides the T cell immunoregulatory activity, Och inhibited T cells activation as evidenced by the IL-2 reduction from PMA/ionomycin-stimulated Jurkat cell line and in addition, the compound killed macrophages in a dose-dependent manner (p < 0.05). However, Och caused no hemolysis (p < 0.05). These data implicate that Och, which has anti-arthritic activity based on the Th2 dominance as well as macrophage removal, can be safely administered into the blood circulation for treatment of the arthritis caused by C. albicans. Thus, it can be concluded that Och would be an ideal immunologically evaluated agent for treating of Candida arthritis.

  11. The novel arylindolylmaleimide PDA-66 displays pronounced antiproliferative effects in acute lymphoblastic leukemia cells

    PubMed Central

    2014-01-01

    Background Prognosis of adult patients suffering from acute lymphoblastic leukemia (ALL) is still unsatisfactory. Targeted therapy via inhibition of deregulated signaling pathways appears to be a promising therapeutic option for the treatment of ALL. Herein, we evaluated the influence of a novel arylindolylmaleimide (PDA-66), a potential GSK3β inhibitor, on several ALL cell lines. Methods ALL cell lines (SEM, RS4;11, Jurkat and MOLT4) were exposed to different concentrations of PDA-66. Subsequently, proliferation, metabolic activity, apoptosis and necrosis, cell cycle distribution and protein expression of Wnt and PI3K/Akt signaling pathways were analyzed at different time points. Results PDA-66 inhibited the proliferation of ALL cells significantly by reduction of metabolic activity. The 72 h IC50 values ranged between 0.41 to 1.28 μM PDA-66. Additionally, caspase activated induction of apoptosis could be detected in the analyzed cell lines. PDA-66 influenced the cell cycle distribution of ALL cell lines differently. While RS4;11 and MOLT4 cells were found to be arrested in G2 phase, SEM cells showed an increased cell cycle in G0/1 phase. Conclusion PDA-66 displays significant antileukemic activity in ALL cells and classifies as candidate for further evaluation as a potential drug in targeted therapy of ALL. PMID:24502201

  12. Fluorescence microscopy imaging of electroperturbation in mammalian cells

    NASA Astrophysics Data System (ADS)

    Sun, Yinghua; Vernier, P. Thomas; Behrend, Matthew; Wang, Jingjing; Thu, Mya Mya; Gundersen, Martin A.; Marcu, Laura

    2006-03-01

    We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation-the effects of nanosecond, megavolt-per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes.

  13. Endothelial cell micropatterning: Methods, effects, and applications

    PubMed Central

    Anderson, Deirdre E.J.; Hinds, Monica T.

    2012-01-01

    The effects of flow on endothelial cells have been widely examined for the ability of fluid shear stress to alter cell morphology and function; however, the effects of endothelial cell morphology without flow have only recently been observed. An increase in lithographic techniques in cell culture spurred a corresponding increase in research aiming to confine cell morphology. These studies lead to a better understanding of how morphology and cytoskeletal configuration affect the structure and function of the cells. This review examines endothelial cell micropatterning research by exploring both the many alternative methods used to alter endothelial cell morphology and the resulting changes in cellular shape and phenotype. Micropatterning induced changes in endothelial cell proliferation, apoptosis, cytoskeletal organization, mechanical properties, and cell functionality. Finally, the ways these cellular manipulation techniques have been applied to biomedical engineering research, including angiogenesis, cell migration, and tissue engineering, is discussed. PMID:21761242

  14. Comparison of the effects of FK-506, cyclosporin A and rapamycin on IL-2 production.

    PubMed Central

    Henderson, D J; Naya, I; Bundick, R V; Smith, G M; Schmidt, J A

    1991-01-01

    The immunosuppressive compounds FK-506, cyclosporin A (CsA) and rapamycin inhibit both the human and mouse mixed lymphocyte reactions (MLR) with IC50s of 2-5 x 10(-10) M for FK-506 and rapamycin and 10(-8) M for CsA. FK-506 and CsA were also potent inhibitors of A23187/PMA-stimulated IL-2 production by Jurkat and HuT-78 cells but had no effect on the response of mouse CTLL cells to IL-2. IC50 values for inhibition of IL-2 production closely matched those for inhibition of the MLR and both drugs were active only during the first 4-6 hr following stimulation. In contrast, rapamycin was a poor inhibitor of IL-2 production, although it inhibited cellular responses to IL-2. The IC50 values for these two activities indicated that neither alone accounted for rapamycin inhibition of the MLR. FK-506 and CsA affected IL-2 gene transcription in Jurkat cells by the same mechanism. Both inhibited the appearance of the transcription factor, NFAT, whereas rapamycin did not. The appearance of another transcription factor, NFK beta, was unaffected by all three drugs. The effects of FK-506 and CsA on IL-2 gene expression, therefore, are similar even though the two drugs act through distinct cytosolic receptors. Images Figure 4 PMID:1715317

  15. Selective protection by hsp 70 against cytotoxic drug-, but not Fas-induced T-cell apoptosis

    PubMed Central

    CREAGH, E M; COTTER, T G

    1999-01-01

    The phenomenon of heat-shock (HS) protection to many cytotoxic insults has previously been described; however, the specific molecular mechanism underlying this HS-mediated protection remains undefined. To gain insight into this protective mechanism, heat-shocked Jurkat T cells were treated with a range of cytotoxic agents. Those against which HS conferred protection (camptothecin and actinomycin D) were compared with agents against which HS showed no protective effect (anti-Fas monoclonal antibody (mAb)). Reactive oxygen species (ROS) production was found to be an event common to apoptosis induced by camptothecin and actinomycin D, whereas Fas-mediated apoptosis was shown to occur via a ROS-independent mechanism. The selective protection observed against these agents was found to be mimicked by pretreatment with antioxidant compounds. Furthermore, this antioxidant protection appears to be occurring downstream of ROS production. Experiments were extended using heat-shock protein (hsp) 70 gene-transfected Jurkat T cells to confirm that the protective effects observed were caused by hsp 70 synthesis rather than any other cellular response to HS. Bcl-2 expression levels were also examined to determine whether any correlation existed between Bcl-2- and hsp 70-mediated protection. PMID:10447712

  16. KU135, a Novel Novobiocin-Derived C-Terminal Inhibitor of the 90-kDa Heat Shock Protein, Exerts Potent Antiproliferative Effects in Human Leukemic Cells

    PubMed Central

    Shelton, Shary N.; Shawgo, Mary E.; Matthews, Shawna B.; Lu, Yuanming; Donnelly, Alison C.; Szabla, Kristen; Tanol, Mehmet; Vielhauer, George A.; Rajewski, Roger A.; Matts, Robert L.; Blagg, Brian S. J.

    2009-01-01

    The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Consequently, there is considerable interest in developing chemotherapeutic drugs that specifically disrupt the function of Hsp90. Here, we investigated the extent to which a novel novobiocin-derived C-terminal Hsp90 inhibitor, designated KU135, induced antiproliferative effects in Jurkat T-lymphocytes. The results indicated that KU135 bound directly to Hsp90, caused the degradation of known Hsp90 client proteins, and induced more potent antiproliferative effects than the established N-terminal Hsp90 inhibitor 17-allylamino-demethoxygeldanamycin (17-AAG). Closer examination of the cellular response to KU135 and 17-AAG revealed that only 17-AAG induced a strong up-regulation of Hsp70 and Hsp90. In addition, KU135 caused wild-type cells to undergo G2/M arrest, whereas cells treated with 17-AAG accumulated in G1. Furthermore, KU135 but not 17-AAG was found to be a potent inducer of mitochondria-mediated apoptosis as evidenced, in part, by the fact that cell death was inhibited to a similar extent by Bcl-2/Bcl-xL overexpression or the depletion of apoptotic protease-activating factor-1 (Apaf-1). Together, these data suggest that KU135 inhibits cell proliferation by regulating signaling pathways that are mechanistically different from those targeted by 17-AAG and as such represents a novel opportunity for Hsp90 inhibition. PMID:19741006

  17. Anticancer activity of the new photosensitizers: dose and cell type dependence

    NASA Astrophysics Data System (ADS)

    Gyulkhandanyan, Grigor V.; Ghambaryan, Sona S.; Amelyan, Gayane V.; Ghazaryan, Robert K.; Haroutiunian, Samvel G.; Gyulkhandanyan, Aram G.; Gasparyan, Gennadi H.

    2005-04-01

    The necessity of researches of antitumor efficiency of new photosensitizers (PS) is explained by the opportunity of their application in photodynamic therapy of tumors. PS, selectively accumulated in cancer cells and activated by the light, generate the active oxygen species that cause apoptosis. Earlier, it was shown that PS chlorin e6 (0.3-0.5 μg/ml) induces rat embryo fibroblast-like cell apoptosis. In present work antitumor activity of the new photosensitizers, water-soluble cationic porphyrins and their metal complexes, is investigated. The dose-dependent destruction of cancer cells was shown on PC-12 (pheochromocytoma, rat adrenal gland) and Jurkat (human lymphoma) cell lines. Meso-tetra-[4-N-(2 `- oxyethyl) pyridyl] porphyrin (TOEPyP) and chlorin e6 possessed the same toxicity at LD50 dose on PC-12 cell line, whereas phototoxicity of TOEPyP was 3 times less compared to chlorin e6(LD50=0.2 and 0.075 μg/ml accordingly). The results have shown weak photosensitizing effect of Zn-and Ag-derivatives of TOEPyP on PC-12 cell line. TOEPyP and Zn-TOEPyP (0.1 - 50 μg/ml) were non-toxic for Jurkat cell line, whereas Ag-TOEPyP was toxic at 10 μg/ml (LD90). TOEPyP and chlorin e6 have shown phototoxic effect in the same dose range (LD50=0.5 and 0.3 μg/ml accordingly). The investigation of toxic and phototoxic effects of the new porphyrins revealed significantly different sensitivity of various cell lines to PSs.

  18. The effect of dendritic cells on the retinal cell transplantation

    SciTech Connect

    Oishi, Akio; Nagai, Takayuki; Mandai, Michiko Takahashi, Masayo; Yoshimura, Nagahisa

    2007-11-16

    The potential of bone marrow cell-derived immature dendritic cells (myeloid iDCs) in modulating the efficacy of retinal cell transplantation therapy was investigated. (1) In vitro, myeloid iDCs but not BMCs enhanced the survival and proliferation of embryonic retinal cells, and the expression of various neurotrophic factors by myeloid iDCs was confirmed with RT-PCR. (2) In subretinal transplantation, neonatal retinal cells co-transplanted with myeloid iDCs showed higher survival rate compared to those transplanted without myeloid iDCs. (3) CD8 T-cells reactive against donor retinal cells were significantly increased in the mice with transplantation of retinal cells alone. These results suggested the beneficial effects of the use of myeloid iDCs in retinal cell transplantation therapy.

  19. A passive-flow microfluidic device for imaging latent HIV activation dynamics in single T cells

    PubMed Central

    Gearhart, Larisa M.; Miller-Jensen, Kathryn

    2015-01-01

    Quantifying cell-to-cell variability in drug response dynamics is important when evaluating therapeutic efficacy. For example, optimizing latency reversing agents (LRAs) for use in a clinical “activate-and-kill” strategy to purge the latent HIV reservoir in patients requires minimizing heterogeneous viral activation dynamics. To evaluate how heterogeneity in latent HIV activation varies across a range of LRAs, we tracked drug-induced response dynamics in single cells via live-cell imaging using a latent HIV–GFP reporter virus in a clonal Jurkat T cell line. To enable these studies in suspension cells, we designed a simple method to capture an array of single Jurkat T cells using a passive-flow microfluidic device. Our device, which does not require external pumps or tubing, can trap hundreds of cells within minutes with a high retention rate over 12 hours of imaging. Using this device, we quantified heterogeneity in viral activation stimulated by transcription factor (TF) activators and histone deacetylase (HDAC) inhibitors. Generally, TF activators resulted in both faster onset of viral activation and faster rates of production, while HDAC inhibitors resulted in more uniform onset times, but more heterogeneous rates of production. Finally, we demonstrated that while onset time of viral gene expression and rate of viral production together predict total HIV activation, rate and onset time were not correlated within the same individual cell, suggesting that these features are regulated independently. Overall, our results reveal drug-specific patterns of noisy HIV activation dynamics not previously identified in static single-cell assays, which may require consideration for the most effective activate-and-kill regime. PMID:26138068

  20. A passive-flow microfluidic device for imaging latent HIV activation dynamics in single T cells.

    PubMed

    Ramji, Ramesh; Wong, Victor C; Chavali, Arvind K; Gearhart, Larisa M; Miller-Jensen, Kathryn

    2015-09-01

    Quantifying cell-to-cell variability in drug response dynamics is important when evaluating therapeutic efficacy. For example, optimizing latency reversing agents (LRAs) for use in a clinical "activate-and-kill" strategy to purge the latent HIV reservoir in patients requires minimizing heterogeneous viral activation dynamics. To evaluate how heterogeneity in latent HIV activation varies across a range of LRAs, we tracked drug-induced response dynamics in single cells via live-cell imaging using a latent HIV-GFP reporter virus in a clonal Jurkat T cell line. To enable these studies in suspension cells, we designed a simple method to capture an array of single Jurkat T cells using a passive-flow microfluidic device. Our device, which does not require external pumps or tubing, can trap hundreds of cells within minutes with a high retention rate over 12 hours of imaging. Using this device, we quantified heterogeneity in viral activation stimulated by transcription factor (TF) activators and histone deacetylase (HDAC) inhibitors. Generally, TF activators resulted in both faster onset of viral activation and faster rates of production, while HDAC inhibitors resulted in more uniform onset times, but more heterogeneous rates of production. Finally, we demonstrated that while onset time of viral gene expression and rate of viral production together predict total HIV activation, rate and onset time were not correlated within the same individual cell, suggesting that these features are regulated independently. Overall, our results reveal drug-specific patterns of noisy HIV activation dynamics not previously identified in static single-cell assays, which may require consideration for the most effective activate-and-kill regime.

  1. CXC chemokine ligand 16, inversely correlated with CD99 expression in Hodgkin Reed-Sternberg cells, is widely expressed in diverse types of lymphomas.

    PubMed

    Liu, Fang; Zhang, Yu; Tang, Hongping; Zhou, Xinhua; Wu, Ziqing; Tang, Dongsheng; Zhao, Tong

    2013-08-01

    The present study examined a correlation between CXC chemokine ligand 16 (CXCL16) and cell differentiation antigen 99 (CD99) expression and investigated the role of CXCL16 in human lymphoma cell lines and clinical samples. Cytokine antibody arrays were used to measure the differentially expressed cytokines in tumor tissues. The expression of CXCL16 and CD99 was analyzed by quantitative PCR (qPCR) and western blotting, while the pathways involved were assessed by western blotting and enzyme-linked immunosorbent assay (ELISA). The expression of CXCL16 was investigated in 9 lymphoma cell lines (L428, RPMI-8226, KM3, Jurkat, OCI-Ly1, OCI-Ly8, OCI-Ly10, Karpass299 and Raji) as well as in clinical lymphoma samples using qPCR, western blotting and immunochemistry. Soluble CXCL16 (sCXCL16) levels were measured by ELISA and proliferation was analyzed by CCK‑8 proliferation assays. CXCL16 was one of the upregulated chemokines when lymphoma cells where transferred from in vitro to in vivo conditions. The increased expression and secretion of CXCL16 paralleled with a decrease of mCD99L2 and was accompanied by NF-κB pathway activation and vice versa. CXCL16 was expressed in all 9 lymphoma cell lines with the highest levels in the Hodgkin lymphoma (HL) cell line L428, the plasma cell-derived cell lines RPMI‑8226 and KM3 and the T leukemia-derived cell line Jurkat. Higher levels of sCXCL16 were secreted by L428 cells, the diffuse large B-cell lymphoma (DLBCL)-derived cell lines (OCI-Ly1, OCI-Ly8 and OCI-Ly10) and Jurkat cells. CXCL16 was widely expressed in clinical samples of lymphoma patients with higher levels in HL compared to non-Hodgkin lymphoma. Human recombinant CXCL16 had no significant effect on L428 cell proliferation, but was able to stimulate CD4+ T lymphocytes to proliferate. CXCL16, inversely correlated with CD99 expression in Hodgkin Reed-Sternberg (H/RS) cells, is widely expressed in diverse types of lymphomas.

  2. Relationship Between Pak-Mediated Cell Death and Stress-Activated Kinase Signaling Breast Cancer

    DTIC Science & Technology

    2001-02-01

    Jurkat: T-lymphoblast cell line; HeLa: human cervical carcinoma; CHO : chinese hamster ovary ; ZR75, MDA23 1, SKBR-3: human breast cancer cell lines 16 RhoA...activation in Jurkat cells . J Immunol 1998 Jan 1; 160(1):7-1 1 12 Genomic Locus of GEF/H1/KIAA0651 GI 11427616: 870353 864048 861008 858631 858224...1: Schematic representation of the genomic locus of GEF-H1i/KIAA0651 as deduced from the working draft sequence of the GI 11427616 contig derived from

  3. The role of heat shock protein 90 in the regulation of tumor cell apoptosis.

    PubMed

    Kaigorodova, E V; Ryazantseva, N V; Novitskii, V V; Belkina, M V; Maroshkina, A N

    2011-02-01

    Programmed death of Jurkat tumor cells was studied under conditions of culturing with 17-AAG selective inhibitor of heat shock protein with a molecular weight of 90 kDa and etoposide. Apoptosis realization was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Activity of caspase-3 was evaluated spectrophotometrically. Inhibition of heat shock protein with a molecular weight of 90 kDa activated the apoptotic program in Jurkat tumor cells and etoposide-induced apoptosis. The heat shock protein with a molecular weight of 90 kDa acted as apoptosis inhibitor in tumor cells.

  4. Peripheral benzodiazepine receptor ligands reverse apoptosis resistance of cancer cells in vitro and in vivo.

    PubMed

    Decaudin, Didier; Castedo, Maria; Nemati, Fariba; Beurdeley-Thomas, Arnaud; De Pinieux, Gonzague; Caron, Antoine; Pouillart, Pierre; Wijdenes, John; Rouillard, Dany; Kroemer, Guido; Poupon, Marie-France

    2002-03-01

    The mitochondrial peripheral benzodiazepine receptor (mPBR) is involved in a functional structure designated as the permeability transition pore, which controls apoptosis. Binding of Fas/APO-1/CD95 triggers a prototypic apoptosis-inducing pathway. Using four different human tumor cell lines (T-cell Jurkat, neuroblastoma SHEP, osteosarcoma 143N2, and glioblastoma SNB79 cell lines), all of which express CD95 and mPBR, we investigated the potential role of mPBR ligands in CD95-induced apoptosis. We show that, in vitro, the three mPBR ligands tested (RO5-4864, PK11195, and diazepam) enhanced apoptosis induced by anti-CD95 antibody in Jurkat cells, as demonstrated by mitochondrial transmembrane potential drop and DNA fragmentation. In contrast, RO5-4864, but not PK11195 or diazepam, enhanced anti-CD95 apoptosis in all other cell lines. These effects were obtained in Bcl-2-overexpressing SHEP cell lines, but not in Bcl-X(L) SHEP cell lines. Enhancement of anti-CD95 antibody-induced apoptosis by RO5-4864 was characterized by an increased mitochondrial release of cytochrome c and Smac/DIABLO proteins and an enhanced activation of caspases 9 and 3, suggesting a mitochondrion-dependent mechanism. Preincubation of cells with the different mPBR ligands or anti-CD95 did not affect the levels of expression of either mPBR or CD95. In vivo, we found that the RO5-4864 mPBR ligand significantly increased the growth inhibition induced by two chemotherapeutic agents, etoposide and ifosfamide, using two human small cell lung cancers xenografted into nude mice. Peripheral benzodiazepine receptor ligands may therefore act as chemosensitizing agents for the treatment of human neoplasms.

  5. Enumeration and viability of rare cells in a microfluidic disk via positive selection approach.

    PubMed

    Chen, Ken-Chao; Pan, Yu-Cheng; Chen, Chen-Lin; Lin, Ching-Hung; Huang, Chiun-Sheng; Wo, Andrew M

    2012-10-15

    Recent studies have shown that specific rare cells in the blood can serve as an indicator of cancer prognosis, among other purposes. This article demonstrates the concept of separating and detecting rare cells from peripheral blood mononuclear cells via an economical microfluidic disk with a model system. MCF7, labeled with magnetic beads, was used to simulate circulating tumor cells as a target. Jurkat clone E6-1 was used to simulate leukocytes or other cells abundant in human blood. A tailored multistage magnet maximized the magnetic field to ensure optimal trapping efficiency. Results indicate that the yield of detected MCF7 was consistent at approximately 80% when fewer than hundreds of MCF7 cells were mixed in greater than 1 million Jurkat cells. The 80% yield also held for 10 MCF7 in 100 million Jurkat (rarity of 10(7)). Compared with the results from autoMACS, the performance was at least 20% higher and was more independent of the number of Jurkat. The viability of the enriched cells was approximately 90 ± 20%, showing that this method caused little damage to trapped cells. The microfluidic disk should be applicable for separation and detection of various rare cells, such as circulating tumor cells and circulating endothelial cells in human blood.

  6. Immunosuppressive effects of apoptotic cells

    NASA Astrophysics Data System (ADS)

    Voll, Reinhard E.; Herrmann, Martin; Roth, Edith A.; Stach, Christian; Kalden, Joachim R.; Girkontaite, Irute

    1997-11-01

    Apoptotic cell death is important in the development and homeostasis of multicellular organisms and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage,. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.

  7. Aronia melanocarpa juice induces a redox-sensitive p73-related caspase 3-dependent apoptosis in human leukemia cells.

    PubMed

    Sharif, Tanveer; Alhosin, Mahmoud; Auger, Cyril; Minker, Carole; Kim, Jong-Hun; Etienne-Selloum, Nelly; Bories, Pierre; Gronemeyer, Hinrich; Lobstein, Annelise; Bronner, Christian; Fuhrmann, Guy; Schini-Kerth, Valérie B

    2012-01-01

    Polyphenols are natural compounds widely present in fruits and vegetables, which have antimutagenic and anticancer properties. The aim of the present study was to determine the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (AMJ) containing 7.15 g/L of polyphenols in the acute lymphoblastic leukemia Jurkat cell line, and, if so, to clarify the underlying mechanism and to identify the active polyphenols involved. AMJ inhibited cell proliferation, which was associated with cell cycle arrest in G(2)/M phase, and caused the induction of apoptosis. These effects were associated with an upregulation of the expression of tumor suppressor p73 and active caspase 3, and a downregulation of the expression of cyclin B1 and the epigenetic integrator UHRF1. AMJ significantly increased the formation of reactive oxygen species (ROS), decreased the mitochondrial membrane potential and caused the release of cytochrome c into the cytoplasm. Treatment with intracellular ROS scavengers prevented the AMJ-induced apoptosis and upregulation of the expression of p73 and active caspase 3. The fractionation of the AMJ and the use of identified isolated compounds indicated that the anticancer activity was associated predominantly with chlorogenic acids, some cyanidin glycosides, and derivatives of quercetin. AMJ treatment also induced apoptosis of different human lymphoblastic leukemia cells (HSB-2, Molt-4 and CCRF-CEM). In addition, AMJ exerted a strong pro-apoptotic effect in human primary lymphoblastic leukemia cells but not in human normal primary T-lymphocytes. Thus, the present findings indicate that AMJ exhibits strong anticancer activity through a redox-sensitive mechanism in the p53-deficient Jurkat cells and that this effect involves several types of polyphenols. They further suggest that AMJ has chemotherapeutic properties against acute lymphoblastic leukemia by selectively targeting lymphoblast-derived tumor cells.

  8. Activated T cells exhibit increased uptake of silicon phthalocyanine Pc 4 and increased susceptibility to Pc 4-photodynamic therapy-mediated cell death.

    PubMed

    Soler, David C; Ohtola, Jennifer; Sugiyama, Hideaki; Rodriguez, Myriam E; Han, Ling; Oleinick, Nancy L; Lam, Minh; Baron, Elma D; Cooper, Kevin D; McCormick, Thomas S

    2016-06-08

    Photodynamic therapy (PDT) is an emerging treatment for malignant and inflammatory dermal disorders. Photoirradiation of the silicon phthalocyanine (Pc) 4 photosensitizer with red light generates singlet oxygen and other reactive oxygen species to induce cell death. We previously reported that Pc 4-PDT elicited cell death in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro, and furthermore that Jurkat cells were more sensitive than A431 cells to treatment. In this study, we examined the effectiveness of Pc 4-PDT on primary human CD3(+) T cells in vitro. Fluorometric analyses of lysed T cells confirmed the dose-dependent uptake of Pc 4 in non-stimulated and stimulated T cells. Flow cytometric analyses measuring annexin V and propidium iodide (PI) demonstrated a dose-dependent increase of T cell apoptosis (6.6-59.9%) at Pc 4 doses ranging from 0-300 nM. Following T cell stimulation through the T cell receptor using a combination of anti-CD3 and anti-CD28 antibodies, activated T cells exhibited increased susceptibility to Pc 4-PDT-induced apoptosis (10.6-81.2%) as determined by Pc 4 fluorescence in each cell, in both non-stimulated and stimulated T cells, Pc 4 uptake increased with Pc 4 dose up to 300 nM as assessed by flow cytometry. The mean fluorescence intensity (MFI) of Pc 4 uptake measured in stimulated T cells was significantly increased over the uptake of resting T cells at each dose of Pc 4 tested (50, 100, 150 and 300 nM, p < 0.001 between 50 and 150 nM, n = 8). Treg uptake was diminished relative to other T cells. Cutaneous T cell lymphoma (CTCL) T cells appeared to take up somewhat more Pc 4 than normal resting T cells at 100 and 150 nm Pc 4. Confocal imaging revealed that Pc 4 localized in cytoplasmic organelles, with approximately half of the Pc 4 co-localized with mitochondria in T cells. Thus, Pc 4-PDT exerts an enhanced apoptotic effect on activated CD3(+) T cells that may be exploited in targeting T cell-mediated skin

  9. Targeting TRPM2 Channels Impairs Radiation-Induced Cell Cycle Arrest and Fosters Cell Death of T Cell Leukemia Cells in a Bcl-2-Dependent Manner

    PubMed Central

    Klumpp, Dominik; Misovic, Milan; Szteyn, Kalina; Shumilina, Ekaterina; Rudner, Justine; Huber, Stephan M.

    2016-01-01

    Messenger RNA data of lymphohematopoietic cancer lines suggest a correlation between expression of the cation channel TRPM2 and the antiapoptotic protein Bcl-2. The latter is overexpressed in various tumor entities and mediates therapy resistance. Here, we analyzed the crosstalk between Bcl-2 and TRPM2 channels in T cell leukemia cells during oxidative stress as conferred by ionizing radiation (IR). To this end, the effects of TRPM2 inhibition or knock-down on plasma membrane currents, Ca2+ signaling, mitochondrial superoxide anion formation, and cell cycle progression were compared between irradiated (0–10 Gy) Bcl-2-overexpressing and empty vector-transfected Jurkat cells. As a result, IR stimulated a TRPM2-mediated Ca2+-entry, which was higher in Bcl-2-overexpressing than in control cells and which contributed to IR-induced G2/M cell cycle arrest. TRPM2 inhibition induced a release from G2/M arrest resulting in cell death. Collectively, this data suggests a pivotal function of TRPM2 in the DNA damage response of T cell leukemia cells. Apoptosis-resistant Bcl-2-overexpressing cells even can afford higher TRPM2 activity without risking a hazardous Ca2+-overload-induced mitochondrial superoxide anion formation. PMID:26839633

  10. Gallic Acid Inhibits Proliferation and Induces Apoptosis in Lymphoblastic Leukemia Cell Line (C121)

    PubMed Central

    Sourani, Zahra; Pourgheysari, Batoul; Beshkar, Pezhman; Shirzad, Hedayatollah; Shirzad, Moein

    2016-01-01

    Leukemia is known as the world’s fifth most prevalent cancer. New cytotoxic drugs have created considerable progress in the treatment, but side effects are still the important cause of mortality. Plant derivatives have been recently considered as important sources for the treatment of various diseases, including cancer. Gallic acid (GA) is a polyhydroxyphenolic compound with a wide range of biological functions. The aim of the present study was to evaluate the effect of GA on proliferation inhibition and apoptosis induction of a lymphoblastic leukemia cell line. Jurkat cell (C121) line was cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) with different concentrations of GA (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM) for 24, 48 and 72 hours. The effect of GA on cell viability was measured using MTS assay. Induction of apoptosis was evaluated with Annexin V-FITC/PI kit and flow cytometry. Data were analyzed by SPSS version 20 using Kruskal-Wallis and Dunn’s multiple comparison tests. Decline of cell viability to less than 50% was observed at 60.3±1.6, 50.9±1.5, and 30.9±2.8 μM concentration after 24, 48, and 72 hours incubation, respectively. All concentrations of GA (10, 30, 50 and 80 μM) enhanced apoptosis compared to the control (P<0.05). The results demonstrate that the polyphenolic compound, GA, is effective in inhibition of proliferation and induction of apoptosis in Jurkat cell line. It is recommended to study the mechanism of apoptosis induction in future investigations. PMID:27853333

  11. Ionizing radiation damage to cells: effects of cell cycle redistribution.

    PubMed

    Chen, P L; Brenner, D J; Sachs, R K

    1995-04-01

    If a population of cycling cells is exposed to a fixed dose of ionizing radiation delivered over time T, it is sometimes observed that increasing T increases the amount of cell killing. This is essentially because at first the radiation preferentially kills cells in a sensitive portion of the cycle and the surviving, more resistant cells then have time to reach more sensitive stages. We refer to this effect as population resensitization, caused by redistribution within the cell cycle. We investigate the effect theoretically by employing the McKendrick-von Foerster equation for age-structured proliferating cell populations, generalized by introducing a radiation damage term. Within our formalism, we show that population resensitization occurs whenever: (a) prior to irradiation the cell population has the stable age-distribution approached asymptotically by an unirradiated population, and (b) T is sufficiently small. Examples and other cases are outlined. The methods of Volterra integral equations, renewal theory, and positive semigroup theory are applied. The effect of varying T is evaluated by considering the ultimate amplitude of the stable age-distribution population at times much greater than both the irradiation duration and the average cell-cycle time. The main biological limitations of the formalism are the following: considering only radiation damage which is not subject to enzymatic repair or quadratic misrepair, using an overly naive method of ensuring loss of cell cycle synchrony, neglecting nonlinear effects such as density inhibition of growth, and neglecting radiatively induced perturbations of the cell cycle. Possible methods for removing these limitations are briefly discussed.

  12. Effects of curvature and cell-cell interaction on cell adhesion in microvessels.

    PubMed

    Yan, W W; Liu, Y; Fu, B M

    2010-10-01

    It has been found that both circulating blood cells and tumor cells are more easily adherent to curved microvessels than straight ones. This motivated us to investigate numerically the effect of the curvature of the curved vessel on cell adhesion. In this study, the fluid dynamics was carried out by the lattice Boltzmann method (LBM), and the cell dynamics was governed by the Newton's law of translation and rotation. The adhesive dynamics model involved the effect of receptor-ligand bonds between circulating cells and endothelial cells (ECs). It is found that the curved vessel would increase the simultaneous bond number, and the probability of cell adhesion is increased consequently. The interaction between traveling cells would also affect the cell adhesion significantly. For two-cell case, the simultaneous bond number of the rear cell is increased significantly, and the curvature of microvessel further enhances the probability of cell adhesion.

  13. Cellular Silica Encapsulation for Development of Robust Cell Based Biosensors

    NASA Astrophysics Data System (ADS)

    Johnston, Robert; Rogelj, Snezna; Harper, Jason; Tartis, Michaelann

    2014-03-01

    In order to detect chemical and biological threats both on the battlefield and in civilian life, development of portable, robust detection systems capable of real-time identification of the chemical and biological agents are needed. Living cell-based sensors have proven effective as sensitive, specific, near real-time detectors; however, living cell-based sensors require frequent cell replenishment due to cell sensitivity to the ex-vivo environment, which limits sensor stability. Incorporation of living cells within a biocompatible matrix that provides mechanical protection and maintains access to the external environment may facilitate the development of long-term stable cell-based biosensors. We are exploring the use of a novel Chemical Vapor into Liquid (CViL) deposition process for whole cell encapsulation in silica. In CViL, the high vapor pressure of common silica alkoxides is utilized to deliver silica into an aqueous medium, creating a silica sol. Mixing of cells with the resulting silica sol facilitates encapsulation of cells in silica while minimizing cell contact with the cytotoxic products of silica generating reactions. Using fluorescence microscopy analysis with multiple silica specific markers, encapsulation of multiple eukaryotic cell types (Saccharomyces cerevisiae, Jurkat, HeLa, and U87 cells) with CViL generated silica is shown, providing a foundation for development of long -term stable cell-based biosensors with diverse sensing capabilities.

  14. Argonaute 2 immunoprecipitation revealed large tumor suppressor kinase 1 as a novel proapoptotic target of miR-21 in T cells.

    PubMed

    Teteloshvili, Nato; Smigielska-Czepiel, Katarzyna; Yuan, Ye; Seitz, Annika; de Jong, Debora; Rutgers, Bea; Jellema, Pytrick; van der Lei, Roelof Jan; Slezak-Prochazka, Izabella; Brouwer, Elisabeth; Boots, Annemieke M H; Kroesen, Bart-Jan; van den Berg, Anke; Kluiver, Joost

    2017-02-01

    MicroRNA (miR)-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many types of cancers. The exact mechanisms underlying the antiapoptotic effects of miR-21 are not well understood. In this study, we used the Jurkat T-cell line as a model to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth which could be explained by an increase in apoptosis. MicroRNA target gene identification by AGO2 RNA-immunoprecipitation followed by gene expression microarray (RIP-Chip) resulted in the identification of 72 predicted miR-21 target genes that were at least twofold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were at least twofold more enriched in the AGO2-IP fraction of miR-21 overexpressing cells as compared to AGO2-IP fraction of control cells. The target gene for which the AGO2-IP enrichment was most prominently increased upon miR-21 overexpression was the proapoptotic protein LATS1. Luciferase reporter assays and western blot analysis confirmed targeting of LATS1 by miR-21. qRT-PCR analysis in primary T cells showed an inverse expression pattern between LATS1 transcript levels and miR-21 upon T-cell stimulation. Finally, LATS1 knockdown partially rescued the miR-21 inhibition-induced impaired cell growth. Collectively, these data identify LATS1 as a miR-21 target important for the antiapoptotic function of miR-21 in T cells and likely also in many types of cancer.

  15. Discriminating in vitro cell fusion from cell aggregation by flow cytometry combined with fluorescence resonance energy transfer.

    PubMed

    Huerta, Leonor; López-Balderas, Nayali; Larralde, Carlos; Lamoyi, Edmundo

    2006-12-01

    Expression of fusion proteins in the plasma membrane enables cells to bind and fuse with surrounding cells to form syncytia. Cell fusion can have important functional outcomes for the interacting cells, as syncytia formation does in AIDS pathogenesis. Studies on cell fusion would be facilitated by a quantitative method able to discriminate between cellular aggregates and bona fide fused cells in a cell population. Flow cytometry with fluorescence resonance energy transfer is applied here for analyzing fusion of HIV-1 envelope-expressing cells with CD4+ Jurkat cells. Fusion partners were labeled with the vital lipophilic fluorescent probes DiO (green) and DiI (red) and FRET is manifested by an enhancement of the DiI red fluorescence intensity in double fluorescent cells, thus allowing discrimination between fused and aggregated cells. The inhibitory effect of anti-CD4 monoclonal antibodies and the inhibitory peptide T-20 upon cell fusion were readily quantified by this technique. This method allows the distinction of fused and aggregated cells even when they are at low frequencies.

  16. Signalling pathways induced by protease-activated receptors and integrins in T cells.

    PubMed

    Bar-Shavit, Rachel; Maoz, Miriam; Yongjun, Yin; Groysman, Maya; Dekel, Idit; Katzav, Shulamit

    2002-01-01

    Recent characterization of the thrombin receptor indicates that it plays a role in T-cell signalling pathways. However, little is known regarding the signalling events following stimulation of additional members of the protease-activated receptor (PAR) family, i.e. PAR2 and PAR3. Most of the postligand cascades are largely unknown. Here, we illustrate that in Jurkat T-leukaemic cells, activation of PAR1, PAR2 and PAR3 induce tyrosine phosphorylation of Vav1. This response was impaired in Jurkat T cells deficient in p56lck (JCaM1.6). Activation of PARs also led to an increase in tyrosine phosphorylation of ZAP-70 and SLP-76, two key proteins in T-cell receptor (TCR) signalling. We also demonstrated that p56lck is meaningful for integrin signalling. Thus, JCaM1.6 cells exhibited a marked reduction in their adherence to fibronectin-coated plates, as compared to the level of adherence of Jurkat T cells. While the phosphorylation of Vav1 in T cells is augmented following adhesion, no additional increase was noted following treatment of the adhered cells with PARs. Altogether, we have identified key components in the postligand-signalling cascade of PARs and integrins. Furthermore, we have identified Lck as a critical and possibly upstream component of PAR-induced Vav1 phosphorylation, as well as integrin activation, in Jurkat T cells.

  17. TFDP3 confers chemoresistance in minimal residual disease within childhood T-cell acute lymphoblastic leukemia

    PubMed Central

    Chu, Ming; Yin, Kailin; Dong, Yujun; Wang, Pingzhang; Xue, Yun; Zhou, Peng; Wang, Yuqi; Wang, Yuedan

    2017-01-01

    Acquired drug resistance in childhood T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem. In this study, a novel gene therapy target for childhood T-ALL to overcome chemoresistance was discovered: TFDP3 increased in the minimal residual disease (MRD) positive childhood T-ALL patients. Then, we established a preclinical model of resistance to induction therapy to examine the functional relevance of TFDP3 to chemoresistance in MRD derived from Jurkat/E6-1. Jurkat xenografts in NOD/SCID mice were exposed to a four drug combination (VXLD) of vincristine (VCR), dexamethasone (DEX), L-asparaginase (L-asp) and daunorubicin (DNR). During the 4-week VXLD treatment, the level of TFDP3 increased 4-fold. High expression of TFDP3 was identified in the re-emerging lines (Jurkat/MRD) with increased chemoresistance, which is correlated with partially promoter demethylation of TFDP3. Downregulation of TFDP3 by RNA interference reversed chemoresistance in Jurkat/MRD accompanied by reinstated E2F1 activity that coincided with increased levels of p53, p73, and associated proapoptotic target genes. Importantly, TFDP3 silencing in vivo induced apparent benefit to overcome chemoresistance in combination with VXLD treatment. Collectively, TFDP3 confers chemoresistance in MRD within childhood T-ALL, indicating that TFDP3 is a potential gene therapy target for residual cancer. PMID:27902457

  18. Effect of aging on stem cells

    PubMed Central

    Ahmed, Abu Shufian Ishtiaq; Sheng, Matilda HC; Wasnik, Samiksha; Baylink, David J; Lau, Kin-Hing William

    2017-01-01

    Pluripotent stem cells have the remarkable self-renewal ability and are capable of differentiating into multiple diverse cells. There is increasing evidence that the aging process can have adverse effects on stem cells. As stem cells age, their renewal ability deteriorates and their ability to differentiate into the various cell types is altered. Accordingly, it is suggested aging-induced deterioration of stem cell functions may play a key role in the pathophysiology of the various aging-associated disorders. Understanding the role of the aging process in deterioration of stem cell function is crucial, not only in understanding the pathophysiology of aging-associated disorders, but also in future development of novel effective stem cell-based therapies to treat aging-associated diseases. This review article first focuses on the basis of the various aging disease-related stem cell dysfunction. It then addresses the several concepts on the potential mechanism that causes aging-related stem cell dysfunction. It also briefly discusses the current potential therapies under development for aging-associated stem cell defects. PMID:28261550

  19. Inhibition of apoptosis in human immunodeficiency virus-infected cells enhances virus production and facilitates persistent infection.

    PubMed Central

    Antoni, B A; Sabbatini, P; Rabson, A B; White, E

    1995-01-01

    Apoptosis is one of several mechanisms by which human immunodeficiency virus type 1 (HIV-1) exerts its cytopathic effects. CD4+ Jurkat T-cell lines overexpressing the adenovirus E1B 19K protein, a potent inhibitor of apoptosis, were used to examine the consequences of inhibition of apoptosis during acute and chronic HIV-1 infections. E1B 19K protein expression inhibited HIV-induced apoptosis, enhanced virus production, and established high levels of persistent viral infection. One E1B 19K-expressing line appeared to undergo HIV-induced death via a nonapoptotic mechanism, illustrating that HIV infection results in lymphocyte depletion through multiple pathways. Increased virus production associated with sustained cell viability suggests that therapeutic approaches involving inhibition of HIV-induced programmed cell death may be problematic. PMID:7884884

  20. Effects of stress waves on cells

    SciTech Connect

    Campbell, H L; Da Silva, L B; Visuri, S R

    1998-03-02

    Laser induced stress waves are being used in a variety of medical applications, including drug delivery and targeted tissue disruption. Stress waves can also be an undesirable side effect in laser procedures such as ophthalmology and angioplasty. Thus, a study of the effects of stress waves on a cellular level is useful. Thermoelastic stress waves were produced using a Q-switched frequency-doubled Nd:YAG laser (@.=532nm) with a pulse duration of 4 ns. The laser radiation was delivered to an absorbing media. A thermoelastic stress wave was produced in the absorbing media and propagated into plated cells. The energy per pulse delivered to a sample and the spot size were varied. Stress waves were quantified. We assayed for cell viability and damage using two methods. The laser parameters within which cells maintain viability were investigated and thresholds for cell damage were defined. A comparison of cell damage thresholds for different cell lines was made.

  1. Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-κB, NFAT and AP-1.

    PubMed

    Jutz, Sabrina; Leitner, Judith; Schmetterer, Klaus; Doel-Perez, Iago; Majdic, Otto; Grabmeier-Pfistershammer, Katharina; Paster, Wolfgang; Huppa, Johannes B; Steinberger, Peter

    2016-03-01

    Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.

  2. Fast and accurate detection of cancer cell using a versatile three-channel plasmonic sensor

    NASA Astrophysics Data System (ADS)

    Hoseinian, M.; Ahmadi, A. R.; Bolorizadeh, M. A.

    2016-09-01

    Surface Plasmon Resonance (SPR) optical fiber sensors can be used as cost-effective small sized biosensors that are relatively simple to operate. Additionally, these instruments are label-free, hence rendering them highly sensitive to biological measurements. In this study, a three-channel microstructure optical fiber plasmonic-based portable biosensor is designed and analyzed using Finite Element Method. The proposed system is capable of determining changes in sample's refractive index with precision of order one thousandth. The biosensor measures three absorption resonance wavelengths of the analytes simultaneously. This property is one of the main advantages of the proposed biosensor since it reduces the error in the measured wavelength and enhances the accuracy of the results up to 10-5 m/RIU by reducing noise. In this paper, Jurkat cell, an indicator cell for leukemia cancer, is considered as the analyte; and its absorption resonance wavelengths as well as sensitivity in each channel are determined.

  3. Effects of Nonequilibrium Plasmas on Eukaryotic Cells

    DTIC Science & Technology

    2009-05-01

    atmospheric pre Effects on the viabil Second, we report on ( planaria ), to see if preliminary conclusio regeneration process, plume generated by...has any effect on cells regeneration following a wound (cut). The model organism in our studies is a multi-cellular worm, " planaria ", which is capable...concluded that exposure to this type of plasma neither helps nor hinders growth/cell regeneration in planaria . These results could have the following

  4. Modulation of intracellular calcium concentrations and T cell activation by prickly pear polyphenols.

    PubMed

    Aires, Virginie; Adote, Sylvie; Hichami, Aziz; Moutairou, Kabirou; Boustani, Es-Saddik E; Khan, Naim A

    2004-05-01

    Opuntia ficus indica (prickly pear) polyphenolic compounds (OFPC) triggered an increase in [Ca2+]i in human Jurkat T-cell lines. Furthermore, OFPC-induced rise in [Ca2+]i was significantly curtailed in calcium-free buffer (0% Ca2+) as compared to that in 100% Ca2+ medium. Preincubation of cells with tyrphostin A9, an inhibitor of Ca2+ release-activated Ca2+ (CRAC) channels, significantly diminished the OFPC-induced sustained response on the increases in [Ca2+]i. Lanthanum and nifedipine, the respective inhibitors of voltage-dependent and L-type calcium channels, failed to curtail significantly the OFPC-induced calcium response. As OFPC still stimulated increases in [Ca2+]i in 0% Ca2+ medium, the role of intracellular calcium was investigated. Hence, addition of thapsigargin (TG), an inhibitor of Ca2+-ATPase of the endoplasmic reticulum (ER), during the OFPC-induced peak response exerted an additive effect, indicating that the mechanism of action of these two agents are different. Furthermore, U73122, an inhibitor of IP3 production, completely abolished increases in [Ca2+]i, induced by OFPC, suggesting that these polyphenols induce the production of IP3 that recruits calcium from ER pool. Polyphenolic compounds do act extracellularly as addition of fatty acid-free bovine serum albumin (BSA) significantly diminished the rise in [Ca2+]i evoked by the formers. OFPC also induced plasma membrane hyperpolarisation which was reversed by addition of BSA. OFPC were found to curtail the expression of IL-2 mRNA and T-cell blastogenesis. Together these results suggest that OFPC induce increases in [Ca2+]i via ER pool and opening of CRAC channels, and exert immunosuppressive effects in Jurkat T-cells.

  5. Effect of chaetocin on renal cell carcinoma cells and cytokine-induced killer cells.

    PubMed

    Rombo, Roman; Weiher, Hans; Schmidt-Wolf, Ingo G H

    2016-01-01

    We examined the cytotoxic effects of chaetocin on clear cell renal cell carcinoma (ccRCC) cells and the possibility to combine the effects of chaetocin with the effects of cytokine-induced killer cells (CIK) assayed by MTT assay and FACS analysis. Chaetocin is a thiodioxopiperazine produced by fungi belonging to the chaetomiaceae family. In 2007, it was first reported that chaetocin shows potent and selective ex vivo anti-cancer activity by inducing reactive oxygen species. CIK cells are generated from CD3+/CD56- T lymphocytes with double negative CD4-/CD8- phenotype that are isolated from human blood. The addition of distinct interleukins and antibodies results in the generation of CIK cells that are able to specifically target and destroy renal carcinoma cells. The results of this research state that the anti-ccRCC activity of chaetocin is weak and does not show a high grade of selectivity on clear cell renal cell carcinoma cells. Although the CIK cells show a high grade of selective anti-ccRCC activity, this effect could not be improved by the addition of chaetocin. So chaetocin seems to be no suitable agent for specific targeting ccRCC cells or for the combination therapy with CIK cells in renal cancer.

  6. Effect of chaetocin on renal cell carcinoma cells and cytokine-induced killer cells

    PubMed Central

    Rombo, Roman; Weiher, Hans; Schmidt-Wolf, Ingo G.H.

    2016-01-01

    We examined the cytotoxic effects of chaetocin on clear cell renal cell carcinoma (ccRCC) cells and the possibility to combine the effects of chaetocin with the effects of cytokine-induced killer cells (CIK) assayed by MTT assay and FACS analysis. Chaetocin is a thiodioxopiperazine produced by fungi belonging to the chaetomiaceae family. In 2007, it was first reported that chaetocin shows potent and selective ex vivo anti-cancer activity by inducing reactive oxygen species. CIK cells are generated from CD3+/CD56- T lymphocytes with double negative CD4-/CD8- phenotype that are isolated from human blood. The addition of distinct interleukins and antibodies results in the generation of CIK cells that are able to specifically target and destroy renal carcinoma cells. The results of this research state that the anti-ccRCC activity of chaetocin is weak and does not show a high grade of selectivity on clear cell renal cell carcinoma cells. Although the CIK cells show a high grade of selective anti-ccRCC activity, this effect could not be improved by the addition of chaetocin. So chaetocin seems to be no suitable agent for specific targeting ccRCC cells or for the combination therapy with CIK cells in renal cancer. PMID:27141211

  7. Contribution of alpha4beta1 integrin to the antiallergic effect of levocabastine.

    PubMed

    Qasem, Ahmed R; Bucolo, Claudio; Baiula, Monica; Spartà, Antonino; Govoni, Paolo; Bedini, Andrea; Fascì, Domenico; Spampinato, Santi

    2008-09-15

    Levocabastine is an antiallergic drug acting as a histamine H1-receptor antagonist. In allergic conjunctivitis (AC), it may also antagonize up-regulation of the intercellular adhesion molecule-1 (ICAM-1) expressed on epithelial conjunctival cells. However, little is known about its effects on eosinophils, important effector cells in AC. The adhesion molecule integrin alpha(4)beta(1) is expressed in eosinophils; it interacts with the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) in vascular endothelial cells and contributes to eosinophil activation and infiltration in AC. This study provides evidence that in a scintillation proximity assay levocabastine (IC(50) 406 microM), but not the first-generation antihistamine chlorpheniramine, displaced (125)I-FN binding to human integrin alpha(4)beta(1) and, in flow cytometry analysis, levocabastine antagonized the binding of a primary antibody to integrin alpha(4) expressed on the Jurkat cell surface. Levocabastine, but not chlorpheniramine, binds the alpha(4)beta(1) integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vascular endothelial cells (HUVEC) in vitro. Similarly, levocabastine affects alpha(L)beta(2)/ICAM-1-mediated adhesion of Jurkat cells. In a model of AC levocabastine eye drops reduced the clinical aspects of the late-phase reaction and the conjunctival expression of alpha(4)beta(1) integrin by reducing infiltrated eosinophils. We propose that blockade of integrin-mediated cell adhesion might be a target of the antiallergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in AC.

  8. Cannabidiol Reduces Leukemic Cell Size – But Is It Important?

    PubMed Central

    Kalenderoglou, Nikoletta; Macpherson, Tara; Wright, Karen L.

    2017-01-01

    The anti-cancer effect of the plant-derived cannabinoid, cannabidiol, has been widely demonstrated both in vivo and in vitro. However, this body of preclinical work has not been translated into clinical use. Key issues around this failure can be related to narrow dose effects, the cell model used and incomplete efficacy. A model of acute lymphoblastic disease, the Jurkat T cell line, has been used extensively to study the cannabinoid system in the immune system and cannabinoid-induced apoptosis. Using these cells, this study sought to investigate the outcome of those remaining viable cells post-treatment with cannabidiol, both in terms of cell size and tracking any subsequent recovery. The phosphorylation status of the mammalian Target of Rapamycin (mTOR) signaling pathway and the downstream target ribosomal protein S6, were measured. The ability of cannabidiol to exert its effect on cell viability was also evaluated in physiological oxygen conditions. Cannabidiol reduced cell viability incompletely, and slowed the cell cycle with fewer cells in the G2/M phase of the cell cycle. Cannabidiol reduced phosphorylation of mTOR, PKB and S6 pathways related to survival and cell size. The remaining population of viable cells that were cultured in nutrient rich conditions post-treatment were able to proliferate, but did not recover to control cell numbers. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Finally, cells grown in 12% oxygen (physiological normoxia) were more resistant to cannabidiol. In conclusion, these results indicate that cannabidiol causes a reduction in cell size, which persists post-treatment. However, resistance to cannabidiol under physiological normoxia for these cells would imply that cannabidiol may not be useful in the clinic as an anti-leukemic agent. PMID

  9. Effects of ultrasound upon endothelial cell ultrastructure

    NASA Astrophysics Data System (ADS)

    Rodemer, Claus; Jenne, Jürgen; Fatar, Marc; Hennerici, Michael G.; Meairs, Stephen

    2012-11-01

    A number of new brain applications for therapeutic ultrasound are emerging including drug delivery through BBB opening, enhancement of angiogenesis, sonothrombolysis and neuromodulation. Safety remains important as alterations in the cytoskeleton and tight junctions of endothelial cells have been described. In this study we characterize the in vitro effects of ultrasound on cell morphology using a new human brain cell line (hCMEC/D3). Changes in ultrastructure were analyzed with antibodies against tubulin, actin and catenin. Transport was analyzed by measuring transferrin uptake. No significant changes were seen after continuous wave ultrasound treatment of hCMEC/D3 cells grown in Opticell{trade mark, serif} chambers. We could not observe disassembled actin stress fibers or variations in the microtubule network. However, severe damage occurred in cells cultured in petri dishes.

  10. Detection of cells captured with antigens on shear horizontal surface-acoustic-wave sensors.

    PubMed

    Hao, Hsu-Chao; Chang, Hwan-You; Wang, Tsung-Pao; Yao, Da-Jeng

    2013-02-01

    Techniques to separate cells are widely applied in immunology. The technique to separate a specific antigen on a microfluidic platform involves the use of a shear horizontal surface-acoustic-wave (SH-SAW) sensor. With specific antibodies conjugated onto the surface of the SH-SAW sensors, this technique can serve to identify specific cells in bodily fluids. Jurkat cells, used as a target in this work, provide a model of cells in small abundance (1:1000) for isolation and purification with the ultimate goal of targeting even more dilute cells. T cells were separated from a mixed-cell medium on a chip (Jurkat cells/K562 cells, 1/1000). A novel microchamber was developed to capture cells during the purification, which required a large biosample. Cell detection was demonstrated through the performance of genetic identification on the chip.

  11. Physiology of Continuous Bone Marrow Culture Derived Permanent Granulocyte-Macrophage Progenitor Cells

    DTIC Science & Technology

    1983-08-01

    capable of differentiating to mature neutrophillic granulocytes and granulocyte-macrophage progenitor cells . Several T- cell lines including K45, JURKAT...CEM, K230 have been screened for produc- tion of Interleukin-3 by assay of supernatant from cell lines for proliferation of mouse IL-3 dependent...hematopoietic progeni- tor cell lines Lines 45 and 230 produce low levels of activity. "In contrast, IL-2 (T- cell growth factor) dependent human T- cell

  12. Effect of glucocorticosteroids on epidermal Langerhans cells

    PubMed Central

    1982-01-01

    The effects of topical and systemic administration of various glucocorticoids on the density of epidermal Langerhans cells (LC) were studied in guinea pigs. Glucocorticoids, such as betamethasone dipropionate and valerate, caused a marked decrease in LC demonstrable by staining for cell membrane ATPase activity and Ia antigens. By electronmicroscopy, LC also showed morphologic alterations. The observed decrements in LC density correlated with the concentration and known vasoconstrictive potency of the glucocorticoids administered. The anti-inflammatory action of glucocorticoids in skin disorders may, at least in part, be through their ability to alter epidermal LC, thus interfering with the antigen-presenting functions of these cells. PMID:6459400

  13. Ethanol cytotoxic effect on trophoblast cells.

    PubMed

    Clave, S; Joya, X; Salat-Batlle, J; Garcia-Algar, O; Vall, O

    2014-03-03

    Prenatal ethanol exposure may cause both, altered fetal neurodevelopment and impaired placental function. These disturbances can lead to growth retardation, which is one of the most prevalent features in Fetal Alcohol Syndrome (FAS). It is not known whether there is a specific pattern of cytotoxicity caused by ethanol that can be extrapolated to other cell types. The aim of this study was to determine the cytotoxic effects caused by sustained exposure of trophoblast cells to ethanol. The cytotoxic effect of sustained exposure to standard doses of ethanol on an in vitro human trophoblast cell line, JEG3, was examined. Viable cell count by exclusion method, total protein concentration, lactate dehydrogenase (LDH) activity and activation of apoptotic markers (P-H2AX, caspase-3 and PARP-1) were determined. Sustained exposure to ethanol decreased viable cell count and total protein concentration. LDH activity did not increased in exposed cells but apoptotic markers were detected. In addition, there was a dose-dependent relationship between ethanol concentration and apoptotic pathways activation. Sustained ethanol exposure causes cellular cytotoxicity by apoptotic pathways induction as a result of DNA damage. This apoptotic induction may partially explain the altered function of placental cells and the damage previously detected in other tissues.

  14. Soluble Fas ligand released by colon adenocarcinoma cells induces host lymphocyte apoptosis: an active mode of immune evasion in colon cancer

    PubMed Central

    Song, E; Chen, J; Ouyang, N; Su, F; Wang, M; Heemann, U

    2001-01-01

    Expression of membrane-bound Fas ligand (mFasL) on colon cancer cells serves as a potential mechanism to inhibit host immune function by inducing apoptosis of host lymphocytes. Membrane-bound FasL can be cleaved and released as a soluble mediator (sFasL), which may spread the apoptosis induction effect. Our study examined whether colon adenocarcinoma cells release sFasL, and induce apoptosis of host lymphocytes without direct cell–cell contact. In 12 consecutive patients with colon adenocarcinoma mFasL was identified in the tumours, sFasL was measured in the sera and apoptosis identified in tumour-infiltrating and peripheral blood lymphocytes. To analyse the function of sFasL, colon cancer cells were primarily cultured; sFasL was isolated from supernatants, measured, incubated with Fas-bearing Jurkat cells, and the resulting apoptosis was analysed. Serum levels of sFasL were significantly elevated in all colon cancer patients with mFasL expression in tumour tissues (n = 8). In these patients, the number of apoptotic lymphocytes was significantly increased within tumour and peripheral blood. Furthermore, sFasL was present in the corresponding supernatants and induced apoptosis of Jurkat cells in a dose-dependent manner. These findings suggest that mFasL-positive colon cancer cells release sFasL, and thus may induce apoptosis of host lymphocytes as a potential mechanism for immune evasion. © 2001 Cancer Research Campaignhttp://www.bjcancer.com PMID:11592778

  15. T-Cell Activation under Hypoxic Conditions Enhances IFN-γ Secretion

    PubMed Central

    Roman, Jessica; Rangasamy, Tirumalai; Guo, Jia; Sugunan, Siva; Meednu, Nida; Packirisamy, Gopinath; Shimoda, Larissa A.; Golding, Amit; Semenza, Gregg; Georas, Steve N.

    2010-01-01

    Secondary lymphoid organs and peripheral tissues are characterized by hypoxic microenvironments, both in the steady state and during inflammation. Although hypoxia regulates T-cell metabolism and survival, very little is known about whether or how hypoxia influences T-cell activation. We stimulated mouse CD4+ T cells in vitro with antibodies directed against the T-cell receptor (CD3) and CD28 under normoxic (20% O2) and hypoxic (1% O2) conditions. Here we report that stimulation under hypoxic conditions augments the secretion of effector CD4+ T-cell cytokines, especially IFN-γ. The enhancing effects of hypoxia on IFN-γ secretion were independent of mouse strain, and were also unaffected using CD4+ T cells from mice lacking one copy of the gene encoding hypoxia-inducible factor-1α. Using T cells from IFN-γ receptor–deficient mice and promoter reporter studies in transiently transfected Jurkat T cells, we found that the enhancing effects of hypoxia on IFN-γ expression were not due to effects on IFN-γ consumption or proximal promoter activity. In contrast, deletion of the transcription factor, nuclear erythroid 2 p45–related factor 2 attenuated the enhancing effect of hypoxia on IFN-γ secretion and other cytokines. We conclude that hypoxia is a previously underappreciated modulator of effector cytokine secretion in CD4+ T cells. PMID:19372249

  16. Transduction of human T cells with a novel T-cell receptor confers anti-HCV reactivity.

    PubMed

    Zhang, Yi; Liu, Yeuying; Moxley, Kelly M; Golden-Mason, Lucy; Hughes, Michael G; Liu, Tongxin; Heemskerk, Mirjam H M; Rosen, Hugo R; Nishimura, Michael I

    2010-07-29

    Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world's population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073-1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4(+) and CD8(+) T cells recognized the HCV NS3:1073-1081 peptide-loaded targets and HCV(+) hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-gamma, IL-2, and TNF-alpha) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8(-) Jurkat cells and CD4(+) PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.

  17. Rapid acquisition of mean Raman spectra of eukaryotic cells for a robust single cell classification.

    PubMed

    Schie, Iwan W; Kiselev, Roman; Krafft, Christoph; Popp, Jürgen

    2016-11-14

    Raman spectroscopy has previously been used to identify eukaryotic and prokaryotic cells. While prokaryotic cells are small in size and can be assessed by a single Raman spectrum, the larger size of eukaryotic cells and their complex organization requires the acquisition of multiple Raman spectra to properly characterize them. A Raman spectrum from a diffraction-limited spot at an arbitrary location within a cell results in spectral variations that affect classification approaches. To probe whole cells with Raman imaging at high spatial resolution is time consuming, because a large number of Raman spectra need to be collected, resulting in low cell throughput and impairing statistical analysis due to low cell numbers. Here we propose a method to overcome the effects of cellular heterogeneity by acquiring integrated Raman spectra covering a large portion of a cell. The acquired spectrum represents the mean macromolecular composition of a cell with an exposure time that is comparable to acquisition of a single Raman spectrum. Data sets were collected from T lymphocyte Jurkat cells, and pancreatic cell lines Capan1 and MiaPaca2. Cell classification by support vector machines was compared for single spectra, spectra of images and integrated Raman spectra of cells. The integrated approach provides better and more stable prediction for individual cells, and in the current implementation, the mean macromolecular information of a cell can be acquired faster than with the acquisition of individual spectra from a comparable region. It is expected that this approach will have a major impact on the implementation of Raman based cell classification.

  18. Simulated Red Blood Cell Motion in Microvessel Bifurcations: Effects of Cell-Cell Interactions on Cell Partitioning

    PubMed Central

    Barber, Jared O.; Restrepo, Juan M.; Secomb, Timothy W.

    2013-01-01

    Partitioning of red blood cell (RBC) fluxes between the branches of a diverging microvessel bifurcation is generally not proportional to the flow rates, as RBCs preferentially enter the higher-flow branch. A two-dimensional model for RBC motion and deformation is used to investigate the effects of cell-cell mechanical interactions on RBC partitioning in bifurcations. The RBC membrane and cytoplasm are represented by sets of viscoelastic elements immersed in a low Reynolds number flow. Several types of two-cell interactions that can affect partitioning are found. In the most frequent interactions, a `trade-off' occurs, in which a cell entering one branch causes a following cell to enter the other branch. Other types of interactions include `herding,' where the leading cell is caused to enter the same branch as the following cell, and `following,' where the trailing cell is caused to enter the same branch as the leading cell. The combined effect of these cell-cell interactions is a tendency towards more uniform partitioning, which results from the trade-off effect but is reduced by the herding and following effects. With increasing hematocrit, the frequency of interactions increases, and more uniform partitioning results. This prediction is consistent with experimental observations on how hematocrit affects RBC partitioning. PMID:23555330

  19. Normal Development and Function of T Cells in Proline Rich 7 (Prr7) Deficient Mice

    PubMed Central

    Hrdinka, Matous; Sudan, Kritika; Just, Sissy; Drobek, Ales; Stepanek, Ondrej; Schlüter, Dirk; Reinhold, Dirk; Jordan, Bryen A.; Gintschel, Patricia; Schraven, Burkhart; Kreutz, Michael R.

    2016-01-01

    Transmembrane adaptor proteins (TRAPs) are important organisers for the transduction of immunoreceptor-mediated signals. Prr7 is a TRAP that regulates T cell receptor (TCR) signalling and potently induces cell death when overexpressed in human Jurkat T cells. Whether endogenous Prr7 has a similar functional role is currently unknown. To address this issue, we analysed the development and function of the immune system in Prr7 knockout mice. We found that loss of Prr7 partially impairs development of single positive CD4+ T cells in the thymus but has no effect on the development of other T cell subpopulations, B cells, NK cells, or NKT cells. Moreover, Prr7 does not affect the TCR signalling pathway as T cells derived from Prr7 knockout and wild-type animals and stimulated in vitro express the same levels of the activation marker CD69, and retain their ability to proliferate and activate induced cell death programs. Importantly, Prr7 knockout mice retained the capacity to mount a protective immune response when challenged with Listeria monocytogenes infection in vivo. In addition, T cell effector functions (activation, migration, and reactivation) were normal following induction of experimental autoimmune encephalomyelitis (EAE) in Prr7 knockout mice. Collectively, our work shows that loss of Prr7 does not result in a major immune system phenotype and suggests that Prr7 has a dispensable function for TCR signalling. PMID:27657535

  20. Adrenal chromaffin cells do not swell when exposed to nanosecond electric pulses.

    PubMed

    Craviso, Gale L; Fisher, Christa; Chatterjee, Indira; Vernier, P Thomas

    2015-06-01

    High intensity, nanosecond duration electric pulses (NEPs) permeabilize plasma membranes causing osmotic cell swelling that can elicit a wide variety of cellular effects. This study examined the possibility that cell swelling is the mechanism by which 5 ns NEPs trigger the release of catecholamines from neuroendocrine adrenal chromaffin cells. Swelling was assessed by comparing measurements of cell area obtained from bright field images of the cells before and at 10s intervals following exposure of the cells to 5 ns pulses at a field intensity of 5-6 MV/m. The results indicated that chromaffin cells do not swell in response to a single pulse or a train of ten pulses delivered at repetition frequencies of 10 Hz and 1 kHz. Swelling was also not observed in response to a train of 50 pulses whereas Jurkat T lymphoblast cell area increased 15% on average under the same NEP exposure conditions. These results demonstrating that chromaffin cells do not undergo swelling when exposed to 5 ns NEPs have important implications regarding the mechanism by which these pulses stimulate the release of catecholamines from these cells, namely that catecholamine secretion is most likely not caused by cell swelling.

  1. Ceramide mediates caspase-independent programmed cell death.

    PubMed

    Thon, Lutz; Möhlig, Heike; Mathieu, Sabine; Lange, Arne; Bulanova, Elena; Winoto-Morbach, Supandi; Schütze, Stefan; Bulfone-Paus, Silvia; Adam, Dieter

    2005-12-01

    Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase-dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase-independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase-independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor-interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase-independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl-2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.

  2. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation

    PubMed Central

    Huynh, Mai-Lan N.; Fadok, Valerie A.; Henson, Peter M.

    2002-01-01

    Ingestion of apoptotic cells in vitro by macrophages induces TGF-β1 secretion, resulting in an anti-inflammatory effect and suppression of proinflammatory mediators. Here, we show in vivo that direct instillation of apoptotic cells enhanced the resolution of acute inflammation. This enhancement appeared to require phosphatidylserine (PS) on the apoptotic cells and local induction of TGF-β1. Working with thioglycollate-stimulated peritonea or LPS-stimulated lungs, we examined the effect of apoptotic cell uptake on TGF-β1 induction. Viable or opsonized apoptotic human Jurkat T cells, or apoptotic PLB-985 cells, human monomyelocytes that do not express PS during apoptosis, failed to induce TGF-β1. PS liposomes, or PS directly transferred onto the PLB-985 surface membranes, restored the TGF-β1 induction. Apoptotic cell instillation into LPS-stimulated lungs reduced proinflammatory chemokine levels in the bronchoalveolar lavage fluid (BALF). Additionally, total inflammatory cell counts in the BALF were markedly reduced 1–5 days after apoptotic cell instillation, an effect that could be reversed by opsonization or coinstillation of TGF-β1 neutralizing antibody. This reduction resulted from early decrease in neutrophils and later decreases in lymphocytes and macrophages. In conclusion, apoptotic cell recognition and clearance, via exposure of PS and ligation of its receptor, induce TGF-β1 secretion, resulting in accelerated resolution of inflammation. PMID:11781349

  3. Cytotoxic and pro-apoptotic activities of cynaropicrin, a sesquiterpene lactone, on the viability of leukocyte cancer cell lines.

    PubMed

    Cho, Jae Youl; Kim, Ae Ra; Jung, Jee H; Chun, Taehoon; Rhee, Man Hee; Yoo, Eun Sook

    2004-05-25

    Cynaropicrin, a sesquiterpene lactone from Saussurea lappa, has been reported to possess immunomodulatory effects on cytokine release, nitric oxide production and immunosuppressive effects. In this study, we have examined cytotoxic effect of cynaropicrin against several types of cell lines such as macrophages, eosinophils, fibroblasts and lymphocytes. Cynaropicrin potently inhibited the proliferation of leukocyte cancer cell lines, such as U937, Eol-1 and Jurkat T cells, but some other cells such as Chang liver cells and human fibroblast cell lines were not strongly suppressed by cynaropicrin treatment. The cytotoxic effect of cynaropicrin was due to inducing apoptosis and cell cycle arrest at G1/S phase, according to flow-cytometric, DNA fragmentation and morphological analyses using U937 cells. Evidence that combination treatment with l-cysteine and N-acetyl-l-cysteine, reactive oxygen species scavengers, or rottlerin (1-[6-[(3-acetyl-2,4,6-trihydroxy-5-methylphenyl)methyl]-5,7-dihydroxy-2, 2-dimethyl-2H-1-benzopyran-8-yl]-3-phenyl-2-propen-1-one), a specific protein kinase (PK) Cdelta inhibitor, abolished cynaropicrin-mediated cytotoxicity and morphological change, and that cynaropicrin-induced proteolytic cleavage of PKCdelta suggests that reactive oxygen species and PKCdelta may play an important role in mediating pro-apoptotic activity by cynaropicrin. Taken together, these results indicate that cynaropicrin may be a potential anticancer agent against some leukocyte cancer cells such as lymphoma or leukemia, through pro-apoptotic activity.

  4. Effect of Carbon Nanotubes on Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Chen, Michelle; Ahmed, Asma; Black, Melanie; Kawamoto, Nicole; Lucas, Jessica; Pagala, Armie; Pham, Tram; Stankiewicz, Sara; Chen, Howard

    2010-03-01

    Carbon Nanotubes possess extraordinary electrical, mechanical, and thermal properties. Research on applying the carbon nanotubes for ultrasensitive detection, disease diagnosis, and drug delivery is rapidly developing. While the fundamental and technological findings on carbon nanotubes show great promise, it is extremely important to investigate the effect of the carbon nanotubes on human health. In our experiments, we introduce purified carbon nanotubes in suspension to ovary cells cultured from Hamsters. These cells are chosen since they show robust morphological changes associated with cytotoxicity that can easily be observed under a light microscope. We will discuss the toxicity of carbon nanotubes by characterizing the cell morphology and viability as a function of time and the concentration of carbon nanotube suspension.

  5. Photo-Induced Effect on Bacterial Cells

    NASA Astrophysics Data System (ADS)

    El Batanouny, M. H.; Amin, Rehab M.; Naga, M. I.; Ibrahim, M. K.

    2010-04-01

    Bacterial resistance against antibiotics is an increasing problem in medicine. This stimulates study of other bactericidal regimens, one of which is photodynamic therapy (PDT), which involves the killing of bacterial species by low power laser light (LLL) in the presence of photosensitizing agent. It has already been shown that, various gram- negative and gram-positive bacteria can be killed by photodynamic therapy in vitro, using exogenous sensitizers. The mechanisms of laser action on bacteria are not adequately understood. Here, PDT on H. pylori, as an example of gram negative bacteria was studied. The ultra structure changes of the organism after PDT were examined under electron microscope. Neither Irradiation with laser without sensitizer nor sensitizing without laser has any lethal effect on bacterial cells. However, the successful lethal photosensitization was achieved by applying certain laser dose with the corresponding concentration of the photosensitizer. On the other hand, PDT has no significant effect on the genomic DNA of the cells.

  6. Analysis of CCR7 mediated T cell transfectant migration using a microfluidic gradient generator.

    PubMed

    Wu, Xun; Wu, Jiandong; Li, Hongzhao; Legler, Daniel F; Marshall, Aaron J; Lin, Francis

    2015-04-01

    T lymphocyte migration is crucial for adaptive immunity. Manipulation of signaling molecules controlling cell migration combined with in-vitro cell migration analysis provides a powerful research approach. Microfluidic devices, which can precisely configure chemoattractant gradients and allow quantitative single cell analysis, have been increasingly applied to cell migration and chemotaxis studies. However, there are a very limited number of published studies involving microfluidic migration analysis of genetically manipulated immune cells. In this study, we describe a simple microfluidic method for quantitative analysis of T cells expressing transfected chemokine receptors and other cell migration signaling probes. Using this method, we demonstrated chemotaxis of Jurkat transfectants expressing wild-type or C-terminus mutated CCR7 within a gradient of chemokine CCL19, and characterized the difference in transfectant migration mediated by wild-type and mutant CCR7. The EGFP-tagged CCR7 allows identification of CCR7-expressing transfectants in cell migration analysis and microscopy assessment of CCR7 dynamics. Collectively, our study demonstrated the effective use of the microfluidic method for studying CCR7 mediated T cell transfectant migration. We envision this developed method will provide a useful platform to functionally test various signaling mechanisms at the cell migration level.

  7. Effect of puerarin on human choriocarcinoma cells

    PubMed Central

    Lidao, Bao; Yi, Wang; Ruilian, Ma; Xianhua, Ren; Agula, B

    2015-01-01

    Objective To discuss the effect of puerarin on human choriocarcinoma cells. Methods Survival rates under puerarin monotherapy, fluorouracil (5-FU) monotherapy and puerarin in combination with 5-FU were detected by MTT assay. Apoptotic morphology was observed with Hoechst 33258 staining. Apoptosis rates were detected with flow cytometry. Expressions of AKT, mechanistic target of rapamycin (mTOR), and P70S6K mRNAs and phosphorylated proteins were detected by RT-PCR and Western blot. Tumor-bearing mice were administered puerarin and puerarin+5-FU, and serum levels of β-human chorionic gonadotropin (β-HCG) were measured. Results Proliferation inhibition and apoptosis rates of JEG-3 cells were positively correlated with puerarin concentration, which increased in the puerarin+5-FU group. Expression levels of AKT, mTOR, P70S6K mRNAs, and phosphorylated proteins decreased significantly after action of puerarin at different concentrations. With increasing puerarin concentration, expression of cleaved-caspase-3 in JEG-3 cells increased, whereas that of Bcl-2 decreased. Puerarin significantly inhibited tumor growth in choriocarcinoma-bearing SCID mice. Serum β-HCG levels were significantly lower than those of control group after administration. Magnitude of β-HCG decline was positively correlated with concentration.. Conclusion Puerarin+5-FU inhibited proliferation of JEG-3 choriocarcinoma cells and promoted their apoptosis, being associated with the mTOR signaling pathway. PMID:28352705

  8. Plantaricin A, a peptide pheromone produced by Lactobacillus plantarum, permeabilizes the cell membrane of both normal and cancerous lymphocytes and neuronal cells.

    PubMed

    Sand, Sverre L; Oppegård, Camilla; Ohara, Shinya; Iijima, Toshio; Naderi, Soheil; Blomhoff, Heidi K; Nissen-Meyer, Jon; Sand, Olav

    2010-07-01

    Antimicrobial peptides produced by multicellular organisms protect against pathogenic microorganisms, whereas such peptides produced by bacteria provide an ecological advantage over competitors. Certain antimicrobial peptides of metazoan origin are also toxic to eukaryotic cells, with preference for a variety of cancerous cells. Plantaricin A (PlnA) is a peptide pheromone with membrane permeabilizing strain-specific antibacterial activity, produced by Lactobacillus plantarum C11. Recently, we have reported that PlnA also permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant. To investigate if preferential effect on cancerous cells is a general feature of PlnA, we have studied effects of the peptide on normal and cancerous lymphocytes and neuronal cells. Normal human B and T cells, Reh cells (from human B cell leukemia), and Jurkat cells (from human T cell leukemia) were studied by flow cytometry to detect morphological changes (scatter) and viability (propidium iodide uptake), and by patch clamp recordings to monitor membrane conductance. Ca(2+) imaging based on a combination of fluo-4 and fura-red was used to monitor PlnA-induced membrane permeabilization in normal rat cortical neurons and glial cells, PC12 cells (from a rat adrenal chromaffin tumor), and murine N2A cells (from a spinal cord tumor). All the tested cell types were affected by 10-100 microM PlnA, whereas concentrations below 10 microM had no significant effect. We conclude that normal and cancerous lymphocytes and neuronal cells show similar sensitivity to PlnA.

  9. Herbal Formulation C168 Attenuates Proliferation and Induces Apoptosis in HCT 116 Human Colorectal Carcinoma Cells: Role of Oxidative Stress and DNA Damage

    PubMed Central

    Leong, Lek Mun; Chan, Kok Meng; Hamid, Asmah; Latip, Jalifah; Rajab, Nor Fadilah

    2016-01-01

    The use of herbal formulations has gained scientific interest, particularly in cancer treatment. In this study, the herbal formulation of interest, denoted as C168, is a mixture of eight genera of plants. This study aims to investigate the antiproliferative effect of C168 methanol extract (CME) on various cancer cells and its underlying mechanism of action on the most responsive cell line, namely, HCT 116 cells. CME exerted antiproliferative activities on HCT 116 colorectal carcinoma cells and HepG2 hepatocellular carcinoma cells but not on CCD-841-CoN normal colon epithelial cells, Jurkat E6.1 lymphoblastic leukemic cells, and V79-4 Chinese hamster lung fibroblasts. Further investigation on HCT 116 cells showed that CME induced G2/M cell-cycle arrest and apoptosis. Treatment of CME induced oxidative stress in HCT 116 cells by increasing the superoxide anion level and decreasing the intracellular glutathione. CME also increased tail moment value and H2AX phosphorylation in HCT 116 cells, suggesting DNA damage as an early signal of CME induced apoptosis. Loss of mitochondrial membrane potential in CME-treated cells also indicated the involvement of mitochondria in CME induced apoptosis. This study indicated the selectivity of CME toward colon cancer cells with the involvement of oxidative damage as its possible mechanism of action. PMID:26884792

  10. Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses

    PubMed Central

    Ibey, Bennett L.; Ullery, Jody; Pakhomova, Olga N.; Roth, Caleb C.; Semenov, Iurri; Beier, Hope T.; Tarango, Melissa; Xiao, Shu; Schoenbach, Karl; Pakhomov, Andrei G.

    2014-01-01

    Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, Propidium Iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (15 minutes) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF. PMID:24332942

  11. Pantethine Alters Lipid Composition and Cholesterol Content of Membrane Rafts, With Down-Regulation of CXCL12-Induced T Cell Migration.

    PubMed

    van Gijsel-Bonnello, Manuel; Acar, Niyazi; Molino, Yves; Bretillon, Lionel; Khrestchatisky, Michel; de Reggi, Max; Gharib, Bouchra

    2015-10-01

    Pantethine, a natural low-molecular-weight thiol, shows a broad activity in a large range of essential cellular pathways. It has been long known as a hypolipidemic and hypocholesterolemic agent. We have recently shown that it exerts a neuroprotective action in mouse models of cerebral malaria and Parkinson's disease through multiple mechanisms. In the present study, we looked at its effects on membrane lipid rafts that serve as platforms for molecules engaged in cell activity, therefore providing a target against inappropriate cell response leading to a chronic inflammation. We found that pantethine-treated cells showed a significant change in raft fatty acid composition and cholesterol content, with ultimate downregulation of cell adhesion, CXCL12-driven chemotaxis, and transendothelial migration of various T cell types, including human Jurkat cell line and circulating effector T cells. The mechanisms involved include the alteration of the following: (i) CXCL12 binding to its target cells; (ii) membrane dynamics of CXCR4 and CXCR7, the two CXCL12 receptors; and (iii) cell redox status, a crucial determinant in the regulation of the chemokine system. In addition, we considered the linker for activation of T cells molecule to show that pantethine effects were associated with the displacement from the rafts of the acylated signaling molecules which had their palmitoylation level reduced.. In conclusion, the results presented here, together with previously published findings, indicate that due to its pleiotropic action, pantethine can downregulate the multifaceted process leading to pathogenic T cell activation and migration.

  12. Immunoinhibitory adapter protein Src homology domain 3 lymphocyte protein 2 (SLy2) regulates actin dynamics and B cell spreading.

    PubMed

    von Holleben, Max; Gohla, Antje; Janssen, Klaus-Peter; Iritani, Brian M; Beer-Hammer, Sandra

    2011-04-15

    Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.

  13. Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells

    PubMed Central

    Ventoso, Iván; Kochetov, Alex; Montaner, David; Dopazo, Joaquín; Santoyo, Javier

    2012-01-01

    In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed. PMID

  14. Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells

    NASA Astrophysics Data System (ADS)

    Su, Xuantao; Qiu, Yuanyuan; Marquez-Curtis, Leah; Gupta, Manisha; Capjack, Clarence E.; Rozmus, Wojciech; Janowska-Wieczorek, Anna; Tsui, Ying Y.

    2011-06-01

    A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

  15. Molecular Characterization of a Fully Human Chimeric T-Cell Antigen Receptor for Tumor-Associated Antigen EpCAM

    PubMed Central

    Shirasu, Naoto; Yamada, Hiromi; Shibaguchi, Hirotomo; Kuroki, Motomu; Kuroki, Masahide

    2012-01-01

    The transduction of T cells to express chimeric T-cell antigen receptor (CAR) is an attractive strategy for adaptive immunotherapy for cancer, because the CAR can redirect the recognition specificity of T cells to tumor-associated antigens (TAAs) on the surface of target cells, thereby avoiding the limitations of HLA restriction. However, there are considerable problems with the clinical application of CAR, mostly due to its xenogeneic components, which could be immunogenic in humans. Moreover, while extensive studies on the CARs have been performed, the detailed molecular mechanisms underlying the activation of CAR-grafted T cells remain unclear. In order to eliminate potential immunogenicity and investigate the molecular basis of the CAR-mediated T-cell activation, we constructed a novel CAR (CAR57-28ζ) specific for one of the most important TAAs, epithelial cell adhesion molecule (EpCAM), using only human-derived genes. We revealed that in Jurkat T cells, lentivirally expressed CAR57-28ζ can transmit the T-cell-activating signals sufficient to induce IL-2 production upon EpCAM stimulation. An immunofluorescent analysis clearly showed that the CAR57-28ζ induces the formation of signaling clusters containing endogenous CD3ζ at the CAR/EpCAM interaction interface. These results suggest that this CAR gene may be safely and effectively applied for adaptive T-cell immunotherapy. PMID:22547929

  16. Oleyl group-functionalized insulating gate transistors for measuring extracellular pH of floating cells

    PubMed Central

    Imaizumi, Yuki; Goda, Tatsuro; Toya, Yutaro; Matsumoto, Akira; Miyahara, Yuji

    2016-01-01

    Abstract The extracellular ionic microenvironment has a close relationship to biological activities such as by cellular respiration, cancer development, and immune response. A system composed of ion-sensitive field-effect transistors (ISFET), cells, and program-controlled fluidics has enabled the acquisition of real-time information about the integrity of the cell membrane via pH measurement. Here we aimed to extend this system toward floating cells such as T lymphocytes for investigating complement activation and pharmacokinetics through alternations in the plasma membrane integrity. We functionalized the surface of tantalum oxide gate insulator of ISFET with oleyl-tethered phosphonic acid for interacting with the plasma membranes of floating cells without affecting the cell signaling. The surface modification was characterized by X-ray photoelectron spectroscopy and water contact angle measurements. The Nernst response of −37.8 mV/pH was obtained for the surface-modified ISFET at 37 °C. The oleyl group-functionalized gate insulator successfully captured Jurkat T cells in a fluidic condition without acute cytotoxicity. The system was able to record the time course of pH changes at the cells/ISFET interface during the process of instant addition and withdrawal of ammonium chloride. Further, the plasma membrane injury of floating cells after exposure by detergent Triton™ X-100 was successfully determined using the modified ISFET with enhanced sensitivity as compared with conventional hemolysis assays. PMID:27877886

  17. A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death

    PubMed Central

    Furness, SGB; Hare, DL; Kourakis, A; Turnley, AM; Wookey, PJ

    2016-01-01

    We have discovered that the accumulation of an anti-calcitonin receptor (anti-CTR) antibody conjugated to a fluorophore (mAb2C4:AF568) provides a robust signal for cells undergoing apoptotic programmed cell death (PCD). PCD is an absolute requirement for normal development of metazoan organisms. PCD is a hallmark of common diseases such as cardiovascular disease and tissue rejection in graft versus host pathologies, and chemotherapeutics work by increasing PCD. This robust signal or high fluorescent events were verified by confocal microscopy and flow cytometry in several cell lines and a primary culture in which PCD had been induced. In Jurkat cells, GBM-L2 and MG63 cells, the percentage undergoing PCD that were positive for both mAb2C4:AF568 and annexin V ranged between 70 and >90%. In MG63 cells induced for the preapoptotic cell stress response (PACSR), the normal expression of α-tubulin, a key structural component of the cytoskeleton, and accumulation of mAb2C4:AF568 were mutually exclusive. Our data support a model in which CTR is upregulated during PACSR and recycles to the plasma membrane with apoptosis. In cells committed to apoptosis (α-tubulin negative), there is accumulation of the CTR-ligand mAb2C4:AF568 generating a high fluorescent event. The reagent mAb2C4:AF568 effectively identifies a novel event linked to apoptosis. PMID:27777788

  18. Cell Biology of Thiazide Bone Effects

    NASA Astrophysics Data System (ADS)

    Gamba, Gerardo; Riccardi, Daniela

    2008-09-01

    The thiazide-sensitive Na+:Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian kidney. The activity of NCC is not only related to salt metabolism, but also to calcium and magnesium homeostasis due to the inverse relationship between NCC activity and calcium reabsorption. Hence, the thiazide-type diuretics that specifically block NCC have been used for years, not only for treatment of hypertension and edematous disease, but also for the management of renal stone disease. Epidemiological studies have shown that chronic thiazide treatment is associated with higher bone mineral density and reduced risk of bone fractures, which can only partly be explained in terms of their effects on the kidney. In this regard, we have recently shown that NCC is expressed in bone cells and that inhibition of NCC in bone, either by thiazides or by reduction of NCC protein with specific siRNA, is associated with increased mineralization in vitro. These observations open a field of study to begin to understand the cell biology of the beneficial effects of thiazides in bone.

  19. Hemodynamic effects of red blood cell aggregation.

    PubMed

    Baskurt, Oguz K; Meiselman, Herbert J

    2007-01-01

    The influence of red blood cell (RBC) aggregation on blood flow in vivo has been under debate since early 1900's, yet a full understanding has still has not been reached. Enhanced RBC aggregation is well known to increase blood viscosity measured in rotational viscometers. However, it has been demonstrated that RBC aggregation may decrease flow resistance in cylindrical tubes, due to the formation of a cell-poor zone near the tube wall which results from the enhanced central accumulation of RBC. There is also extensive discussion regarding the effects of RBC aggregation on in vivo blood flow resistance. Several groups have reported increased microcirculatory flow resistance with enhanced RBC aggregation in experiments that utilized intravital microscopy. Alternatively, whole organ studies revealed that flow resistance may be significantly decreased if RBC aggregation is enhanced. Recently, new techniques have been developed to achieve well-controlled, graded alterations in RBC aggregation without influencing suspending phase properties. Studies using this technique revealed that the effects of RBC aggregation are determined by the degree of aggregation changes, and that this relationship can be explained by different hemodynamic mechanisms.

  20. The effects of fluoride on cell migration, cell proliferation, and cell metabolism in GH4C1 pituitary tumour cells.

    PubMed

    Mendoza-Schulz, A; Solano-Agama, C; Arreola-Mendoza, L; Reyes-Márquez, B; Barbier, O; Del Razo, L M; Mendoza-Garrido, M E

    2009-10-28

    The consumption of drinking water rich in fluoride has toxic effects on the central nervous system. In cell biology research, fluoride is currently used as a phosphatase inhibitor. The aim of the present study was to evaluate the effect of fluoride on different physiological processes in GH4C1 pituitary tumour cells. We used a range of different fluoride concentrations, from levels below normal human serum concentrations (0.23 and 1.2 micromol/L) to those observed in chronically exposed persons (10.7 micromol/L) and above (107 and 1072 micromol/L). Treatment of 10.7 micromol/L fluoride resulted in a discrete induction of DNA synthesis, without a change in cell number. Cell migration, a behaviour stimulated by growth factors, was increased in cells treated with 2.4 micromol/L. At this fluoride concentration, changes in phosphorylation status of both cytoskeletal and cytosolic protein fractions, as well as in actin cytoskeletal arrangements were observed. The GH4C1 fluoride treated cells had significantly less cellular protein than control cells, suggesting an effect of fluoride on hormone secretion and protein synthesis in this endocrine cell. The bioreduction of MTT was significantly increased with a wide range of fluoride concentrations. With the highest fluoride concentration, 1072 micromol/L, all of the analysed parameters were significantly reduced, suggesting that this dose is highly toxic in GH4C1 cells. Our results show that biologically relevant concentrations of fluoride are capable of increasing cell migration in tumour cells, suggesting that exposure to fluoride could stimulate tumour invasion.

  1. Restoration of WNT4 inhibits cell growth in leukemia-derived cell lines

    PubMed Central

    2013-01-01

    Background WNT signaling pathways are significantly altered during cancer development. Vertebrates possess two classes of WNT signaling pathways: the “canonical” WNT/β-catenin signaling pathway, and the “non-canonical” pathways including WNT/Ca2+ and WNT/Planar cell polarity [PCP] signaling. WNT4 influences hematopoietic progenitor cell expansion and survival; however, WNT4 function in cancer development and the resulting implications for oncogenesis are poorly understood. The aim of this study was twofold: first, to determine the expression of WNT4 in mature peripheral blood cells and diverse leukemia-derived cells including cell lines from hematopoietic neoplasms and cells from patients with leukemia; second, to identify the effect of this ligand on the proliferation and apoptosis of the blast-derived cell lines BJAB, Jurkat, CEM, K562, and HL60. Methods We determined WNT4 expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) and T- and B-lymphocytes from healthy individuals, as well as from five leukemia-derived cell lines and blasts derived from patients with leukemia. To analyze the effect of WNT4 on cell proliferation, PBMCs and cell lines were exposed to a commercially available WNT4 recombinant human protein. Furthermore, WNT4 expression was restored in BJAB cells using an inducible lentiviral expression system. Cell viability and proliferation were measured by the addition of WST-1 to cell cultures and counting cells; in addition, the progression of the cell cycle and the amount of apoptosis were analyzed in the absence or presence of WNT4. Finally, the expression of WNT-pathway target genes was measured by qRT-PCR. Results WNT4 expression was severely reduced in leukemia-derived cell lines and blasts derived from patients with leukemia. The exposure of cell lines to WNT4 recombinant protein significantly inhibited cell proliferation; inducing WNT4 expression in BJAB

  2. Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines

    PubMed Central

    Montaner, Beatriz; Navarro, Sira; Piqué, Maria; Vilaseca, Marta; Martinell, Marc; Giralt, Ernest; Gil, Joan; Pérez-Tomás, Ricardo

    2000-01-01

    The effects of supernatant from the bacterial strain Serratia marcescens 2170 (CS-2170) on the viability of different haematopoietic cancer cell lines (Jurkat, NSO, HL-60 and Ramos) and nonmalignant cells (NIH-3T3 and MDCK) was studied. We examined whether this cytotoxic effect was due to apoptosis, and we purified the molecule responsible for this effect and determined its chemical structure.Using an MTT assay we showed a rapid (4 h) decrease in the number of viable cells. This cytotoxic effect was due to apoptosis, according to the fragmentation pattern of DNA, Hoechst 33342 staining and FACS analysis of the phosphatidylserine externalization. This apoptosis was blocked by using the caspase inhibitor Z-VAD.fmk, indicating the involvement of caspases.Prodigiosin is a red pigment produced by various bacteria including S. marcescens. Using mutants of S. marcescens (OF, WF and 933) that do not synthesize prodigiosin, we further showed that prodigiosin is involved in this apoptosis. This evidence was corroborated by spectroscopic analysis of prodigiosin isolated from S. marcescens.These results indicate that prodigiosin, an immunosuppressor, induces apoptosis in haematopoietic cancer cells with no marked toxicity in nonmalignant cells, raising the possibility of its therapeutic use as an antineoplastic drug. PMID:11015311

  3. G2/M Cell Cycle Arrest and Tumor Selective Apoptosis of Acute Leukemia Cells by a Promising Benzophenone Thiosemicarbazone Compound

    PubMed Central

    Cabrera, Maia; Gomez, Natalia; Remes Lenicov, Federico; Echeverría, Emiliana; Shayo, Carina; Moglioni, Albertina; Fernández, Natalia; Davio, Carlos

    2015-01-01

    Anti-mitotic therapies have been considered a hallmark in strategies against abnormally proliferating cells. Focusing on the extensively studied family of thiosemicarbazone (TSC) compounds, we have previously identified 4,4’-dimethoxybenzophenone thiosemicarbazone (T44Bf) as a promising pharmacological compound in a panel of human leukemia cell lines (HL60, U937, KG1a and Jurkat). Present findings indicate that T44Bf-mediated antiproliferative effects are associated with a reversible chronic mitotic arrest caused by defects in chromosome alignment, followed by induced programmed cell death. Furthermore, T44Bf selectively induces apoptosis in leukemia cell lines when compared to normal peripheral blood mononuclear cells. The underlying mechanism of action involves the activation of the mitochondria signaling pathway, with loss of mitochondrial membrane potential and sustained phosphorylation of anti-apoptotic protein Bcl-xL as well as increased Bcl-2 (enhanced phosphorylated fraction) and pro-apoptotic protein Bad levels. In addition, ERK signaling pathway activation was found to be a requisite for T44Bf apoptotic activity. Our findings further describe a novel activity for a benzophenone thiosemicarbazone and propose T44Bf as a promising anti-mitotic prototype to develop chemotherapeutic agents to treat acute leukemia malignancies. PMID:26360247

  4. New betulinic acid derivatives induce potent and selective antiproliferative activity through cell cycle arrest at the S phase and caspase dependent apoptosis in human cancer cells.

    PubMed

    Santos, Rita C; Salvador, Jorge A R; Cortés, Roldán; Pachón, Gisela; Marín, Silvia; Cascante, Marta

    2011-06-01

    New semisynthetic derivatives of betulinic acid (BA) RS01, RS02 and RS03 with 18-45 times improved cytotoxic activity against HepG2 cells, were tested for their ability to induce apoptosis and cell cycle arrest in HepG2, HeLa and Jurkat cells. All the compounds induced significant increase in the population at the S phase more effectively than BA. RS01, RS02 and RS03 were also found to be potent inducers of apoptosis with RS01 being markedly more potent than BA, suggesting that the introduction of the imidazolyl moiety is crucial for enhancing the induction of apoptosis and the cell cycle arrest. The mechanism of apoptosis induction has been studied in HepG2 cells and found to be mediated by activation of the postmitochondrial caspases-9 and -3 cascade and possibly by mitochondrial amplification loop involving caspase-8. These facts were corroborated by detection of mitochondrial cytochrome c release and DNA fragmentation. Because RS01, RS02 and RS03 exhibited significant improved antitumor activity with respect to BA, they may be promising new agents for the treatment of cancer. In particular, RS01 is the most promising compound with an IC(50) value 45 times lower than BA on HepG2 cells and 61 times lower than the one found for the non-tumoral Chang liver cells.

  5. Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element.

    PubMed Central

    Low, K G; Chu, H M; Schwartz, P M; Daniels, G M; Melner, M H; Comb, M J

    1994-01-01

    Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes. This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate. Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation. Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription. Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation. In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A. Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax. Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3. Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax. Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax. Images PMID:8007991

  6. Lysophosphatidic acid possesses dual action in cell proliferation.

    PubMed Central

    Tigyi, G; Dyer, D L; Miledi, R

    1994-01-01

    Lysophosphatidic acid (LPA) induces mitogenic responses in cultured fibroblasts through a pertussis toxin-sensitive signaling pathway. In contrast, we have shown that LPA inhibits the proliferation of Sp2/0-Ag14 myeloma cells. To resolve this apparent controversy, LPA-elicited responses in cell proliferation and the underlying second messenger mechanisms were compared in Sp2/0-Ag14 myeloma and NIH 3T3 fibroblast cells. The antimitogenic response was not elicited by micromolar concentrations of phosphatidic acid, phosphatidylglycerol, or diacylglycerol. In NIH 3T3 and Sp2 cells, LPA elicited an increase in inositol trisphosphate and a subsequent transient increase in free cytoplasmic Ca2+. Unlike the mitogenic response in NIH 3T3 cells, the antimitogenic effect was not affected by pertussis toxin; on the contrary, it was accompanied by an increase in cAMP. In Sp2 cells, cAMP analogs, forskolin, and isobutylmethylxanthine inhibited cell proliferation and enhanced LPA action in an additive manner, suggesting that an LPA-elicited increase in cAMP-mediated signaling was responsible for the antimitogenic response. In addition to the mitogenic response in fibroblasts and the antimitogenic response in tumor cell lines, there are some cell types (Jurkat T-cell lymphoma and primary astrocytes) in which LPA is ineffective in altering cell proliferation. The cell-type-specific dual action of LPA suggests that this endogenous lipid mediator when released from activated cells might play an important role as a regulator, rather than a ubiquitous inducer, of cell proliferation. Images PMID:8127904

  7. Interleukin-8 enhances the effect of colchicine on cell death.

    PubMed

    Yokoyama, Chikako; Yajima, Chika; Machida, Tetsuro; Kawahito, Yuji; Uchida, Marie; Hisatomi, Hisashi

    2017-03-25

    Pro-inflammatory cytokines are known to be generated in tumors and play important roles in angiogenesis, mitosis, and tumor progression. However, few studies have investigated the synergistic effects of pro-inflammatory cytokines and anticancer drugs on cell death. In the present study, we examined the combined effects of pro-inflammatory cytokines and colchicine on cell death of cancer cells. Colchicine induces G2/M arrest in the cell cycle by binding to tubulin, one of the main constituents of microtubules. SUIT-2 human pancreatic cancer cell line cells overexpressing pro-inflammatory cytokines, including interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α, were treated with colchicine. The effect of colchicine on cell death was enhanced in cells overexpressing IL-8. Moreover, the effect of colchicine on cell death was enhanced in cells overexpressing two IL-8 up-regulators, NF-κB and IL-6, but not in cells overexpressing an IL-8 down-regulator, splicing factor proline/glutamine-rich (SFPQ). Synergistic effects of IL-8 and colchicine were also observed in cells overexpressing IL-8 isoforms lacking the signal peptide. Therefore, IL-8 appeared to function as an enhancer of cell death in cancer cells treated with colchicine. The present results suggest a new role for IL-8 related to cell death of cancer cells.

  8. Interleukin-1-independent activation of human T lymphocytes stimulated by anti-CD3 and a Hodgkin's disease cell line with accessory cell activity.

    PubMed

    Ellis, T M; McMannis, J D; Chua, A O; Gubler, U; Fisher, R I

    1988-10-15

    Antibodies directed against the human T cell receptor or the closely associated CD3 molecule stimulate polyclonal T cell proliferation via mechanisms that mimic a primary immune response. We have investigated the requirement for IL-1 production in anti-CD3 (OKT3)-mediated mitogenesis using a Hodgkin's disease cell line (L428) as the accessory cell. L428 cells did not produce detectable IL-1 following stimulation with lipopolysaccharide or phorbol ester (PMA), nor did they transcribe detectable levels of mRNA for IL-1 alpha or beta after such treatment. Despite their inability to produce IL-1, as few as 1 X 10(4) L428 cells reconstituted the proliferative response of accessory cell-depleted T cells to anti-CD3. Although larger numbers of non-rosette-forming (E-) cells were required for maximal responsiveness to anti-CD3, the maximal degree of proliferation was higher with E- cells than with L428 cells. L428-mediated T cell proliferation did not result from residual accessory cells in the responding population or an allogeneic effect since L428 cells were also capable of providing accessory cell activity for the anti-CD3-dependent generation of IL-2 by the Jurkat T cell line. Although the mechanism by which L428 cells provide accessory functions remains incompletely characterized, the ability of anti-HLA-DR F(ab')2 fragments to completely abrogate L428 and monocyte-mediated anti-CD3 mitogenesis, despite the addition of exogenous IL-1, provides evidence for the participation HLA-DR molecules in this response. These data indicate that anti-CD3-induced proliferation of unprimed human T lymphocytes can occur independently of IL-1 production by accessory cells and may involve the participation of HLA-DR molecules.

  9. A clinoptilolite effect on cell media and the consequent effects on tumor cells in vitro.

    PubMed

    Katic, Masa; Bosnjak, Berislav; Gall-Troselj, Koraljka; Dikic, Ivan; Pavelic, Kresimir

    2006-05-01

    Clinoptilolite is a nontoxic natural zeolite with properties of an ion-exchanger and adsorbent. Earlier studies showed that clinoptilolite could be an adjuvant in cancer therapy. The aim of this study was to define effects of clinoptilolite in cell media on cell viability and activity of key proteins regulating cell survival, cell division and stress response. The number of viable cells, DNA synthesis and activity of EGF-R, PKB/Akt and NF?B was reduced, while apoptosis was increased in cells that were cultured in medium supplemneted with clinoptilolite. These results might be due to adsorbtion of some serum components such as EGF to clinoptilolite. In treated medium without serum the predominant role of clinoptilolite is that of cation exchange, likely affecting calcium levels and calcium-dependent signalling pathways. These results are in line with other data that confirm enhanced apoptosis in cells incubated in treated medium. Together, data presented here demonstrate that clinoptilolite affects cellular microenvironment through mechanisms that are dependent on adsorptive and ion-exchange characteristics of this material.

  10. Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells.

    PubMed

    Badal, Sujan; Her, Yeng F; Maher, L James

    2015-09-04

    Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics.

  11. Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells*

    PubMed Central

    Badal, Sujan; Her, Yeng F.; Maher, L. James

    2015-01-01

    Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics. PMID:26205818

  12. Effects of spaceflight on levels and activity of immune cells

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, Gerald; Berry, Wallace D.; Mandel, Adrian D.; Konstantinova, Irena V.; Taylor, Gerald R.

    1990-01-01

    Experiments were carried out on cells from rats that had been flown on Soviet Biosputnik Cosmos 1887 to explore the effects of speceflight on immune responses. Rat bone marrow cells were examined for their response to colony stimulating factor-M. Rat spleen and bone marrow cells were stained with antibodies directed against cell surface antigenic markers. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell, and interleukin-2 receptor cell surface antigens. A small increase in the percentage of cells staining positively for helper-T-cell antigens was also noted. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin.

  13. The bystander effect in photodynamic inactivation of cells.

    PubMed

    Dahle, J; Bagdonas, S; Kaalhus, O; Olsen, G; Steen, H B; Moan, J

    2000-07-26

    Treatment of MDCK II cells with the lipophilic photosensitizer tetra(3-hydroxyphenyl)porphyrin and light was found to induce a rapid apoptotic response in a large fraction of the cells. Furthermore, the distribution of apoptotic cells in microcolonies of eight cells was found to be different from the binomial distribution, indicating that the cells are not inactivated independently, but that a bystander effect is involved in cell killing by photodynamic treatment. The observation of a bystander effect disagrees with the common view that cells are inactivated only by direct damage and indicates that communication between cells in a colony plays a role in photosensitized induction of apoptosis. The degree of bystander effect was higher for cells dying by necrosis than for cell dying by apoptosis.

  14. The role of TRPV1 in the CD4+ T cell-mediated inflammatory response of allergic rhinitis

    PubMed Central

    Son, Hye Ran; Rhee, Yun-Hee; Kim, Eun Hee; Kim, Ji Hye; Bae, Jun-Sang; Chung, Young-Jun; Chung, Phil-Sang; Raz, Eyal; Mo, Ji-Hun

    2016-01-01

    Transient receptor potential vanilloid 1 (TRPV1), which has been identified as a molecular target for the activation of sensory neurons by various painful stimuli, was reported to regulate the signaling and activation of CD4+ T cells. However, the role of TRPV1 in CD4+ T cell in allergic rhinitis remains poorly understood. In this study, TRPV1 expression was localized in CD4+ T cells. Both knockout and chemical inhibition of TRPV1 suppressed Th2/Th17 cytokine production in CD4 T cells and Jurkat T cells, respectively, and can suppress T cell receptor signaling pathways including NF-κB, MAP kinase, and NFAT. In TRPV1 knockout allergic rhinitis (AR) mice, eosinophil infiltration, Th2/Th17 cytokines in the nasal mucosa, and total and ova-specific IgE levels in serum decreased, compared with wild-type AR mice. The TRPV1 antagonists, BCTC or theobromine, showed similar inhibitory immunologic effects on AR mice models. In addition, the number of TRPV1+/CD4+ inflammatory cells increased in the nasal mucosa of patients with AR, compared with that of control subjects. Thus, TRPV1 activation on CD4+ T cells is involved in T cell receptor signaling, and it could be a novel therapeutic target in AR. PMID:26700618

  15. Toxoplasma gondii inhibits cytochrome c-induced caspase activation in its host cell by interference with holo-apoptosome assembly

    PubMed Central

    Graumann, Kristin; Schaumburg, Frieder; Reubold, Thomas F.; Hippe, Diana; Eschenburg, Susanne; Lüder, Carsten G. K.

    2015-01-01

    Inhibition of programmed cell death pathways of mammalian cells often facilitates the sustained survival of intracellular microorganisms. The apicomplexan parasite Toxoplasma gondii is a master regulator of host cell apoptotic pathways. Here, we have characterized a novel anti-apoptotic activity of T. gondii. Using a cell-free cytosolic extract model, we show that T. gondii interferes with the activities of caspase 9 and caspase 3/7 which have been induced by exogenous cytochrome c and dATP. Proteolytic cleavage of caspases 9 and 3 is also diminished suggesting inhibition of holo-apoptosome function. Parasite infection of Jurkat T cells and subsequent triggering of apoptosome formation by exogenous cytochrome c in vitro and in vivo indicated that T. gondii also interferes with caspase activation in infected cells. Importantly, parasite inhibition of cytochrome c-induced caspase activation considerably contributes to the overall anti-apoptotic activity of T. gondii as observed in staurosporine-treated cells. Co-immunoprecipitation showed that T. gondii abolishes binding of caspase 9 to Apaf-1 whereas the interaction of cytochrome c with Apaf-1 remains unchanged. Finally, T. gondii lysate mimics the effect of viable parasites and prevents holo-apoptosome functionality in a reconstituted in vitro system comprising recombinant Apaf-1 and caspase 9. Beside inhibition of cytochrome c release from host cell mitochondria, T. gondii thus also targets the holo-apoptosome assembly as a second mean to efficiently inhibit the caspase-dependent intrinsic cell death pathway. PMID:28357287

  16. The role of TRPV1 in the CD4+ T cell-mediated inflammatory response of allergic rhinitis.

    PubMed

    Samivel, Ramachandran; Kim, Dae Woo; Son, Hye Ran; Rhee, Yun-Hee; Kim, Eun Hee; Kim, Ji Hye; Bae, Jun-Sang; Chung, Young-Jun; Chung, Phil-Sang; Raz, Eyal; Mo, Ji-Hun

    2016-01-05

    Transient receptor potential vanilloid 1 (TRPV1), which has been identified as a molecular target for the activation of sensory neurons by various painful stimuli, was reported to regulate the signaling and activation of CD4+ T cells. However, the role of TRPV1 in CD4+ T cell in allergic rhinitis remains poorly understood. In this study, TRPV1 expression was localized in CD4+ T cells. Both knockout and chemical inhibition of TRPV1 suppressed Th2/Th17 cytokine production in CD4 T cells and Jurkat T cells, respectively, and can suppress T cell receptor signaling pathways including NF-κB, MAP kinase, and NFAT. In TRPV1 knockout allergic rhinitis (AR) mice, eosinophil infiltration, Th2/Th17 cytokines in the nasal mucosa, and total and ova-specific IgE levels in serum decreased, compared with wild-type AR mice. The TRPV1 antagonists, BCTC or theobromine, showed similar inhibitory immunologic effects on AR mice models. In addition, the number of TRPV1+/CD4+ inflammatory cells increased in the nasal mucosa of patients with AR, compared with that of control subjects. Thus, TRPV1 activation on CD4+ T cells is involved in T cell receptor signaling, and it could be a novel therapeutic target in AR.

  17. The physiological concentration of ferrous iron (II) alters the inhibitory effect of hydrogen peroxide on CD45, LAR and PTP1B phosphatases.

    PubMed

    Kuban-Jankowska, Alicja; Gorska, Magdalena; Jaremko, Lukasz; Jaremko, Mariusz; Tuszynski, Jack A; Wozniak, Michal

    2015-12-01

    Hydrogen peroxide is an important regulator of protein tyrosine phosphatase activity via reversible oxidation. However, the role of iron in this reaction has not been yet elucidated. Here we compare the influence of hydrogen peroxide and the ferrous iron (reagent for Fenton reaction) on the enzymatic activity of recombinant CD45, LAR, PTP1B phosphatases and cellular CD45 in Jurkat cells. The obtained results show that ferrous iron (II) is potent inhibitor of CD45, LAR and PTP1B, but the inhibitory effect is concentration dependent. We found that the higher concentrations of ferrous iron (II) increase the inactivation of CD45, LAR and PTP1B phosphatase caused by hydrogen peroxide, but the addition of the physiological concentration (500 nM) of ferrous iron (II) has even a slightly preventive effect on the phosphatase activity against hydrogen peroxide.

  18. Study of radiation effects on mammalian cells in vitro

    NASA Technical Reports Server (NTRS)

    Sinclair, W. K.

    1968-01-01

    Radiation effect on single cells and cell populations of Chinese hamster lung tissue is studied in vitro. The rate and position as the cell progresses through the generation cycle shows division delay, changes in some biochemical processes in the cell, chromosomal changes, colony size changes, and loss of reproductive capacity.

  19. Effects of nanotopography on stem cell phenotypes

    PubMed Central

    Ravichandran, Rajeswari; Liao, Susan; Ng, Clarisse CH; Chan, Casey K; Raghunath, Michael; Ramakrishna, Seeram

    2009-01-01

    Stem cells are unspecialized cells that can self renew indefinitely and differentiate into several somatic cells given the correct environmental cues. In the stem cell niche, stem cell-extracellular matrix (ECM) interactions are crucial for different cellular functions, such as adhesion, proliferation, and differentiation. Recently, in addition to chemical surface modifications, the importance of nanometric scale surface topography and roughness of biomaterials has increasingly becoming recognized as a crucial factor for cell survival and host tissue acceptance in synthetic ECMs. This review describes the influence of nanotopography on stem cell phenotypes. PMID:21607108

  20. The interaction of the carbon nanoparticles with human cell plasma membrane

    NASA Astrophysics Data System (ADS)

    Overchuk, M.; Prylutska, S.; Bilyy, Rostyslav; Prylutsky, Yu.; Ritter, U.

    2013-09-01

    The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or photosensitizers. In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells). We have shown, that carbon nanoparticles do not have any hemolytic effects, if judged by the dynamics of acidic hemolysis, although they are capable of permeabilizating the cells and facilitating the internalization of propidium iodide into the nuclei.

  1. Lack of Fas/CD95 surface expression in highly proliferative leukemic cell lines correlates with loss of CtBP/BARS and redirection of the protein toward giant lysosomal structures.

    PubMed

    Monleón, Inmaculada; Iturralde, María; Martínez-Lorenzo, María José; Monteagudo, Luis; Lasierra, Pilar; Larrad, Luis; Piñeiro, Andrés; Naval, Javier; Alava, María Angeles; Anel, Alberto

    2002-07-01

    Fas/CD95 is a type-I membrane glycoprotein, which inducesapoptotic cell death when ligated by its physiological ligand. We generated previously hyperproliferative sublines derived from the human T-cell leukemia Jurkat, Jurkat-ws and Jurkat-hp, which lost Fas/CD95 surface expression. We have now observed that the total amount of Fas protein is similar in the sublines and in the parental cells, indicating that in the sublines Fas remains in an intracellular compartment. We have found that the protein is directed toward lysosomes in the sublines, where it is degraded. This defect in the secretory pathway correlates with loss of polyunsaturated fatty acids from cellular lipids, and with the lack of expression of endophilin-I and CtBP/BARS, enzymes that regulate vesicle fission by catalyzing the acylation of arachidonate into lysophosphatidic acid. In addition, great multillamer bodies, which contained acid phosphatase activity, absent in the parental Jurkat cells, were observed by transmission electron microscopy in the sublines.

  2. Short polyhistidine peptides penetrate effectively into Nicotiana tabacum-cultured cells and Saccharomyces cerevisiae cells.

    PubMed

    Kimura, Sayaka; Kawano, Tsuyoshi; Iwasaki, Takashi

    2017-01-01

    The polyhistidine peptides (PHPs) have been previously reported as novel cell-penetrating peptides and are efficiently internalized into mammal cells; however, penetration of PHPs into other cell types is unknown. In this study, the cellular uptake of PHPs in plant and yeast cells was found to be dependent on the number of histidines, and short PHPs (H6-H10 peptides) showed effective internalization. The H8 peptide showed the highest cell-penetrating capacity and localized to vacuoles in plant and yeast cells. Low-temperature conditions inhibited significantly the cellular uptake of short PHPs by both cells. However, net charge neutralization of PHPs also completely inhibited cellular uptake by plant cells, but not by yeast cells. These results indicate that short PHPs penetrate effectively into plant and yeast cells by similar mechanism with the exception of net charge dependency. The findings show the short PHPs are promising candidates for new delivery tools into plant and yeast cells.

  3. Prostaglandin synthesis in human T cells: its partial inhibition by lectins and anti-CD3 antibodies as a possible step in T cell activation.

    PubMed

    Aussel, C; Mary, D; Fehlmann, M

    1987-05-15

    The human leukemic T cell line Jurkat was used to study arachidonic acid (AA) metabolism. We demonstrated that Jurkat cells are able to convert AA into prostaglandins (PG) and thromboxanes. The presence of tritiated 6-keto-PGF1 alpha, PGE2, PGA2 (B2), and thromboxane B2 in the culture medium was shown either by thin-layer chromatography after a 4-hr incubation period of [3H]AA-prelabeled Jurkat cells or by using specific radioimmuno assays. PG synthesis was inhibited by both indomethacin and niflumic acid, two cyclooxygenase inhibitors. AA metabolism through the cyclooxygenase pathway was followed during T cell activation. T cells were activated by lectins or anti-CD3 monoclonal antibodies (mAb) to trigger the T3-Ti complex and by 12-0-tetradecanoylphorbol 13-acetate (TPA) to mimic IL 1-dependent pathways. Our results show that lectins and anti-CD3 mAb both reduce the amount of PG released by the cells, whereas TPA did not. We confirmed that a combination of TPA and lectins or TPA and anti-CD3 mAb is necessary to obtain full activation of Jurkat cells if this event is monitored by using measurement of IL 2 synthesis. In addition, lectins and anti-CD3 mAb can be replaced by the cyclooxygenase inhibitors indomethacin or niflumic acid. Indeed, a combination of TPA and one of these two drugs induced maximal IL 2 synthesis. These results thus suggest that a reduction in PG synthesis might be a prerequisite to allow the cascade of events involved in T cell activation.

  4. Differentiation state determines neural effects on microvascular endothelial cells

    SciTech Connect

    Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  5. The Effect of Spaceflight on Cartilage Cell Cycle and Differentiation

    NASA Technical Reports Server (NTRS)

    Doty, Stephen B.; Stiner, Dalina; Telford, William G.

    2000-01-01

    In vivo studies have shown that spaceflight results in loss of bone and muscle. In an effort to understand the mechanisms of these changes, cell cultures of cartilage, bone and muscle have been subjected to spaceflight to study the microgravity effects on differentiated cells. However it now seems possible that the cell differentiation process itself may be the event(s) most affected by spaceflight. For example, osteoblast-like cells have been shown to have reduced cellular activity in microgravity due to an underdifferentiated state (Carmeliet, et al, 1997). And reduced human lymphocyte growth in spaceflight was related to increased apoptosis (Lewis, et al, 1998). Which brings us to the question of whether reduced cellular activity in space is due to an effect on the differentiated cell, an effect on the cell cycle and cell proliferation, or an effect on cell death. This question has not been specifically addressed on previous flights and was the question behind die present study.

  6. Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples.

    PubMed

    Paryan, Mahdi; Mohammadi-Yeganeh, Samira; Samiee, Siamak Mirab; Soleimani, Masoud; Arefian, Ehsan; Azadmanesh, Keyhan; Poopak, Behzad; Mostafavi, Ehsan; Karimipoor, Morteza; Mahdian, Reza

    2013-10-01

    In diagnostic research challenges, quantitative real-time PCR (QPCR) has been widely utilized in gene expression analysis because of its sensitivity, accuracy, reproducibility, and most importantly, quantitativeness. Real-time PCR base kits are wildly applicable in cancer signaling pathways, especially in cancer investigations. T-cell acute lymphoblastic leukemia (T-ALL) is a type of leukemia that is more common in older children and teenagers. Deregulation of the Notch signaling pathway promotes proliferation and inhibits apoptosis of the lymphoblastic T cells. The aim of this study was to investigate the effect of Notch signaling activation on the expression of target genes using real-time QPCR and further use this method in clinical examination after validation. Two T-ALL cell lines, Jurkat and Molt-4, were used as models for activation of the Notch signaling via over-expression of the Notch1 intracellular domain. Expression analysis was performed for six downstream target genes (NCSTN, APH1, PSEN1, ADAM17, NOTCH1 and C-MYC) which play critical roles in the Notch signaling pathway. The results showed significant difference in the expression of target genes in the deregulated Notch signaling pathway. These results were also verified in 12 clinical samples bearing over-expression of the Notch signaling pathway. Identification of such downstream Notch target genes, which have not been studied inclusively, provides insights into the mechanisms of the Notch function in T cell leukemia, and may help identify novel diagnoses and therapeutic targets in acute lymphoblastic leukemia.

  7. Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis

    PubMed Central

    Lu, Tsung Chi; Zhao, Guan- Hao; Chen, Yao Yun; Chien, Chia-Ying; Huang, Chi-Hung; Lin, Kwang Hui; Chen, Shen Liang

    2016-01-01

    Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors. PMID:27390612

  8. Bioactivity of the Murex Homeopathic Remedy and of Extracts from an Australian Muricid Mollusc against Human Cancer Cells

    PubMed Central

    Benkendorff, Kirsten; McIver, Cassandra M.; Abbott, Catherine A.

    2011-01-01

    Marine molluscs from the family Muricidae are the source of a homeopathic remedy Murex, which is used to treat a range of conditions, including cancer. The aim of this study was to evaluate the in vitro bioactivity of egg mass extracts of the Australian muricid Dicathais orbita, in comparison to the Murex remedy, against human carcinoma and lymphoma cells. Liquid chromatography coupled with mass spectrometry (LC-MS) was used to characterize the chemical composition of the extracts and homeopathic remedy, focusing on biologically active brominated indoles. The MTS (tetrazolium salt) colorimetric assay was used to determine effects on cell viability, while necrosis and apoptosis induction were investigated using flow cytometry (propidium iodide and Annexin-V staining, resp.). Cells were treated with varying concentrations (1–0.01 mg/mL) of crude and semi-purified extracts or preparations (dilute 1 M and concentrated 4 mg/mL) from the Murex remedy (4 h). The Murex remedy showed little biological activity against the majority of cell lines tested. In contrast, the D. orbita egg extracts significantly decreased cell viability in the majority of carcinoma cell lines. Flow cytometry revealed these extracts induce necrosis in HT29 colorectal cancer cells, whereas apoptosis was induced in Jurkat cells. These findings highlight the biomedical potential of Muricidae extracts in the development of a natural therapy for the treatment of neoplastic tumors and lymphomas. PMID:19491143

  9. Atmospheric effects on solar-cell calibration and evaluation

    SciTech Connect

    Bird, R.E.; Hulstrom, R.L.

    1981-12-01

    Results are presented that illustrate atmospheric effects on cell short currents and calibration numbers for silicon, gallium arsenide, and cadmium sulfide cells. Rigorous radiative transfer codes are used in this analysis to illustrate the effects of precipitable water, turbidity, air mass, and global normal irradiance compared with direct normal irradiance on cell performance. Precipitable water is shown to have a relatively large effect on GaAs (5%) as compared to a small effect (2%) on other cells. The quantitative effects of air mass and turbidity are illustrated. It was found that under some atmospheric conditions global calibration methods have a greater dependence on air mass than direct normal calibrations methods.

  10. Effects of radiation on rat respiratory epithelial cells: Critical target cell populations and the importance of cell-cell interactions

    NASA Astrophysics Data System (ADS)

    Terzaghi-Howe, M.; Ford, J.

    1994-10-01

    The oncongenic effects of radiation on rat respiratory tissues are modulated in vivo within the intact tissue. The degree of modulation as well as the mechanism whereby modulation occurs appears to be different for different types of ionizing radiations. A combined cell culture -in vivo model is described. This model has been developed to evaluate the influence of the host and tissue environment on development and expression of the neoplastic phenotype in irradiated rat trachea. Our data indicates that the potentially oncogenic effects of neutrons, X Rays, and α-particles are different depending on the exposure conditions employed and the conditions under which exposed cells are maintained following exposure.

  11. Effect of spaceflight on natural killer cell activity

    NASA Technical Reports Server (NTRS)

    Rykova, Marina P.; Sonnenfeld, Gerald; Lesniak, A. T.; Taylor, Gerald R.; Meshkov, Dimitrii O.; Mandel, Adrian D.; Medvedev, Andrei E.; Berry, Wallace D.; Fuchs, Boris B.; Konstantinova, Irina V.

    1992-01-01

    The effects of spaceflight on immune cell function were determined in rats flown on Cosmos 2044. Control groups included vivarium, synchronous, and antiorthostatically suspended rats. The ability of natural killer cells to lyse two different target cell lines was determined. Spleen and bone marrow cells obtained from flight rats showed significantly inhibited cytotoxicity for YAC-1 target cells compared with cells from synchronous control rats. This could have been due to exposure of the rats to microgravity. Antiorthostatic suspension did not affect the level of cytotoxicity from spleen cells of suspended rats for YAC-1 cells. On the other hand, cells from rats flown in space showed no significant differences from vivarium and synchronous control rats in cytotoxicity for K-562 target cells. Binding of natural killer cells to K-562 target cells was unaffected by spaceflight. Antiorthostatic suspension resulted in higher levels of cytotoxicity from spleen cells for Cr-51-labeled K-562 cells. The results indicate differential effects of spaceflight on function of natural killer cells. This shows that spaceflight has selective effects on the immune response.

  12. Impact of Exogenous Galectin-9 on Human T Cells

    PubMed Central

    Lhuillier, Claire; Barjon, Clément; Niki, Toshiro; Gelin, Aurore; Praz, Françoise; Morales, Olivier; Souquere, Sylvie; Hirashima, Mitsuomi; Wei, Ming; Dellis, Olivier; Busson, Pierre

    2015-01-01

    Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca2+) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca2+ mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca2+ mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4+ T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca2+ mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4+ T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex. PMID:25947381

  13. Novel anticancer compound [trifluoromethyl-substituted pyrazole N-nucleoside] inhibits FLT3 activity to induce differentiation in acute myeloid leukemia cells.

    PubMed

    Saleh, Ayman M; Taha, Mutasem O; Aziz, Mohammad A; Al-Qudah, Mahmoud A; AbuTayeh, Reem F; Rizvi, Syed A

    2016-06-01

    Anticancer properties of chemically synthesized compounds have continuously been optimized for better efficacy and selectivity. Derivatives of heterocyclic compounds are well known to have selective antiproliferative effect against many types of cancer. In this study, we investigated the ability of an indigenously synthesized anticancer molecule, G-11 [1-(2",3",4",6"-Tetra-O-acetyl-β-D-glucopyranosyl)-4-(3'-trifluoromethylphenylhydrazono)-3-trifluoromethyl-1,4-dihydropyrazol-5-one], to cause selective cytotoxicity and induce differentiation in the acute myeloid leukemia HL-60 cells. G-11 was able to exert cytotoxic effect on hematological (Jurkat, U937, K562, HL-60, CCRF-SB) and solid tumor (MCF-7, HepG2, HeLa, Caco-2) cell lines, with IC50 values significantly lower than noncancerous cells (HEK-293, BJ and Vero) and normal peripheral blood mononuclear cells. G-11 induced differentiation of HL-60 cells to granulocytes and monocytes/macrophages by inhibiting the activation of FLT3 (CD135 tyrosine kinase). ITD-FLT3 mutation found in many acute myeloid leukemia patients could also be targeted by G-11 as exhibited by its inhibitory effect on MOLM-13 and MV4-11 cell lines. Molecular docking studies suggest the involvement of Leu616, Asp698, Cys694 and Cys828 residues in binding of G-11 to FLT3. The ability of G-11 to cause selective cytotoxicity and induce differentiation in cancer cells could be clinically relevant for therapeutic gains.

  14. Eurycomanone and eurycomanol from Eurycoma longifolia Jack as regulators of signaling pathways involved in proliferation, cell death and inflammation.

    PubMed

    Hajjouli, Shéhérazade; Chateauvieux, Sébastien; Teiten, Marie-Hélène; Orlikova, Barbora; Schumacher, Marc; Dicato, Mario; Choo, Chee-Yan; Diederich, Marc

    2014-09-16

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

  15. Cancer cell line identification by short tandem repeat profiling: power and limitations.

    PubMed

    Parson, Walther; Kirchebner, Romana; Mühlmann, Roswitha; Renner, Kathrin; Kofler, Anita; Schmidt, Stefan; Kofler, Reinhard

    2005-03-01

    Cancer cell lines are used worldwide in biological research, and data interpretation depends on unambiguous attribution of the respective cell line to its original source. Short-tandem-repeat (STR) profiling (DNA fingerprinting) is the method of choice for this purpose; however, the genetic stability of cell lines under various experimental conditions is not well defined. We tested the effect of long-term culture, subcloning, and generation of drug-resistant subclones on fingerprinting profiles in four widely used leukemia cell lines. The DNA fingerprinting profile remained unaltered in two of them (U937 and K562) throughout 12 months in culture, and the vast majority of subclones derived therefrom by limiting dilution after long-term culture revealed the same profile, indicating a high degree of stability and clonotypic homogeneity. In contrast, two other cell lines (CCRF-CEM and Jurkat) showed marked alterations in DNA fingerprinting profiles during long-term culture. Limiting dilution subcloning revealed extensive clonotypic heterogeneity with subclones differing in up to eight STR loci from the parental culture. Similar heterogeneity was observed in subclones generated by selection culture for drug resistance where DNA fingerprinting proved useful in identifying possible resistance mechanisms. Thus, common tissue culture procedures may dramatically affect the fingerprinting profile of certain cell lines and thus render definition of their origin difficult.

  16. Synthesis and Antiproliferative Effect of Ethyl 4-[4-(4-Substituted Piperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxalinecarboxylate Derivatives on Human Leukemia Cells.

    PubMed

    Desplat, Vanessa; Vincenzi, Marian; Lucas, Romain; Moreau, Stéphane; Savrimoutou, Solène; Rubio, Sandra; Pinaud, Noël; Bigat, David; Enriquez, Elodie; Marchivie, Mathieu; Routier, Sylvain; Sonnet, Pascal; Rossi, Filomena; Ronga, Luisa; Guillon, Jean

    2017-02-20

    Acute leukemia is a hematological malignancy with high incidence and recurrence rates and is characterized by an accumulation of blasts in bone marrow due to proliferation of immature lymphoid or myeloid cells associated with a blockade of differentiation. The heterogeneity of leukemia led us to look for new specific molecules for leukemia subtypes or for therapy-resistant cases. Among heterocyclic derivatives that attracted attention due to their wide range of biological activities, we focused our interest on the pyrrolo[1,2-a]quinoxaline heterocyclic framework that has been previously identified as an interesting scaffold for antiproliferative activities against various human cancer cell lines. In this work, new ethyl 4-[4-(4-substituted piperidin-1-yl)]benzylpyrrolo[1,2-a]quinoxalinecarboxylate derivatives (1 a-o) were designed, synthesized, and evaluated against five different leukemia cell lines, including Jurkat and U266 (lymphoid cell lines) and K562, U937, and HL60 (myeloid cell lines), as well as on normal human peripheral blood mononuclear cells (PBMCs). This new pyrrolo[1,2-a]quinoxaline series showed interesting cytotoxic potential against all tested leukemia cell lines. In particular, pyrroloquinoxalines 1 a and 1 m,n seem to be interesting due to their high activity against leukemia and their low activity against normal hematopoietic cells, leading to a high index of selectivity.

  17. 3,3'-Diindolylmethane induces G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia cells.

    PubMed

    Shorey, Lyndsey E; Hagman, Amanda M; Williams, David E; Ho, Emily; Dashwood, Roderick H; Benninghoff, Abby D

    2012-01-01

    Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G(1) phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL.

  18. Human mesenchymal stem cells promote survival of T cells in a quiescent state.

    PubMed

    Benvenuto, Federica; Ferrari, Stefania; Gerdoni, Ezio; Gualandi, Francesca; Frassoni, Francesco; Pistoia, Vito; Mancardi, Gianluigi; Uccelli, Antonio

    2007-07-01

    Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response, as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells, we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast, rescue from AICD was not associated with a significant change of Bcl-2, an inhibitor of apoptosis induced by cell stress. Accordingly, MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis, a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall, MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state, providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article.

  19. Effect of glutamate analogues on brain tumor cell lines.

    PubMed

    Campbell, G L; Bartel, R; Freidman, H S; Bigner, D D

    1985-10-01

    Glutamate analogues have been used in many different experimental approaches in neurobiology. A small number of these analogues have been classified as gliotoxic. We have examined the effect of seven glutamate analogues (five gliotoxic and two neurotoxic) on the growth and viability of four human glioma cell lines, one human medulloblastoma cell line, and one human sarcoma cell line. Aminoadipic acid and homocysteic acid predominantly affected the growth of two glioma cell lines in the presence of 4 mM glutamine. Phosphonobutyric acid predominantly affected the other two glioma cell lines and the medulloblastoma cell line in the presence of 4 mM glutamine. In medium containing no glutamine, all three analogues had marked effects on all the cell lines except the sarcoma cell line. These effects were dose dependent. We postulate that these results can in part be explained on the basis of metabolic compartmentalization.

  20. WNT5A expression is regulated by the status of its promoter methylation in leukaemia and can inhibit leukemic cell malignant proliferation.

    PubMed

    Deng, Gang; Li, Zhao Quan; Zhao, Chen; Yuan, Yuan; Niu, Chang Chun; Zhao, Chen; Pan, Jing; Si, Wei Ke

    2011-02-01

    Although down-regulation of WNT5A expression has been reported in some types of leukaemias, the level of WNT5A expression has not been assessed in leukaemia complete remission (CR) cases, the relationship among WNT5A expression level, the status of its promoter methylation, and the curative effect of leukaemia has not been reported, and the effect of WNT5A on cell proliferation has not been assessed. In this study, we analyzed WNT5A expression in various kinds of leukaemia cases, leukaemia CR cases, non-malignant hematopoietic (NMH) cases, as well as in leukemic cell lines and CD34+ cells. The methylation status of the WNT5A promoter and the levels of the Wnt5a protein were also studied. We also investigated the effect of Wnt5a on leukemic cell proliferation. WNT5A expression level was higher in NMH but lower in leukaemia cases compared to that in CR-cases (P<0.01), and was expressed at low level in leukemic cell lines K562, U937 and Jurkat. Wnt5a protein was positive in NMH, CR cases and CD34+, but negative in leukaemia cases. WNT5A promoter was methylated in leukaemia cases and all leukemic cell lines, but not in NMH and CR cases. WNT5A expression was up-regulated after exposure to the demethylating agent 5-Aza-2'-deoxycytidine (Aza) in the K562, U937, Jurkat leukemic cell lines and in 83.3% (10/12) of CR patients after cure, respectively. The increased Wnt5a protein can inhibit K562 malignant proliferation and arrest cell cycle at the G2/M phase after exposure to Aza. These results indicate that WNT5A expression was restored in complete remission cases due to demethylation, and Wnt5a can inhibit leukaemic cell proliferation. We propose that WNT5A can act as a suppressor factor in leukemogenesis and can be used as a potential marker for curative effect assessment in leukaemia.

  1. Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells

    SciTech Connect

    Duan, Lincan; Shen, Hongmei; Zhao, Guangqiang; Yang, Runxiang; Cai, Xinyi; Zhang, Lijuan; Jin, Congguo; Huang, Yunchao

    2014-04-18

    Highlights: • Disulfiram and copper synergistically inhibit lung cancer cell proliferation. • Lung cancer cell colony formation ability is inhibited by Disulfiram/copper. • Disulfiram/copper increases the sensitivity of cisplatin to lung cancer cells. • Lung cancer stem cells are specifically targeted by Disulfiram/copper complex. - Abstract: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition of cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients.

  2. CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes.

    PubMed

    Ruggiero, G; Martinez Cáceres, E; Voordouw, A; Noteboom, E; Graf, D; Kroczek, R A; Spits, H

    1996-05-15

    Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.

  3. Heat shock protein 90-mediated inactivation of nuclear factor-κB switches autophagy to apoptosis through becn1 transcriptional inhibition in selenite-induced NB4 cells.

    PubMed

    Jiang, Qian; Wang, Yuhan; Li, Tianjiao; Shi, Kejian; Li, Zhushi; Ma, Yushi; Li, Feng; Luo, Hui; Yang, Yang; Xu, Caimin

    2011-04-15

    Autophagy can protect cells while also contributing to cell damage, but the precise interplay between apoptosis and autophagy and the contribution of autophagy to cell death are still not clear. Previous studies have shown that supranutritional doses of sodium selenite promote apoptosis in human leukemia NB4 cells. Here, we report that selenite treatment triggers opposite patterns of autophagy in the NB4, HL60, and Jurkat leukemia cell lines during apoptosis and provide evidence that the suppressive effect of selenite on autophagy in NB4 cells is due to the decreased expression of the chaperone protein Hsp90 (heat shock protein 90), suggesting a novel regulatory function of Hsp90 in apoptosis and autophagy. Excessive or insufficient expression indicates that Hsp90 protects NB4 cells from selenite-induced apoptosis, and selenite-induced decreases in the expression of Hsp90, especially in NB4 cells, inhibit the activities of the IκB kinase/nuclear factor-κB (IKK/NF-κB) signaling pathway, leading to less nuclear translocation and inactivation of NF-κB and the subsequent weak binding of the becn1 promoter, which facilitates the transition from autophagy to apoptosis. Taken together, our observations provide novel insights into the mechanisms underlying the balance between apoptosis and autophagy, and we also identified Hsp90-NF-κB-Beclin1 as a potential biological pathway for signaling the switch from autophagy to apoptosis in selenite-treated NB4 cells.

  4. Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells.

    PubMed

    Haarmann, Axel; Nehen, Mathias; Deiß, Annika; Buttmann, Mathias

    2015-08-13

    Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1β-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1β-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1β-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.

  5. Localized electroporation effect on adherent cells in modified electric cell-substrate impedance sensing circuits

    NASA Astrophysics Data System (ADS)

    Kim, Yu Jin; Ram Song, Ka; Kim, Hee-Dae; Park, Bum Chul; Kim, Young Keun; Kang, Chi Jung

    2016-10-01

    Electroporation is a physical transfection method for introducing foreign genes or drugs into cells. It does not require toxic reagents or transfection vectors. However, its applications have been limited because of cell damage and nonspecific transport. Here, we present an effective method for selective and localized electroporation using atomic force microscopy. This electroporation method is applied to adherent cells on substrates, instead of conventionally used suspended cells, and offers relatively effective cell transfection. Moreover, this method enables localized transfection into targeted areas at the single-cell level.

  6. Effects of space flight exposure on cell growth, tumorigenicity and gene expression in cancer cells

    NASA Astrophysics Data System (ADS)

    Yang, Cheng; Li, Yuehui; Zhang, Zhijie; Luo, Chen; Tong, Yongqing; Zhou, Guohua; Xie, Pingli; Hu, Jinyue; Li, Guancheng

    2008-12-01

    It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the "Shen Zhou IV" spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.

  7. Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

    SciTech Connect

    Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia . E-mail: lombardi@pharm.unipmn.it

    2005-12-30

    The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.

  8. Effect of Static Magnetic Field on Cell Migration

    NASA Astrophysics Data System (ADS)

    Hashimoto, Yuichiro; Kawasumi, Masashi; Saito, Masao

    The effect of magnetic field on cell has long been investigated, but there are few quantitative investigations of the migration of cells. Cell-migration is important as one of the fundamental activities of the cell. This study proposes a method to evaluate quantitatively the cell-diffusion constant and the effect of static magnetic field on cell migration. The cell-lines are neuroblastoma (NG108-15), fibroblastoma (NIH/3T3) and osteoblastoma (MC3T3-E1). The static magnetic field of 30 mT or 120 mT is impressed by a permanent magnet in vertical or horizontal direction to the dish. It is shown that the cell-diffusion constant can represent the cell migration as the cell activity. It is found that the cell migration is enhanced by exposure to the magnetic field, depending on the kind of cell. It is conjectured that the effect of static magnetic field affects the cell migration, which is at the downstream of the information transmission.

  9. Effects of oxalate on IMCD cells: a line of mouse inner medullary collecting duct cells.

    PubMed

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Chandhoke, Paramjit S; Koul, Hari K

    2004-12-01

    Oxalate, a metabolic end product and a major constituent of the majority of renal stones, has been shown to be toxic to renal epithelial cells of cortical origin. However, it is unknown whether inner medullary collecting duct (IMCD) cells that are physiologically exposed to higher concentrations of oxalate also behave in a similar manner. In the present study, we examined the effects of oxalate on IMCD cells. IMCD cells from the mouse were maintained in DMEM/F12 media supplemented with fetal bovine serum and antibiotics. Exposure of IMCD cells to oxalate produced time- and concentration-dependent changes in the light microscopic appearance of the cells. Long-term exposure to oxalate resulted in alterations in cell viability, with net cell loss after exposure to concentrations of 2 mM or greater. The production of free radicals was directly related to the exposure time and the concentration of oxalate. Crystal formation occurred in less than 1 h and cells in proximity to crystals would lose membrane integrity. Compared with IMCD cells, LLC-PK1 cells as well as HK-2 cells showed significant toxicity starting at lower oxalate concentrations (0.4 mM or greater). These results provide the first direct demonstration of toxic effects of oxalate in IMCD cells, a line of renal epithelial cells of the inner medullary collecting duct, and suggest that the cells lining the collecting duct are relatively resistant to oxalate toxicity.

  10. Thermal effects in IR-laser-irradiated living cells

    NASA Astrophysics Data System (ADS)

    Meier, Thomas H.; Rueck, Angelika C.; Scalfi-Happ, Claudia; Hug, Hubert; Schneider, Marion E.

    2003-10-01

    Irradiation of cell-layers with focussed 2.8 μm ir-laser allows to control the cell temperature from room temperature up to 100°C. Temperatures were calculated for a cell culture model and verified experimentally by thermal mapping of the cell-surrounding medium by means of thermochromic liquid crystals (TLC). Irradiation power and time were varied and associated biological effects like necrosis and apoptosis were observed with respect to the irradiation dosis.

  11. The effect of internal stresses on solar cell efficiency

    NASA Technical Reports Server (NTRS)

    Weizer, Victor G.

    1987-01-01

    Diffusion induced stresses in silicon are shown to result in large localized changes in the minority carrier mobility which in turn have a significant effect on cell output. Evidence is given that both compressive and tensile stresses can be generated in either the emitter or the base region. Tensile stresses appear to be much more effective in altering cell performance. While most stress related effects appear to degrade cell efficiency, this is not always the case. Evidence is presented showing that arsenic induced stresses can result in emitter characteristics comparable to those found in the MINP cell without requiring a high degree of surface passivation.

  12. Synthesis and characterization of N-coumaroyltyramine as a potent phytochemical which arrests human transformed cells via inhibiting protein tyrosine kinases.

    PubMed

    Park, Jae B; Schoene, Norberta

    2002-04-12

    Numerous phytochemicals are believed to have beneficial effects on human health. N-Coumaroyltyramine accumulates in plants in response to wounding and pathogen attack. Due to the scarcity of N-coumaroyltyramine, its biological activities have not been studied in human cells. In this study, N-coumaroyltyramine was chemically synthesized and then purified by an HPLC with a UV-visible absorbance detector. Retention times of major peaks were 14.3 and 20.7 min, and the peak at 20.7 min was confirmed by LC-MS as N-coumaroyltyramine with a mass/charge (m/z) unit of 284.1. The synthesis procedure was relatively easy and had an acceptable yield (approximately 55%). The compound exhibited a new activity, suppression of growth of human tumor cells such as U937 and Jurkat cells. In addition, the suppressed growth of the cells was strongly associated with an increased percentage of cells in the S phase of the cell cycle progression. Furthermore, N-coumaroyltyramine was able to inhibit the protein tyrosine kinases including epidermal growth factor receptor (EGFR). This is the first report of the growth suppressing activity of N-coumaroyltyramine and its arrest of cells at the S phase of the cell cycle, possibly by inhibition of protein tyrosine kinases.

  13. Functional gains in energy and cell metabolism after TSPO gene insertion.

    PubMed

    Liu, Guo-Jun; Middleton, Ryan J; Kam, Winnie Wai-Ying; Chin, David Y; Hatty, Claire R; Chan, Ronald H Y; Banati, Richard B

    2017-03-04

    Recent loss-of-function studies in tissue-specific as well as global Tspo (Translocator Protein 18 kDa) knockout mice have not confirmed its long assumed indispensability for the translocation of cholesterol across the mitochondrial inter-membrane space, a rate-limiting step in steroid biosynthesis. Instead, recent studies in global Tspo knockout mice indicate that TSPO may play a more fundamental role in cellular bioenergetics, which may include the indirect down-stream regulation of transport or metabolic functions. To examine whether overexpression of the TSPO protein alters the cellular bioenergetic profile, Jurkat cells with low to absent endogenous expression were transfected with a TSPO construct to create a stable cell line with de novo expression of exogenous TSPO protein. Expression of TSPO was confirmed by RT-qPCR, radioligand binding with [3H]PK11195 and immunocytochemistry with a TSPO antibody. We demonstrate that TSPO gene insertion causes increased transcription of genes involved in the mitochondrial electron transport chain. Furthermore, TSPO insertion increased mitochondrial ATP production as well as cell excitability, reflected in a decrease in patch clamp recorded rectified K channel currents. These functional changes were accompanied by an increase in cell proliferation and motility, which were inhibited by PK11195, a selective ligand for TSPO. We suggest that TSPO may serve a range of functions that can be viewed as downstream regulatory effects of its primary, evolutionary conserved role in cell metabolism and energy production.

  14. Functional gains in energy and cell metabolism after TSPO gene insertion

    PubMed Central

    Chin, David Y.; Hatty, Claire R.; Chan, Ronald H. Y.

    2017-01-01

    ABSTRACT Recent loss-of-function studies in tissue-specific as well as global Tspo (Translocator Protein 18 kDa) knockout mice have not confirmed its long assumed indispensability for the translocation of cholesterol across the mitochondrial inter-membrane space, a rate-limiting step in steroid biosynthesis. Instead, recent studies in global Tspo knockout mice indicate that TSPO may play a more fundamental role in cellular bioenergetics, which may include the indirect down-stream regulation of transport or metabolic functions. To examine whether overexpression of the TSPO protein alters the cellular bioenergetic profile, Jurkat cells with low to absent endogenous expression were transfected with a TSPO construct to create a stable cell line with de novo expression of exogenous TSPO protein. Expression of TSPO was confirmed by RT-qPCR, radioligand binding with [3H]PK11195 and immunocytochemistry with a TSPO antibody. We demonstrate that TSPO gene insertion causes increased transcription of genes involved in the mitochondrial electron transport chain. Furthermore, TSPO insertion increased mitochondrial ATP production as well as cell excitability, reflected in a decrease in patch clamp recorded rectified K channel currents. These functional changes were accompanied by an increase in cell proliferation and motility, which were inhibited by PK11195, a selective ligand for TSPO. We suggest that TSPO may serve a range of functions that can be viewed as downstream regulatory effects of its primary, evolutionary conserved role in cell metabolism and energy production. PMID:28103132

  15. Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells.

    PubMed

    Dissanayake, Niluka M; Current, Kelley M; Obare, Sherine O

    2015-09-30

    In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism's ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the OPEN ACCESS Int. J. Mol. Sci. 2015, 16 23483 transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells.

  16. Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells

    PubMed Central

    Dissanayake, Niluka M.; Current, Kelley M.; Obare, Sherine O.

    2015-01-01

    In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism’s ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells. PMID:26437397

  17. Direct Effect of Zinc on Mitochondrial Apoptogenesis in Prostate Cells

    PubMed Central

    Feng, Pei; Li, Tie-Luo; Guan, Zhi-Xin; Franklin, Renty B.; Costello, Leslie C.

    2015-01-01

    BACKGROUND Prostate epithelial cells uniquely accumulate significantly higher levels of zinc than other mammalian cells. We previously showed that the accumulation of high intracellular zinc levels in specific prostate cells results in the induction of apoptosis and the inhibition of cell growth. The apoptotic effect is due to zinc induction of mitochondrial apoptogenesis. We now report additional studies that corroborate this effect of zinc and provide insight into the mechanism of this unique effect. METHODS The effect of exposure to physiological levels of zinc on apoptosis was determined for three human prostate cell lines (PC-3, BPH, and HPR-1). Zinc-induced apoptosis was identified by DNA fragmentation. The direct effect of zinc on isolated mitochondrial preparations from each cell line was determined. The mitochondrial release of cytochrome c was determined by Western blot. RESULTS Exposure to zinc induced apoptosis in PC-3 and BPH cells but not in HPR-1 cells. The zinc accumulation in PC-3 (4.3 ± 0.3) and BPH (2.8 ± 0.4) was higher than that in HPR-1 cells (1.8 ± 0.1). The apoptotic effect of zinc on PC-3 cells could be observed as early as 4–6 hr of zinc treatment, and this effect was not reversible. The exposure of isolated mitochondria from PC-3 and BPH cells to zinc resulted in the release of cytochrome c; but zinc had no effect on mitochondria from HPR-1 cells. CONCLUSIONS Exposure to zinc induces apoptosis in PC-3 and BPH cells, which accumulate high intracellular levels of zinc, but not in HPR-1 cells, which do not accumulate high levels of zinc. Once initiated, the induction of apoptosis is not reversed by the removal of zinc, i.e., it is an irreversible process. The apoptogenic effect is due to a direct effect of zinc on mitochondria that results in the release of cytochrome c. The cell specificity of zinc induction of apoptogenesis is dependent on the ability of the cells to accumulate high levels of intracellular zinc and on the ability of

  18. Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium.

    PubMed

    Chinthamani, Sreedevi; Odusanwo, Olutayo; Mondal, Nandini; Nelson, Joel; Neelamegham, Sriram; Baker, Olga J

    2012-04-01

    Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS).

  19. Effects of cell cycle on the uptake of water soluble quantum dots by cells

    NASA Astrophysics Data System (ADS)

    Zheng, Shen; Chen, Ji-Yao; Wang, Jun-Yong; Zhou, Lu-Wei; Peng, Qian

    2011-12-01

    Quantum dots (QDs) with excellent optical properties have become powerful candidates for cell imaging. Although numerous reports have studied the uptake of QDs by cells, little information exists on the effects of cell cycle on the cellular QD uptake. In this report, the effects of cell cycle on the uptake of water soluble thiol-capped CdTe QDs by the human cervical carcinoma Hela cell line, human hepatocellular carcinoma QGY7701 cell line, and human embryonic kidney 293T cell line were studied by means of laser scanning confocal microscopy and flow cytometry. All three cell lines show to take up CdTe QDs via endocytosis. After arresting cells at specific phases with pharmacological agents, the cells in G2/M phase take up the most CdTe QDs, probably due to an increased membrane expansion during mitosis; whereas the cells in G1 phase do the least. A mathematical physics model was built to calculate the relative uptake rates of CdTe QDs by cells in different phases of the cell cycle, with the result as the uptake rate in G2/M phase is 2-4 times higher than that in G1 phase for these three cell lines. The results obtained from this study may provide the information useful for intracellular delivery of QDs.

  20. A Pyrazolo[3,4-d]pyrimidine Compound Reduces Cell Viability and Induces Apoptosis in Different Hematological Malignancies

    PubMed Central

    Laurenzana, Ilaria; Caivano, Antonella; La Rocca, Francesco; Trino, Stefania; De Luca, Luciana; D’Alessio, Francesca; Schenone, Silvia; Falco, Geppino; Botta, Maurizio; Del Vecchio, Luigi; Musto, Pellegrino

    2016-01-01

    Molecular targeted therapies are based upon drugs acting on tumors by interfering with specific targets involved in growth and spread of cancer. Many targeted therapies were approved by Food and Drug Administration as standard treatment, others were introduced into preclinical or clinical studies on hematological malignancies (HMs). The development of drug-resistance in some HMs and the lack of effective treatments in other ones emphasized the need for searching new molecular targets and therapeutic agents. The aim of this study was to evaluate the effects of 4c pyrazolo[3,4-d]pyrimidine compound, a Src inhibitor, on lymphoid and myeloid neoplasms. Here, we demonstrated its ability to reduce cell viability, induce apoptosis and cell cycle arrest in lymphoid cell lines such as Jurkat, SKMM1, Derl-2/7, and myeloid cell lines, such as Jurl-MK1. Moreover, we reported a high expression of a Src kinase, Fyn, in these cell lines compared to healthy subjects. This study was a starting point to investigate 4c pyrazolo[3,4-d]pyrimidine compound as a drug for HMs and Src kinases as its potential molecular targets. PMID:27872592

  1. The effects of adiponectin and leptin on human endothelial cell proliferation: a live-cell study.

    PubMed

    Alvarez, Granada; Visitación Bartolomé, M; Miana, María; Jurado-López, Raquel; Martín, Ruben; Zuluaga, Pilar; Martinez-Martinez, Ernesto; Nieto, M Luisa; Alvarez-Sala, Luis A; Millán, Jesús; Lahera, Vicente; Cachofeiro, Victoria

    2012-01-01

    The effect of adiponectin and leptin on the proliferation of the human microvascular endothelial cell line (HMEC-1) was studied in the absence or presence of fetal bovine serum (FBS). The participation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt (PI-3K/Akt) pathways in this effect were evaluated. We studied the effect of both adipokines on the motility, mitosis, proliferation and cell death processes of HMEC-1 cells using live-cell imaging techniques. Adiponectin but not leptin further increased the proliferative effect induced by FBS on HMEC-1. This effect seems to be the consequence of an increase in the mitotic index in adiponectin-treated cells when compared to untreated ones. The presence of either the mitogen-activated protein kinase (MAPK) inhibitor (PD98059), or PI-3K inhibitor (LY294002), reduced the effect of adiponectin in a dose-dependent manner. Neither adipokine was able to affect HMEC-1 proliferation in FBS-free conditions. Duration of mitosis, cell motility and the cell death process were similar in all conditions. These data suggest that adiponectin and leptin exert different effects on endothelial cell function. Adiponectin was able to potentiate proliferation of HMEC-1. This effect involves the activation of both PI3-K/Akt and ERK/MAPK pathways. However, it seems to exert minimal effects on HMEC-1 function in the case of leptin.

  2. Effects of deuterium oxide on cell growth and vesicle speed in RBL-2H3 cells

    PubMed Central

    Triplett, Ashley R.

    2014-01-01

    For the first time we show the effects of deuterium oxide on cell growth and vesicle transport in rat basophilic leukemia (RBL-2H3) cells. RBL-2H3 cells cultured with 15 moles/L deuterium showed decreased cell growth which was attributed to cells not doubling their DNA content. Experimental observations also showed an increase in vesicle speed for cells cultured in deuterium oxide. This increase in vesicle speed was not observed in deuterium oxide cultures treated with a microtubule-destabilizing drug, suggesting that deuterium oxide affects microtubule-dependent vesicle transport. PMID:25237603

  3. Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells.

    PubMed

    Duan, Lincan; Shen, Hongmei; Zhao, Guangqiang; Yang, Runxiang; Cai, Xinyi; Zhang, Lijuan; Jin, Congguo; Huang, Yunchao

    2014-04-18

    Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition of cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients.

  4. Cytotoxic activity of novel palladium-based compounds on leukemia cell lines.

    PubMed

    Antunovic, Maja; Kriznik, Bojana; Ulukaya, Engin; Yilmaz, Veysel T; Mihalic, Katarina C; Madunic, Josip; Marijanovic, Inga

    2015-02-01

    Effective treatment methods for human leukemia are under development, but so far none of them have been found to be completely satisfactory. It was recently reported that palladium complexes have significant anticancer activity as well as lower toxicity compared with some clinically used chemotherapeutics. The anticancer activities of two novel palladium(II) complexes, [Pd(sac)(terpy)](sac)·4H2O and [PdCl(terpy)](sac)·2H2O, were tested against three human leukemia cell lines, Jurkat, MOLT-4, and THP-1, in comparison with cisplatin and adriamycin. The cytotoxic effect of the drugs was determined using the MTT assay. Cell death was assessed using fluorescein isothiocyanate-annexin/propidium iodide staining for flow cytometry. Furthermore, p53 phosphorylation, poly(ADP-ribose) polymerase cleavage, and Bax and Bcl-2 mRNA levels were examined to elucidate the mechanism of cell death induction. Both complexes exhibited a significant dose-dependent antigrowth effect in vitro. The complexes predominately induced apoptosis, but necrosis was also observed. In-vitro results have shown that palladium(II) complexes may be regarded as potential anticancer agents for treating human leukemia. Therefore, further analysis to determine the putative mechanism of action and in-vivo studies on animal models are warranted.

  5. Effects of verteporfin-mediated photodynamic therapy on endothelial cells

    NASA Astrophysics Data System (ADS)

    Kraus, Daniel; Chen, Bin

    2015-03-01

    Photodynamic therapy (PDT) is a treatment modality in which cytotoxic reactive oxygen species are generated from oxygen and other biological molecules when a photosensitizer is activated by light. PDT has been approved for the treatment of cancers and age-related macular degeneration (AMD) due to its effectiveness in cell killing and manageable normal tissue complications. In this study, we characterized the effects of verteporfin-PDT on SVEC mouse endothelial cells and determined its underlying cell death mechanisms. We found that verteporfin was primarily localized in mitochondria and endoplasmic reticulum (ER) in SVEC cells. Light treatment of photosensitized SVEC cells induced a rapid onset of cell apoptosis. In addition to significant structural damages to mitochondria and ER, verteporfin-PDT caused substantial degradation of ER signaling molecules, suggesting ER stress. These results demonstrate that verteporfin-PDT triggered SVEC cell apoptosis by both mitochondrial and ER stress pathways. Results from this study may lead to novel therapeutic approaches to enhance PDT outcome.

  6. Effects of vibrations and shocks on lithium-ion cells

    NASA Astrophysics Data System (ADS)

    Brand, Martin J.; Schuster, Simon F.; Bach, Tobias; Fleder, Elena; Stelz, Manfred; Gläser, Simon; Müller, Jana; Sextl, Gerhard; Jossen, Andreas

    2015-08-01

    Lithium-ion batteries are increasingly used in mobile applications where mechanical vibrations and shocks are a constant companion. This work shows how these mechanical loads affect lithium-ion cells. Therefore pouch and cylindrical cells are stressed with vibrational and shock profiles according to the UN 38.3 standard. Additionally, a vibration test is set up to reflect stress in real-world applications and is carried out for 186 days. The effects of the load profiles on the tested cells are investigated by capacity measurement, impedance spectroscopy, micro-X-ray computed tomography and post mortem analyses. The mechanical stress has no effect on the investigated pouch cells. Although all tested cylindrical cells would pass the standard tests, in certain cells stressed in a vertical position the mandrel dispatched itself and struck against internal components. This caused bruised active materials, short circuits, a damaged current collector and current interrupt device. The investigations are not directly transferrable to all pouch or cylindrical cells but show that the mechanical cell design, especially the fixation of the internal components, determines whether a cell withstands vibrations and shocks. Depending on the cell design and the loading direction, long-term vibrational loads can have additional detrimental effects on lithium-ion cells compared to standard tests.

  7. Effect of hydrocortisone on multipotent human mesenchymal stromal cells.

    PubMed

    Shipunova, N N; Petinati, N A; Drize, N I

    2013-05-01

    We studied the effect of natural glucocorticosteroid hydrocortisone on total cell production, cloning efficiency, and expression of genes important for the function of mesenchymal stromal cells. Addition of hydrocortisone to the culture medium reduces the total cell yield by 2 times and significantly increased cloning efficiency by 2-3 times; this effect was more pronounced in multipotent mesenchymal stromal cells obtained from female donors. Hydrocortisone had no effect on the expression of immunomodulatory factors produced by multipotent mesenchymal stromal cells. Hydrocortisone inhibits the expression of bone differentiation markers, increases the expression of the early adipocyte differentiation marker at the beginning of culturing, and dramatically stimulates the expression of the late adipocyte differentiation marker throughout the culturing period. The findings suggest that hydrocortisone activates multipotent mesenchymal stromal cells.

  8. The effect of cell phones on human health

    NASA Astrophysics Data System (ADS)

    Abu-Isbeih, Ibrahim N.; Saad, Dina

    2011-10-01

    The effect of cell phone radiation on human health is the subject of recent interest and study, as a result of the enormous increase in cell phone usage throughout the world. Cell phones use electromagnetic radiation in the microwave range, which some believe may be harmful to human health. Other digital wireless systems, such as data communication networks, produce similar radiation. The objective of this survey is to review the effects of cell phones on human health: A large body of research exists, both epidemiological and experimental, in non-human animals and in humans, of which the majority shows no definite causative relationship between exposure to cell phones and harmful biological effects in humans. This is often paraphrased simply as the balance of evidence showing no harm to humans from cell phones, although a significant number of individual studies do suggest such a relationship, or are inconclusive.

  9. [Effect of carcinogenic nitrogen-containing compounds on cell metabolism].

    PubMed

    Antropov, V I; Samoĭlov, V O; Slepian, É I

    2012-01-01

    The brown frog (Rana temporaria) skin cells respiration, calcium metabolism and glycolysis, the tree frog (Hyla arborea) skin cells respiration and calcium metabolism were studied under short-term (first hours) and long-term (first days) exposure to nitrogenous compounds [N-nitroso-N-methyl urea (NMU) and thiourea (TU)]. The first direct effect of nitrogenous compounds exposure was cell breathing inhibition occurring in Rana temporaria skin cells after 28 days of exposure, and in Hyla arborea skin cells after 8 days of exposure. These changes were precided by decrease of lactate dehydrogenase (LDH) activity in Rana temporaria skin cells starting 16 days after NMU and TU introduction. The increase of intracellular calcium level was noted in tree frog skin cells 4-8 days after NMU and TU introduction, in brown frogs skin cells this parameter was unchanged.

  10. Effects of trichostatin A on HDAC8 expression, proliferation and cell cycle of Molt-4 cells.

    PubMed

    He, Jing; Liu, Hongli; Chen, Yan

    2006-01-01

    The effects of Trichostatin A (TSA) on histone deacetylase 8 (HDAC8) expression, proliferation and cell cycle arrest in T-lymphoblastic leukemia cell line Molt-4 cells in vitro were investigated. The effect of TSA on the growth of Molt-4 cells was studied by MTT assay. Flow cytometry was used to examine the cell cycle. The expression of HDAC8 was detected by using immunocytochemistry and Western blot. The results showed that proliferation of Molt-4 cells was inhibited in TSA-treated group in a time- and dose-dependent manner. The IC50 of TSA exposures for 24 h and 36 h were 254.3236 and 199.257 microg/L respectively. The cell cycle analysis revealed that Molt-4 was mostly in G0/G1 phase, and after treatment with TSA from 50 to 400 microg/L for 24 h, the percents of G0/G1 cells were decreased and cells were arrested in G2/M phase. Treatment of TSA for 24 h could significantly inhibit the expression of HDAC8 protein in Molt-4 cells (P<0.01). It was concluded that TSA could decrease the expression of HDAC8 in Molt-4 cells, which contributed to the inhibition of proliferation and induction of cell cycle arrest in Molt-4 cells.

  11. Effects of Polyhydroxybutyrate Production on Cell Division

    NASA Technical Reports Server (NTRS)

    Miller, Kathleen; Rahman, Asif; Hadi, Masood Z.

    2015-01-01

    Synthetic biological engineering can be utilized to aide the advancement of improved long-term space flight. The potential to use synthetic biology as a platform to biomanufacture desired equipment on demand using the three dimensional (3D) printer on the International Space Station (ISS) gives long-term NASA missions the flexibility to produce materials as needed on site. Polyhydroxybutyrates (PHBs) are biodegradable, have properties similar to plastics, and can be produced in Escherichia coli using genetic engineering. Using PHBs during space flight could assist mission success by providing a valuable source of biomaterials that can have many potential applications, particularly through 3D printing. It is well documented that during PHB production E. coli cells can become significantly elongated. The elongation of cells reduces the ability of the cells to divide and thus to produce PHB. I aim to better understand cell division during PHB production, through the design, building, and testing of synthetic biological circuits, and identify how to potentially increase yields of PHB with FtsZ overexpression, the gene responsible for cell division. Ultimately, an increase in the yield will allow more products to be created using the 3D printer on the ISS and beyond, thus aiding astronauts in their missions.

  12. Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells.

    PubMed

    Hiwarkar, Prashant; Qasim, Waseem; Ricciardelli, Ida; Gilmour, Kimberly; Quezada, Sergio; Saudemont, Aurore; Amrolia, Persis; Veys, Paul

    2015-12-24

    Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naïve CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rg(null) mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P < .0003). Comparison of CB T cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7(+)CD8(+) T cells and prompt induction of cytotoxic CD8(+) and CD4(+) T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.

  13. Effects of murine natural killer cells on Cryptococcus neoformans

    SciTech Connect

    Nabavi Nouri, N.

    1985-01-01

    Previous data generated by Murphy and McDaniel indicate that normal murine nylon wool nonadherent splenic cells, with the characteristics of natural killer (NK) cells, effectively inhibit the in vitro growth of Cryptococcus neoformans, a yeast-like pathogen. Nylon wood nonadherent cells from spleens of 7-8 week old mice were further fractionated on discontinuous Percoll gradients. The enrichment of NK cells in Percoll fractions 1 and 2 was confirmed by morphological examination, immunofluorescent staining, and by assessing the cytolytic activity of each Percoll cell fraction against YAC-1 targets in the 4 h /sup 51/Cr release assay. Cells isolated from each Percoll fraction were tested for growth inhibitory activity against C neoformans, using an in vitro 18 h growth inhibition assay. The results showed that NK cell enrichment was concomitant with the enrichment of anti-cryptococcal activity the Percoll fractions 1 and 2. An immunolabeling method combined with scanning electron microscopy was used to demonstrate that the effector cells attached to C. neoformans were asialo GM/sub 1/ positive and, therefore, had NK cell characteristics. NK cells have Fc receptors on their surfaces , and are capable of antibody-dependent cell-mediated cytotoxicity (ADCC) against IgG-coated target cells. The author examined the effects of the IgG fraction of rabbit anti-cryptococcal antibody on the NK cell-mediated growth inhibition of C. neoformans. The data indicated that the effector cells involved in antibody-dependent growth inhibition of cryptococci are either NK cells or copurify and coexist in the same population with NK cells.

  14. Effect of acyclovir and interferon on human hematopoietic progenitor cells.

    PubMed Central

    Parker, L M; Lipton, J M; Binder, N; Crawford, E L; Kudisch, M; Levin, M J

    1982-01-01

    Continuous in vitro exposure of human bone marrow cells to acyclovir (approximately 200 microM) or human leukocyte interferon (approximately 250 U/ml) caused 50% inhibition of granulocyte colony-forming cell differentiation. Colonies expressed in the presence of either agent were reduced both in size and number. Erythroid progenitors were more resistant than granulocyte progenitors to the antiproliferative effects of acyclovir. Progenitor cells of patients recovering from cytotoxic chemotherapy were no more sensitive to the effects of acyclovir or interferon than were cells obtained from patients before chemotherapy. PMID:6177284

  15. Effects of cell cycle noise on excitable gene circuits

    NASA Astrophysics Data System (ADS)

    Veliz-Cuba, Alan; Gupta, Chinmaya; Bennett, Matthew R.; Josić, Krešimir; Ott, William

    2016-12-01

    We assess the impact of cell cycle noise on gene circuit dynamics. For bistable genetic switches and excitable circuits, we find that transitions between metastable states most likely occur just after cell division and that this concentration effect intensifies in the presence of transcriptional delay. We explain this concentration effect with a three-states stochastic model. For genetic oscillators, we quantify the temporal correlations between daughter cells induced by cell division. Temporal correlations must be captured properly in order to accurately quantify noise sources within gene networks.

  16. 5,7-Dihydroxyflavone Enhances the Apoptosis-Inducing Potential of TRAIL in Human Tumor Cells via Regulation of Apoptosis-Related Proteins.

    PubMed

    Zhang, Zhenzhen; Ye, Tingmei; Cai, Xueting; Yang, Jie; Lu, Wuguang; Hu, Chunping; Wang, Zhigang; Wang, Xiaoning; Cao, Peng

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer, because it preferentially induces apoptosis in numerous cancer cells with little or no effect on normal cells. 5,7-Dihydroxyflavone is a dietary flavonoid commonly found in many plants. Here we show that the combined treatment with 5,7-dihydroxyflavone and TRAIL at subtoxic concentrations induced strong apoptotic response in human hepatocarcinoma HepG2 cells, acute leukemia Jurkat T cells, and cervical carcinoma HeLa cells. We further investigated the mechanisms by which 5,7-dihydroxyflavone augments TRAIL-induced apoptosis in HepG2 cells. 5,7-Dihydroxyflavone up-regulated the expression of pro-apoptotic protein Bax, attenuated the expression of anti-apoptotic proteins Bcl-2, Mcl-1, and IAPs, and reduced the phosphorylation levels of Akt and STAT3, weakening the anti-apoptotic signals thus facilitating the process of apoptosis. Moreover, 5,7-dihydroxyflavone and TRAIL were well tolerated in mice, and the combination of 5,7-dihydroxyflavone and TRAIL reduced tumor burden in vivo in a HepG2 tumor xenograft model. Interestingly, 5,7-dihydroxyflavone-mediated sensitization to TRAIL-induced cell death was not observed in normal human hepatocytes L-O2. These results suggest that the 5,7-dihydroxyflavone in combination with TRAIL might be used for cancer prevention and/or therapy.

  17. 5,7-Dihydroxyflavone Enhances the Apoptosis-Inducing Potential of TRAIL in Human Tumor Cells via Regulation of Apoptosis-Related Proteins

    PubMed Central

    Zhang, Zhenzhen; Ye, Tingmei; Cai, Xueting; Yang, Jie; Lu, Wuguang; Hu, Chunping; Wang, Zhigang; Wang, Xiaoning; Cao, Peng

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer, because it preferentially induces apoptosis in numerous cancer cells with little or no effect on normal cells. 5,7-Dihydroxyflavone is a dietary flavonoid commonly found in many plants. Here we show that the combined treatment with 5,7-dihydroxyflavone and TRAIL at subtoxic concentrations induced strong apoptotic response in human hepatocarcinoma HepG2 cells, acute leukemia Jurkat T cells, and cervical carcinoma HeLa cells. We further investigated the mechanisms by which 5,7-dihydroxyflavone augments TRAIL-induced apoptosis in HepG2 cells. 5,7-Dihydroxyflavone up-regulated the expression of pro-apoptotic protein Bax, attenuated the expression of anti-apoptotic proteins Bcl-2, Mcl-1, and IAPs, and reduced the phosphorylation levels of Akt and STAT3, weakening the anti-apoptotic signals thus facilitating the process of apoptosis. Moreover, 5,7-dihydroxyflavone and TRAIL were well tolerated in mice, and the combination of 5,7-dihydroxyflavone and TRAIL reduced tumor burden in vivo in a HepG2 tumor xenograft model. Interestingly, 5,7-dihydroxyflavone-mediated sensitization to TRAIL-induced cell death was not observed in normal human hepatocytes L-O2. These results suggest that the 5,7-dihydroxyflavone in combination with TRAIL might be used for cancer prevention and/or therapy. PMID:23533482

  18. Phytosphingosine in combination with TRAIL sensitizes cancer cells to TRAIL through synergistic up-regulation of DR4 and DR5.

    PubMed

    Choi, Soon-Young; Kim, Min-Jung; Chung, Hee Yong; Lee, Su-Jae; Jang, Young-Ju

    2007-01-01

    Sensitization of cancer cells to TRAIL could improve the effectiveness of TRAIL as an anticancer agent. We explored whether TRAIL in combination with phytosphingosine could sensitize cancer cells to TRAIL. The combined treatment enhanced synergistic apoptotic cell death of Jurkat T cells, compared to TRAIL or phytosphingosine alone. Enhanced apoptosis in response to the combination treatment was associated with caspase-8 activation-mediated Bax and Bak activation and mitochondrial dysfunction. The combination treatment also resulted in synergistic up-regulation of TRAIL receptor R1 (DR4) and R2 (DR5). siRNA targeting of DR5 significantly attenuated the combination treatment-induced caspase-8 activation, mitochondrial dysfunction, and apoptotic cell death. Upon stimulation of cells with the combination treatment, NF-kappaB was activated. Moreover, siRNA targeting of NF-kappaB significantly attenuated the combination treatment-induced DR4 and DR5 expression and receptor-mediated caspase-8 activation. These results indicate that phytosphingosine sensitizes cancer cells to TRAIL through the synergistic up-regulation of DR4 and DR5 in an NF-kappaB-dependent fashion resulting in caspase-8 activation and subsequent mitochondrial dysfunction. These findings support the potential application of combination treatment with TRAIL and phytosphingosine in the treatment of cancers that are less sensitive to TRAIL.

  19. Distinct ion channel classes are expressed on the outer nuclear envelope of T- and B-lymphocyte cell lines.

    PubMed Central

    Franco-Obregón, A; Wang, H W; Clapham, D E

    2000-01-01

    The outer nuclear membrane, endoplasmic reticulum, and mitochondrial membrane ion channels are poorly understood, although they are important in the control of compartmental calcium levels, cell division, and apoptosis. Few direct recordings of these ion channels have been made because of the difficulty of accessing these intracellular membranes. Using patch-clamp techniques on isolated nuclei, we measured distinct ion channel classes on the outer nuclear envelope of T-cell (human Jurkat) and BFL5 cell (murine promyelocyte) lines. We first imaged the nuclear envelopes of both Jurkat and FL5 cells with atomic force microscopy to determine the density of pore proteins. The nuclear pore complex was intact at roughly similar densities in both cell types. In patch-clamp recordings of Jurkat nuclear membranes, Cl channels (105 +/- 5 pS) predominated and inactivated with negative pipette potentials. Nucleotides transiently inhibited the anion channel. In contrast, FL5 nuclear channels were cation selective (52 +/- 2 pS), were inactivated with positive membrane potentials, and were insensitive to GTPgammaS applied to the bath. We hypothesize that T- and B-cell nuclear membrane channels are distinct, and that this is perhaps related to their unique roles in the immune system. PMID:10866948

  20. ECRG4 is a negative regulator of caspase-8-mediated apoptosis in human T-leukemia cells.

    PubMed

    Matsuzaki, Junichi; Torigoe, Toshihiko; Hirohashi, Yoshihiko; Kamiguchi, Kenjiro; Tamura, Yasuaki; Tsukahara, Tomohide; Kubo, Terufumi; Takahashi, Akari; Nakazawa, Emiri; Saka, Eri; Yasuda, Kazuyo; Takahashi, Shuji; Sato, Noriyuki

    2012-05-01

    We previously established Fas-resistant variant clones from the human T-cell leukemia lines Jurkat and SUP-T13. Comparative gene expression analysis of the Fas-resistant and Fas-sensitive clones revealed several genes that were aberrantly expressed in the Fas-resistant clones. One of the genes, esophageal cancer-related gene 4 (ECRG4), contained a VDAC2-like domain that might be associated with apoptotic signals. In the present study, we examined the subcellular localization and function of ECRG4 in Fas-mediated apoptosis. By confocal fluorescence microscopy, ECRG4-EGFP fusion protein was detected in mitochondria, endoplasmic reticulum and the Golgi apparatus in gene-transfected HeLa cells. Overexpression of ECRG4 in Fas-sensitive Jurkat cells inhibited mitochondrial membrane permeability transition, leading to resistance against Fas-induced apoptosis. Tumor necrosis factor-alpha-induced apoptosis was also suppressed in ECRG4-overexpressing Jurkat cells. Immunoprecipitation assay demonstrated that ECRG4 is associated with procaspase-8. The inhibitory mechanism included the inhibition of caspase-8 activity and Bid cleavage. Since ECRG4 expression is downregulated in activated T cells, our results suggest that ECRG4 is a novel antiapoptotic gene which is involved in the negative regulation of caspase-8-mediated apoptosis in T cells.

  1. Proton Pump Inhibitors Display Antitumor Effects in Barrett's Adenocarcinoma Cells

    PubMed Central

    Chueca, Eduardo; Apostolova, Nadezda; Esplugues, Juan V.; García-González, María A.; Lanas, Ángel; Piazuelo, Elena

    2016-01-01

    Recent evidence has reported that proton pump inhibitors (PPIs) can exert antineoplastic effects through the disruption of pH homeostasis by inhibiting vacuolar ATPase (H+-VATPase), a proton pump overexpressed in several tumor cells, but this aspect has not been deeply investigated in EAC yet. In the present study, the expression of H+-VATPase was assessed through the metaplasia-dysplasia-adenocarcinoma sequence in Barrett's esophagus (BE) and the antineoplastic effects of PPIs and cellular mechanisms involved were evaluated in vitro. H+-VATPase expression was assessed by immunohistochemistry in paraffined-embedded samples or by immunofluorescence in cultured BE and EAC cell lines. Cells were treated with different concentrations of PPIs and parameters of citotoxicity, oxidative stress, and autophagy were evaluated. H+-VATPase expression was found in all biopsies and cell lines evaluated, showing differences in the location of the pump between the cell lines. Esomeprazole inhibited proliferation and cell invasion and induced apoptosis of EAC cells. Production of reactive oxygen species (ROS) seemed to be involved in the cytotoxic effects observed since the addition of N-acetylcysteine significantly reduced esomeprazole-induced apoptosis in EAC cells. Esomeprazole also reduced intracellular pH of tumor cells, whereas only disturbed the mitochondrial membrane potential in OE33 cells. Esomeprazole induced autophagy in both EAC cells, but also triggered a blockade in autophagic flux in the metastatic cell line. These data provide in vitro evidence supporting the potential use of PPIs as novel antineoplastic drugs for EAC and also shed some light on the mechanisms that trigger PPIs cytotoxic effects, which differ upon the cell line evaluated. PMID:27932981

  2. 3,3′-Diindolylmethane Induces G1 Arrest and Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia Cells

    PubMed Central

    Shorey, Lyndsey E.; Hagman, Amanda M.; Williams, David E.; Ho, Emily; Dashwood, Roderick H.; Benninghoff, Abby D.

    2012-01-01

    Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL. PMID:22514694

  3. Cell phone radiation effects on cytogenetic abnormalities of oral mucosal cells.

    PubMed

    Daroit, Natália Batista; Visioli, Fernanda; Magnusson, Alessandra Selinger; Vieira, Geila Radunz; Rados, Pantelis Varvaki

    2015-01-01

    The aim of this study was to evaluate the effects of exposure to cell phone electromagnetic radiation on the frequency of micronuclei, broken eggs cells, binucleated cells, and karyorrhexis in epithelial cells of the oral mucosa. The sample was composed of 60 cell phone users, who were non-smokers and non-drinkers, and had no clinically visible oral lesions. Cells were obtained from anatomical sites with the highest incidence of oral cancer: lower lip, border of the tongue, and floor of the mouth. The Feulgen reaction was used for quantification of nuclear anomalies in 1,000 cells/slide. A slightly increase in the number of micronucleated cells in the lower lip and in binucleated cells on the floor of the mouth was observed in individuals who used their phones > 60 minutes/week. The analysis also revealed an increased number of broken eggs in the tongue of individuals owning a cell phone for over eight years. Results suggest that exposure to electromagnetic waves emitted by cell phones can increase nuclear abnormalities in individuals who use a cell phone for more than 60 minutes per week and for over eight years. Based on the present findings, we suggest that exposure to electromagnetic radiation emitted by cell phones may interfere with the development of metanuclear anomalies. Therefore, it is demonstrated that, despite a significant increase in these anomalies, the radiation emitted by cell phones among frequent users is within acceptable physiological limits.

  4. The effects of glucocorticoids on cultured human endothelial cells.

    PubMed

    Maca, R D; Fry, G L; Hoak, J C

    1978-04-01

    The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man.

  5. Do cell-autonomous and non-cell-autonomous effects drive the structure of tumor ecosystems?

    PubMed

    Tissot, Tazzio; Ujvari, Beata; Solary, Eric; Lassus, Patrice; Roche, Benjamin; Thomas, Frédéric

    2016-04-01

    By definition, a driver mutation confers a growth advantage to the cancer cell in which it occurs, while a passenger mutation does not: the former is usually considered as the engine of cancer progression, while the latter is not. Actually, the effects of a given mutation depend on the genetic background of the cell in which it appears, thus can differ in the subclones that form a tumor. In addition to cell-autonomous effects generated by the mutations, non-cell-autonomous effects shape the phenotype of a cancer cell. Here, we review the evidence that a network of biological interactions between subclones drives cancer cell adaptation and amplifies intra-tumor heterogeneity. Integrating the role of mutations in tumor ecosystems generates innovative strategies targeting the tumor ecosystem's weaknesses to improve cancer treatment.

  6. Effects of whole genome duplication on cell size and gene expression in mouse embryonic stem cells

    PubMed Central

    IMAI, Hiroyuki; FUJII, Wataru; KUSAKABE, Ken Takeshi; KISO, Yasuo; KANO, Kiyoshi

    2016-01-01

    Alterations in ploidy tend to influence cell physiology, which in the long-term, contribute to species adaptation and evolution. Polyploid cells are observed under physiological conditions in the nerve and liver tissues, and in tumorigenic processes. Although tetraploid cells have been studied in mammalian cells, the basic characteristics and alterations caused by whole genome duplication are still poorly understood. The purpose of this study was to acquire basic knowledge about the effect of whole genome duplication on the cell cycle, cell size, and gene expression. Using flow cytometry, we demonstrate that cell cycle subpopulations in mouse tetraploid embryonic stem cells (TESCs) were similar to those in embryonic stem cells (ESCs). We performed smear preparations and flow cytometric analysis to identify cell size alterations. These indicated that the relative cell volume of TESCs was approximately 2.2–2.5 fold that of ESCs. We also investigated the effect of whole genome duplication on the expression of housekeeping and pluripotency marker genes using quantitative real-time PCR with external RNA. We found that the target transcripts were 2.2 times more abundant in TESCs than those in ESCs. This indicated that gene expression and cell volume increased in parallel. Our findings suggest the existence of a homeostatic mechanism controlling the cytoplasmic transcript levels in accordance with genome volume changes caused by whole genome duplication. PMID:27569766

  7. Cytotoxic, cell agglutinating, and syncytium forming effect of purified lectins from Ricinus communis on cultured cells.

    PubMed

    Koga, M; Ohtsu, M; Funatsu, G

    1979-10-01

    The toxicity of lectins from castor bean (Ricinus communis L.), ricin-D, ricin-E, and castor bean hemagglutinin, was investigated on five cultured cell lines. The differential effect of their constituent polypeptide chains was also investigated using these cell lines. Ricin-D, ricin-E, and castor bean hemagglutinin (CBH) possessed cytoagglutinating activity and cytotoxic activity to all five cell lines. These lectins showed the strongest toxicity to L5178Y cells, which are leukemic cells. The toxic activity of ricin-D was stronger than that of CBH in all cell lines. The constituent polypebtide chains of ricin-D and CBH were separated by DEAE-cellulose chromatography and designated as isoleucine chain and alanine chain denoted by their N-terminal amino acids. Only alanine chain of ricin-D was toxic to cells grown in vitro, whereas isoleucine chain of ricin-D and alanine chain of CBH were not toxic to the cells. Moreover, it was found that both lectins caused syncytium formation in NIH3T3 cells infected with Moloney leukemia virus and this cell fusion activity was shown to be exclusively associated with the alanine chain. Cytotoxic, cell agglutinating, and syncytium forming effect of the lectins is due to binding of the alanine chain of ricin-D to galactose-like residues of the membrane constituents of these cells.

  8. An Investigation of the Growth Inhibitory Capacity of Several Medicinal Plants From Iran on Tumor Cell Lines

    PubMed Central

    Esmaeilbeig, Maryam; Kouhpayeh, Seyed Amin; Amirghofran, Zahra

    2015-01-01

    Background: Traditional herbal medicine is a valuable resource that provides new drugs for cancer treatment. Objectives: In this study we aim to screen and investigate the in vitro anti-tumor activities of ten species of plants commonly grown in Southern Iran. Materials and Methods: We used the MTT colorimetric assay to evaluate the cytotoxic activities of the methanol extracts of these plants on various tumor cell lines. The IC50 was calculated as a scale for this evaluation. Results: Satureja bachtiarica, Satureja hortensis, Thymus vulgaris, Thymus daenensis and Mentha lonigfolia showed the inhibitoriest effects on Jurkat cells with > 80% inhibition at 200 µg/mL. Satureja hortensis (IC50: 66.7 µg/mL) was the most effective. These plants also strongly inhibited K562 cell growth; Satureja bachtiarica (IC50: 28.3 µg/mL), Satureja hortensis (IC50: 52 µg/mL) and Thymus vulgaris (IC50: 87 µg/mL) were the most effective extracts. Cichorium intybus, Rheum ribes, Alhagi pseudalhagi and Glycyrrihza glabra also showed notable effects on the leukemia cell lines. The Raji cell line was mostly inhibited by Satureja bachtiarica and Thymus vulgaris with approximately 40% inhibition at 200µg/ml. The influence of these extracts on solid tumor cell lines was not strong. Fen cells were mostly affected by Glycyrrihza glabra (IC50: 182 µg/mL) and HeLa cells by Satureja hortensis (31.6% growth inhibitory effect at 200 µg/mL). Conclusions: Leukemic cell lines were more sensitive to the extracts than the solid tumor cell lines; Satureja hortensis, Satureja bachtiarica, Thymus vulgaris, Thymus daenensis and Mentha lonigfolia showed remarkable inhibitory potential. PMID:26634114

  9. The effects of acoustic vibration on fibroblast cell migration.

    PubMed

    Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

    2016-12-01

    Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration.

  10. Pharmacodynamic Modeling of Cell Cycle Effects for Gemcitabine and Trabectedin Combinations in Pancreatic Cancer Cells

    PubMed Central

    Miao, Xin; Koch, Gilbert; Ait-Oudhia, Sihem; Straubinger, Robert M.; Jusko, William J.

    2016-01-01

    Combinations of gemcitabine and trabectedin exert modest synergistic cytotoxic effects on two pancreatic cancer cell lines. Here, systems pharmacodynamic (PD) models that integrate cellular response data and extend a prototype model framework were developed to characterize dynamic changes in cell cycle phases of cancer cell subpopulations in response to gemcitabine and trabectedin as single agents and in combination. Extensive experimental data were obtained for two pancreatic cancer cell lines (MiaPaCa-2 and BxPC-3), including cell proliferation rates over 0–120 h of drug exposure, and the fraction of cells in different cell cycle phases or apoptosis. Cell cycle analysis demonstrated that gemcitabine induced cell cycle arrest in S phase, and trabectedin induced transient cell cycle arrest in S phase that progressed to G2/M phase. Over time, cells in the control group accumulated in G0/G1 phase. Systems cell cycle models were developed based on observed mechanisms and were used to characterize both cell proliferation and cell numbers in the sub G1, G0/G1, S, and G2/M phases in the control and drug-treated groups. The proposed mathematical models captured well both single and joint effects of gemcitabine and trabectedin. Interaction parameters were applied to quantify unexplainable drug-drug interaction effects on cell cycle arrest in S phase and in inducing apoptosis. The developed models were able to identify and quantify the different underlying interactions between gemcitabine and trabectedin, and captured well our large datasets in the dimensions of time, drug concentrations, and cellular subpopulations. PMID:27895579

  11. Beyond Warburg effect – dual metabolic nature of cancer cells

    PubMed Central

    Xie, Jiansheng; Wu, Hao; Dai, Chunyan; Pan, Qiangrong; Ding, Zonghui; Hu, Danqing; Ji, Bingyan; Luo, Yan; Hu, Xun

    2014-01-01

    Warburg effect is a dominant phenotype of most cancer cells. Here we show that this phenotype depends on its environment. When cancer cells are under regular culture condition, they show Warburg effect; whereas under lactic acidosis, they show a nonglycolytic phenotype, characterized by a high ratio of oxygen consumption rate over glycolytic rate, negligible lactate production and efficient incorporation of glucose carbon(s) into cellular mass. These two metabolic modes are intimately interrelated, for Warburg effect generates lactic acidosis that promotes a transition to a nonglycolytic mode. This dual metabolic nature confers growth advantage to cancer cells adapting to ever changing microenvironment. PMID:24820099

  12. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  13. Effect of gyromagnetic fields on human prostatic adenocarcinoma cells

    PubMed Central

    Lei, Hongen; Xu, Yongde; Guan, Ruili; Li, Meng; Hui, Yu; Gao, Zhezhu; Yang, Bicheng; Xin, Zhongcheng

    2015-01-01

    Purpose To investigate the biological effect of gyromagnetic fields (GMFs) on cell proliferation and apoptosis of human prostatic adenocarcinoma cells and explore the underlying mechanisms. Methods PC-3 cells were grouped into normal control (NC) and GMF treatment groups. Cell proliferation was analyzed with kit-8 and Ki67 immunofluorescence staining, while cell apoptosis was analyzed with flow cytometry double staining of Annexin V-PE/7-AAD. The Akt and p38 MAPK/Caspase signaling pathways were analyzed by western blotting and immunofluorescence staining, and cell polarization was analyzed with PARD3. Results Cell proliferation and activity of the Akt pathway were significantly decreased by the GMF, while cell apoptosis, activity of p38 MAPK, and PARD3-positive cell number were significantly increased in the GMF group compared to the NC group. Conclusion GMFs inhibit cell proliferation, induce apoptosis, and regulate tumor cell polarity conditions, potentially through down-regulating Akt, activating the p38 MAPK/Caspase pathway, and promoting PARD3 expression in PC-3 cells. PMID:26648740

  14. Millimeter wave induced reversible externalization of phosphatidylserine molecules in cells exposed in vitro.

    PubMed

    Szabo, Imre; Kappelmayer, Janos; Alekseev, Stanislav I; Ziskin, Marvin C

    2006-04-01

    In vitro exposure of refrigerated samples (4 degrees C) of anti-coagulated blood with millimeter waves (MMWs) at incident power densities (IPDs) between 0.55 and 1.23 W/cm2 has been found to induce clot formation. We found a small but statistically significant change in clot size with increasing IPD value. MMW exposure of blood samples starting at room temperature (22 degrees C) did not induce blood coagulation; neither did conventional heating at temperatures up to 40 degrees C. Since cell-free plasma did not clot upon MMW exposure, the role of blood cells was particularly analyzed. Experiments on various mixtures of blood cells with plasma revealed an important role of red blood cells (RBC) in the coagulation process. Plasma coagulation also developed within the MMW beam above dense keratinocyte (HaCaT) monolayers suggesting it lacked cell-type specificity. We hypothesized that alteration of the membrane surface in exposed cells might be responsible for the circumscribed coagulation. The thrombogenic role of externalized phosphatidylserine (PS) molecules is well known. Therefore, we carried out experiments for immunolabeling PS molecules with fluorescein isothiocyanate (FITC)-conjugated Annexin V on exposed cells. Fluorescence microscopy of the adherent human keratinocytes (HaCaT) and murine melanoma cells (B16F10) showed that MMW exposure at an IPD of 1.23 W/cm2 is capable of inducing reversible externalization of PS molecules in cells within the beam area without detectable membrane damage. Nonadherent Jurkat cells exposed to MMW at an IPD of 34.5 mW/cm2 also showed reversible PS externalization with flow cytometry, whether the cell temperature was held constant or permitted to rise. These results suggest that certain biological effects induced by MMWs could be initiated by membrane changes in exposed cells.

  15. Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells.

    PubMed

    Hsu, Hsien-Yeh; Hua, Kuo-Feng; Wu, Wei-Chi; Hsu, Jason; Weng, Shih-Ting; Lin, Tsai-Leng; Liu, Chun-Yi; Hseu, Ruey-Shyang; Huang, Ching-Tsan

    2008-04-01

    Ganoderma lucidum, a medicinal fungus is thought to possess and enhance a variety of human immune functions. An immuno-modulatory protein, Ling Zhi-8 (LZ-8) isolated from G. lucidum exhibited potent mitogenic effects upon human peripheral blood lymphocytes (PBL). However, LZ-8-mediated signal transduction in the regulation of interleukin-2 (IL-2) gene expression within human T cells is largely unknown. Here we cloned the LZ-8 gene of G. lucidum, and expressed the recombinant LZ-8 protein (rLZ-8) by means of a yeast Pichia pastoris protein expression system. We found that rLZ-8 induces IL-2 gene expression via the Src-family protein tyrosine kinase (PTK), via reactive oxygen species (ROS), and differential protein kinase-dependent pathways within human primary T cells and cultured Jurkat T cells. In essence, we have established the nature of the rLZ-8-mediated signal-transduction pathways, such as PTK/protein kinase C (PKC)/ROS, PTK/PLC/PKCalpha/ERK1/2, and PTK/PLC/PKCalpha/p38 pathways in the regulation of IL-2 gene expression within human T cells. Our current results of analyzing rLZ-8-mediated signal transduction in T cells might provide a potential application for rLZ-8 as a pharmacological immune-modulating agent.

  16. Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

    PubMed Central

    2010-01-01

    Background Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. Results The study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ≤5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. Conclusion The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy. PMID:20925932

  17. The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells

    PubMed Central

    Ruiz-Magaña, María J.; Martínez-Aguilar, Rocío; Lucendo, Estefanía; Campillo-Davo, Diana; Schulze-Osthoff, Klaus; Ruiz-Ruiz, Carmen

    2016-01-01

    Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect. PMID:26942461

  18. Effects of flavonoids on the growth and cell cycle of cancer cells.

    PubMed

    Choi, S U; Ryu, S Y; Yoon, S K; Jung, N P; Park, S H; Kim, K H; Choi, E J; Lee, C O

    1999-01-01

    In this study, we investigated the cytotoxicities of flavone (F01), 3-hydroxyflavone (F02), 6- hydroxyflavone (F03), 7-hydroxyflavone (F04), 3,6-dihydroxyflavone (F05), 5,7-dihydroxyflavone (F06) and 5,6,7-trihydroxyflavone (F07) to human cancer cells including P- glycoprotein (Pgp)-expressing HCT15 cells and its multidrug resistant subline, HCT15/CL02 cells. We also examined the effects of those flavonoids on the cell cycle of these cancer cells. HCT15/CL02 cells did not reveal resistance to all the flavonoids tested in comparison with HCT15 cells. In cell cycle analysis, all the flavonoids tested, except F01 and F04, reduced the G0/G1 population of SF295 cells at growth inhibitory concentrations, and increased G2/M (F02, F03 and F06) or S (F05 and F07) populations. In addition, F02 and F03 decreased the G2/M and G0/G1 population, and increased the S and G2/M population in HCT15 cells, respectively. Meanwhile, in HCT15/CL02 cells, F02 and F03 decreased the G0/G1 populations and increased the S population. In conclusion, we deemed that the flavonoids tested had diverse cytotoxic mechanisms, and exerted their cell growth inhibitory or killing activity by distinctive ways in different cells.

  19. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    PubMed

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity.

  20. Effect of tumor cells and tumor microenvironment on NK-cell function.

    PubMed

    Vitale, Massimo; Cantoni, Claudia; Pietra, Gabriella; Mingari, Maria Cristina; Moretta, Lorenzo

    2014-06-01

    The ability of tumors to manage an immune-mediated attack has been recently included in the "next generation" of cancer hallmarks. In solid tumors, the microenvironment that is generated during the first steps of tumor development has a pivotal role in immune regulation. An intricate net of cross-interactions occurring between tumor components, stromal cells, and resident or recruited immune cells skews the possible acute inflammatory response toward an aberrant ineffective chronic inflammatory status that favors the evasion from the host's defenses. Natural killer (NK) cells have powerful cytotoxic activity, but their activity may be eluded by the tumor microenvironment. Immunosubversion, immunoediting or immunoselection of poorly immunogenic tumor cells and interference with tumor infiltration play a major role in evading NK-cell responses to tumors. Tumor cells, tumor-associated fibroblasts and tumor-induced aberrant immune cells (i.e. tolerogenic or suppressive macrophages, dendritic cells (DCs) and T cells) can interfere with NK-cell activation pathways or the complex receptor array that regulate NK-cell activation and antitumor activity. Thus, the definition of tumor microenvironment-related immunosuppressive factors, along with the identification of new classes of tissue-residing NK-like innate lymphoid cells, represent key issues to design effective NK-cell-based therapies of solid tumors.

  1. Juglone exerts antitumor effect in ovarian cancer cells

    PubMed Central

    Fang, Fang; Qin, Yingxin; Qi, Ling; Fang, Qing; Zhao, Liangzhong; Chen, Shuang; Li, Qiang; Zhang, Duo; Wang, Liguo

    2015-01-01

    Objective(s): Juglone is isolated from many species of the Juglandaceae family and used as an anti-viral, anti-bacterial, and anti-tumor therapeutic. Here, we evaluated juglone-induced antitumor effect in ovarian cancer SKOV3 cells. Materials and Methods: MTT assay was performed to examine juglone anti-proliferative effect. Cell cycle and apoptosis were studied using flow cytometry in juglone-treated SKOV3 cells. To investigate molecular mechanism of cell cycle and apoptosis, protein expression levels were measured by Western blot analysis of cyclin D1, Bcl-2, Bax, cytochrome c, caspase-9 and caspase-3. To investigate the motility of juglone-treated SKOV3 cell, Matrigel invasion assay was employed to characterize cell invasion. Also, matrix metalloproteinase-2 (MMP-2) expression levels were detected by western blot. Results: Juglone significantly inhibited SKOV3 cell proliferation as shown by G0/G1 phase arrest, and this effect was mediated by inactivation of cyclin D1 protein (P<0.05). Juglone induced apoptosis in SKOV3 cell which was accompanied by caspase-9 and caspase-3 activation (P<0.05). Juglone decreased Bcl-2 levels and increased Bax and cytochrome c (Cyt c) levels (P<0.05). Juglone sufficiently inhibited invasion while evidently decreased MMP-2 expression (P<0.05). Conclusion: The results suggest that juglone could probably induce apoptosis through mitochondrial pathway and restrained cell invasiveness by decreasing MMP expression. PMID:26221477

  2. The Effect of Titanium Dioxide Nanoparticles on Keratinocyte Cell (KC) and Squamous Cell Carcinoma (SCC-13)

    NASA Astrophysics Data System (ADS)

    Lin, Chienhsiu; Simon, Marcia; Jurukovski, Vladimir; Lee, Wilson; Rafailovich, Miriam

    2009-03-01

    We have studied the effects of TiO2 nanoparticles on cell keratinocyte and SCC (Squamous Cell Carcinoma) cells. We found that the concentration of particles required to adversely affect the cells was many times higher for keratinocyte than SCC cells. Confocal microscope shows that the particles in keratinocyte culture are sequestered in membranes between the cell colonies. The particles penetrated into the cells in the case of the SCC cells. TEM images revealed very few particles in the keratinocyte, many more particles were observed sequestered in vacuole of the SCC cells. These results indicate that the keratinocyte layer behaves very different from the fibroblast layers which are much more sensate to TiO2 nanoparticle damage and may suggest a protection mechanism of the dermal tissue. The effect of UV exposure in the presence of DNA was also investigated. We found that adsorbed proteins, as well as grafted polymer provided a measure of protection against free radical formation. The effects of low level UV exposure when the particles are near in-vitro cell culture will be presented.

  3. Notch1 receptor regulates AKT protein activation loop (Thr308) dephosphorylation through modulation of the PP2A phosphatase in phosphatase and tensin homolog (PTEN)-null T-cell acute lymphoblastic leukemia cells.

    PubMed

    Hales, Eric C; Orr, Steven M; Larson Gedman, Amanda; Taub, Jeffrey W; Matherly, Larry H

    2013-08-02

    Notch1 activating mutations occur in more than 50% of T-cell acute lymphoblastic leukemia (T-ALL) cases and increase expression of Notch1 target genes, some of which activate AKT. HES1 transcriptionally silences phosphatase and tensin homolog (PTEN), resulting in AKT activation, which is reversed by Notch1 inhibition with γ-secretase inhibitors (GSIs). Mutational loss of PTEN is frequent in T-ALL and promotes resistance to GSIs due to AKT activation. GSI treatments increased AKT-Thr(308) phosphorylation and signaling in PTEN-deficient, GSI-resistant T-ALL cell lines (Jurkat, CCRF-CEM, and MOLT3), suggesting that Notch1 represses AKT independent of its PTEN transcriptional effects. AKT-Thr(308) phosphorylation and downstream signaling were also increased by knocking down Notch1 in Jurkat (N1KD) cells. This was blocked by treatment with the AKT inhibitor perifosine. The PI3K inhibitor wortmannin and the protein phosphatase type 2A (PP2A) inhibitor okadaic acid both impacted AKT-Thr(308) phosphorylation to a greater extent in nontargeted control than N1KD cells, suggesting decreased dephosphorylation of AKT-Thr(308) by PP2A in the latter. Phosphorylations of AMP-activated protein kinaseα (AMPKα)-Thr(172) and p70S6K-Thr(389), both PP2A substrates, were also increased in both N1KD and GSI-treated cells and responded to okadaic acid treatment. A transcriptional regulatory mechanism was implied because ectopic expression of dominant-negative mastermind-like protein 1 increased and wild-type HES1 decreased phosphorylation of these PP2A targets. This was independent of changes in PP2A subunit levels or in vitro PP2A activity, but was accompanied by decreased association of PP2A with AKT in N1KD cells. These results suggest that Notch1 can regulate PP2A dephosphorylation of critical cellular regulators including AKT, AMPKα, and p70S6K.

  4. Effects of curine in HL-60 leukemic cells: cell cycle arrest and apoptosis induction.

    PubMed

    Dantas, Bruna Braga; Faheina-Martins, Gláucia Veríssimo; Coulidiati, Tangbadioa Hervé; Bomfim, Caio César Barbosa; da Silva Dias, Celidarque; Barbosa-Filho, José Maria; Araújo, Demetrius Antônio Machado

    2015-04-01

    Curine is a natural alkaloid isolated from Chondrodendron platyphyllum and it has been reported that this alkaloid has vasodilatory and anti-inflammatory effects. The aim of this study is to analyze the cytotoxic effects of curine in cancer cell lines HL-60, K562, and HT-29, and in primary cultures of peripheral blood mononuclear cells (PBMC). Cells were treated with curine (from 3 to 15 µM) for 24 and 48 h. Cell viability was analyzed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry with propidium iodide (PI) assay. To assess the type of cell death induced in HL-60, the cell cycle, morphological, and biochemical alterations were analyzed, which were determined by differential staining with acridine orange/ethidium bromide, and annexin V/PI double-labeling and change in mitochondrial membrane potential assays. Curine demonstrated a potent cytotoxic effect on leukemic cell lines (HL-60 and K562). Its cytotoxic effects in HL-60 cells was related to plasma membrane damage and cell cycle arrest at the G1 phase from 43.4 ± 1.0 to 56.7 ± 1.4 % (p < 0.05). Curine (15 μM) also increased the apoptotic cells number by around 60 % in HL-60 cells and caused phosphatidylserine externalization, inducing about 57 % of apoptosis. Moreover, this alkaloid provoked 20 % of mitochondrial membrane depolarization. We conclude that curine presented a cytotoxic effect and induced apoptosis in HL-60 cells. Thus, it can be considered a promising pharmacological drug.

  5. Antitumoral Effect of Hibiscus sabdariffa on Human Squamous Cell Carcinoma and Multiple Myeloma Cells.

    PubMed

    Malacrida, Alessio; Maggioni, Daniele; Cassetti, Arianna; Nicolini, Gabriella; Cavaletti, Guido; Miloso, Mariarosaria

    2016-10-01

    Cancer is a leading cause of death worldwide. Despite therapeutic improvements, some cancers are still untreatable. Recently there has been an increasing interest in the use of natural substances for cancer prevention and treatment. Hibiscus sabdariffa (HS) is a plant, belonging to Malvaceae family, widespread in South Asia and Central Africa. HS extract (HSE) used in folk medicine, gained researchers' interest thanks to its antioxidant, anti-inflammatory, and chemopreventive properties. In the present study, we initially assessed HSE effect on a panel of human tumor cell lines. Then we focused our study on the following that are most sensitive to HSE action cell lines: Multiple Myeloma (MM) cells (RPMI 8226) and Oral Squamous Cell Carcinoma (OSCC) cells (SCC-25). In both RPMI 8226 and SCC-25 cells, HSE impaired cell growth, exerted a reversible cytostatic effect, and reduced cell motility and invasiveness. We evaluated the involvement of MAPKs ERK1/2 and p38 in HSE effects by using specific inhibitors, U0126 and SB203580, respectively. For both SCC-25 and RPMI 8226, HSE cytostatic effect depends on p38 activation, whereas ERK1/2 modulation is crucial for cell motility and invasiveness. Our results suggest that HSE may be a potential therapeutic agent against MM and OSCC.

  6. Requirements for effective antitumor responses of TCR transduced T cells.

    PubMed

    de Witte, Moniek A; Jorritsma, Annelies; Kaiser, Andrew; van den Boom, Marly D; Dokter, Maarten; Bendle, Gavin M; Haanen, John B A G; Schumacher, Ton N M

    2008-10-01

    Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.

  7. Effects of Benzodiazepines on Acinar and Myoepithelial Cells

    PubMed Central

    Mattioli, Tatiana M. F.; Alanis, Luciana R. A.; Sapelli, Silvana da Silva; de Lima, Antonio A. S.; de Noronha, Lucia; Rosa, Edvaldo A. R.; Althobaiti, Yusuf S.; Almalki, Atiah H.; Sari, Youssef; Ignacio, Sergio A.; Johann, Aline C. B. R.; Gregio, Ana M. T.

    2016-01-01

    Background: Benzodiazepines (BZDs), the most commonly prescribed psychotropic drugs with anxiolytic action, may cause hyposalivation. It has been previously shown that BZDs can cause hypertrophy and decrease the acini cell number. In this study, we investigated the effects of BZDs and pilocarpine on rat parotid glands, specifically on acinar, ductal, and myoepithelial cells. Methods: Ninety male Wistar rats were divided into nine groups. Control groups received a saline solution for 30 days (C30) and 60 days (C60), and pilocarpine (PILO) for 60 days. Experimental groups received lorazepam (L30) and midazolam (M30) for 30 days. Another group (LS60 or MS60) received lorazepam or midazolam for 30 days, respectively, and saline for additional 30 days. Finally, other groups (LP60 or MP60) received either lorazepam or midazolam for 30 days, respectively, and pilocarpine for additional 30 days. The expression of calponin in myoepithelial cells and the proliferating cell nuclear antigen (PCNA) in acinar and ductal cells were evaluated. Results: Animals treated with lorazepam showed an increase in the number of positive staining cells for calponin as compared to control animals (p < 0.05). Midazolam administered with pilocarpine (MP60) induced an increase in the proliferation of acinar and ductal cells and a decrease in the positive staining cells for calponin as compared to midazolam administered with saline (MS60). Conclusion: We found that myoepithelial cells might be more sensitive to the effects of BZD than acinar and ductal cells in rat parotid glands. PMID:27445812

  8. Pockels-effect cell for gas-flow simulation

    NASA Technical Reports Server (NTRS)

    Weimer, D.

    1982-01-01

    A Pockels effect cell using a 75 cu cm DK*P crystal was developed and used as a gas flow simulator. Index of refraction gradients were produced in the cell by the fringing fields of parallel plate electrodes. Calibration curves for the device were obtained for index of refraction gradients in excess of .00025 m.

  9. Effect of EMP fields on cell membrane potentials

    SciTech Connect

    Gailey, P.C.; Easterly, C.E.

    1993-06-01

    A simple model is presented for cell membrane potentials induced during exposure to electromagnetic pulse (EMP). Using calculated values of internal electric field strength induced during EMP exposure, the model predicts that cell membrane potentials of about 100 mV may be induced for time frames on the order of 10 ns. Possible biological effects of these potentials including electroporation area discussed.

  10. Modeling circadian clock-cell cycle interaction effects on cell population growth rates.

    PubMed

    El Cheikh, R; Bernard, S; El Khatib, N

    2014-12-21

    The circadian clock and the cell cycle are two tightly coupled oscillators. Recent analytical studies have shown counter-intuitive effects of circadian gating of the cell cycle on growth rates of proliferating cells which cannot be explained by a molecular model or a population model alone. In this work, we present a combined molecular-population model that studies how coupling the circadian clock to the cell cycle, through the protein WEE1, affects a proliferating cell population. We show that the cell cycle can entrain to the circadian clock with different rational period ratios and characterize multiple domains of entrainment. We show that coupling increases the growth rate for autonomous periods of the cell cycle around 24 h and above 48 h. We study the effect of mutation of circadian genes on the growth rate of cells and show that disruption of the circadian clock can lead to abnormal proliferation. Particularly, we show that Cry 1, Cry 2 mutations decrease the growth rate of cells, Per 2 mutation enhances it and Bmal 1 knockout increases it for autonomous periods of the cell cycle less than 21 h and decreases it elsewhere. Combining a molecular model to a population model offers new insight on the influence of the circadian clock on the growth of a cell population. This can help chronotherapy which takes benefits of physiological rhythms to improve anti-cancer efficacy and tolerance to drugs by administering treatments at a specific time of the day.

  11. The Biological Effects of Dickkopf1 on Small Cell Lung Cancer Cells and Bone Metastasis.

    PubMed

    Pang, Hailin; Ma, Ningqiang; Jiao, Mi; Shen, Weiwei; Xin, Bo; Wang, Tongfei; Zhang, Feng; Liu, Lili; Zhang, Helong

    2017-01-02

    The bone is among the most common sites of metastasis in patients with lung cancer. Over 30%-40% of lung cancers can develop bone metastasis, and no effective therapeutic methods exist in clinic cases. Wnt/β-catenin signaling and Dickkopf1 (DKK1) play important roles in the progression of lung cancer, which preferentially metastasizes to the skeleton. However, the role of DKK1 in osteotropism of small cell lung cancer (SCLC) remains to be elucidated. This study aimed to define the role of DKK1 in SCLC bone metastasis and investigate the underlying mechanisms. Our results demonstrated that the expression level of DKK1 was dramatically higher in bone metastatic SCLC cells (SBC-5 cell line) compared with that in cells without bone metastatic ability (SBC-3 cell line). Therefore, we hypothesized that DKK1 was involved in the bone metastasis of SCLC. We then suppressed the DKK1 expression in SBC-5 cells by RNAi and found that downregulation of DKK1 can inhibit cell proliferation, colony formation, cell migration, and invasion, but increase the apoptosis rate. Downregulation of DKK1 did not affect the cell cycle progression of SBC-5 cells in vitro. In vivo, downregulated DKK1 in SBC-5 cells resulted in attenuated bone metastasis. These results indicated that DKK1 may be an important regulator in bone metastases of SCLC, and targeting DKK1 may be an effective method to prevent and treat skeleton metastases in SCLC cases.

  12. Effects of diluents on cell culture viability measured by automated cell counter

    PubMed Central

    Chen, Aaron; Leith, Matthew; Tu, Roger; Tahim, Gurpreet; Sudra, Anish; Bhargava, Swapnil

    2017-01-01

    Commercially available automated cell counters based on trypan blue dye-exclusion are widely used in industrial cell culture process development and manufacturing to increase throughput and eliminate inherent variability in subjective interpretation associated with manual hemocytometers. When using these cell counters, sample dilution is often necessary to stay within the assay measurement range; however, the effect of time and diluents on cell culture is not well understood. This report presents the adverse effect of phosphate buffered saline as a diluent on cell viability when used in combination with an automated cell counter. The reduced cell viability was attributed to shear stress introduced by the automated cell counter. Furthermore, length of time samples were incubated in phosphate buffered saline also contributed to the observed drop in cell viability. Finally, as erroneous viability measurements can severely impact process decisions and product quality, this report identifies several alternative diluents that can maintain cell culture viability over time in order to ensure accurate representation of cell culture conditions. PMID:28264018

  13. The FRK/RAK-SHB signaling cascade: a versatile signal-transduction pathway that regulates cell survival, differentiation and proliferation.

    PubMed

    Annerén, Cecilia; Lindholm, Cecilia K; Kriz, Vitezslav; Welsh, Michael

    2003-06-01

    Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK/RAK (also named BSK/IYK or GTK). FRK/RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and beta-cells, where they both induce PC12 cell differentiation and beta-cell proliferation. Furthermore, beta-cell apoptosis is augmented by these proteins under conditions that cause beta-cell degeneration. The FRK/RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2. Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development. In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express

  14. Thymoquinone causes multiple effects, including cell death, on dividing plant cells.

    PubMed

    Hassanien, Sameh E; Ramadan, Ahmed M; Azeiz, Ahmed Z Abdel; Mohammed, Rasha A; Hassan, Sabah M; Shokry, Ahmed M; Atef, Ahmed; Kamal, Khalid B H; Rabah, Samar; Sabir, Jamal S M; Abuzinadah, Osama A; El-Domyati, Fotouh M; Martin, Gregory B; Bahieldin, Ahmed

    2013-01-01

    Thymoquinone (TQ) is a major constituent of Nigella sativa oil with reported anti-oxidative activity and anti-inflammatory activity in animal cells. It also inhibits proliferation and induces programmed cell death (apoptosis) in human skin cancer cells. The present study sought to detect the influence of TQ on dividing cells of three plant systems and on expression of Bcl2-associated athanogene-like (BAG-like) genes that might be involved during the process of cell death. BAG genes are known for the regulation of diverse physiological processes in animals, including apoptosis, tumorigenesis, stress responses, and cell division. Synthetic TQ at 0.1mg/mL greatly reduced wheat seed germination rate, whereas 0.2mg/mL completely inhibited germination. An Evans blue assay revealed moderate cell death in the meristematic zone of Glycine max roots after 1h of TQ treatment (0.2mg/mL), with severe cell death occurring in this zone after 2h of treatment. Light microscopy of TQ-treated (0.2mg/mL) onion hairy root tips for 1h revealed anti-mitotic activity and also cell death-associated changes, including nuclear membrane disruption and nuclear fragmentation. Transmission electron microscopy of TQ-treated cells (0.2mg/mL) for 1h revealed shrinkage of the plasma membrane, leakage of cell lysate, degradation of cell walls, enlargement of vacuoles and condensation of nuclei. Expression of one BAG-like gene, previously associated with cell death, was induced 20 min after TQ treatment in Glycine max root tip cells. Thus, TQ has multiple effects, including cell death, on dividing plant cells and plants may serve as a useful system to further investigate the mechanisms underlying the response of eukaryotic cells to TQ.

  15. Identifying and removing the cell-cycle effect from single-cell RNA-Sequencing data

    PubMed Central

    Barron, Martin; Li, Jun

    2016-01-01

    Single-cell RNA-Sequencing (scRNA-Seq) is a revolutionary technique for discovering and describing cell types in heterogeneous tissues, yet its measurement of expression often suffers from large systematic bias. A major source of this bias is the cell cycle, which introduces large within-cell-type heterogeneity that can obscure the differences in expression between cell types. The current method for removing the cell-cycle effect is unable to effectively identify this effect and has a high risk of removing other biological components of interest, compromising downstream analysis. We present ccRemover, a new method that reliably identifies the cell-cycle effect and removes it. ccRemover preserves other biological signals of interest in the data and thus can serve as an important pre-processing step for many scRNA-Seq data analyses. The effectiveness of ccRemover is demonstrated using simulation data and three real scRNA-Seq datasets, where it boosts the performance of existing clustering algorithms in distinguishing between cell types. PMID:27670849

  16. Inhibitory effect of maple syrup on the cell growth and invasion of human colorectal cancer cells.

    PubMed

    Yamamoto, Tetsushi; Uemura, Kentaro; Moriyama, Kaho; Mitamura, Kuniko; Taga, Atsushi

    2015-04-01

    Maple syrup is a natural sweetener consumed by individuals of all ages throughout the world. Maple syrup contains not only carbohydrates such as sucrose but also various components such as organic acids, amino acids, vitamins and phenolic compounds. Recent studies have shown that these phenolic compounds in maple syrup may possess various activities such as decreasing the blood glucose level and an anticancer effect. In this study, we examined the effect of three types of maple syrup, classified by color, on the cell proliferation, migration and invasion of colorectal cancer (CRC) cells in order to investigate whether the maple syrup is suitable as a phytomedicine for cancer treatment. CRC cells that were administered maple syrup showed significantly lower growth rates than cells that were administered sucrose. In addition, administration of maple syrup to CRC cells caused inhibition of cell invasion, while there was no effect on cell migration. Administration of maple syrup clearly inhibited AKT phosphorylation, while there was no effect on ERK phosphorylation. These data suggest that maple syrup might inhibit cell proliferation and invasion through suppression of AKT activation and be suitable as a phytomedicine for CRC treatment, with fewer adverse effects than traditional chemotherapy.

  17. Inhibitory effect of maple syrup on the cell growth and invasion of human colorectal cancer cells

    PubMed Central

    YAMAMOTO, TETSUSHI; UEMURA, KENTARO; MORIYAMA, KAHO; MITAMURA, KUNIKO; TAGA, ATSUSHI

    2015-01-01

    Maple syrup is a natural sweetener consumed by individuals of all ages throughout the world. Maple syrup contains not only carbohydrates such as sucrose but also various components such as organic acids, amino acids, vitamins and phenolic compounds. Recent studies have shown that these phenolic compounds in maple syrup may possess various activities such as decreasing the blood glucose level and an anticancer effect. In this study, we examined the effect of three types of maple syrup, classified by color, on the cell proliferation, migration and invasion of colorectal cancer (CRC) cells in order to investigate whether the maple syrup is suitable as a phytomedicine for cancer treatment. CRC cells that were administered maple syrup showed significantly lower growth rates than cells that were administered sucrose. In addition, administration of maple syrup to CRC cells caused inhibition of cell invasion, while there was no effect on cell migration. Administration of maple syrup clearly inhibited AKT phosphorylation, while there was no effect on ERK phosphorylation. These data suggest that maple syrup might inhibit cell proliferation and invasion through suppression of AKT activation and be suitable as a phytomedicine for CRC treatment, with fewer adverse effects than traditional chemotherapy. PMID:25647359

  18. Effects of simulated microgravity on cell cycle in human endothelial cells

    NASA Astrophysics Data System (ADS)

    Sokolovskaya, Alisa A.; Ignashkova, Tatiana I.; Bochenkova, Anna V.; Moskovtsev, Aleksey A.; Baranov, Victor M.; Kubatiev, Aslan A.

    2014-06-01

    The aim of the current study is to investigate effects of simulated microgravity on the cell cycle of endothelial cells. We analyze changes in the cell cycle after exposure of endothelial-like EA.hy 926 cells to simulated microgravity using a Desktop random positioning machine (RPM). Cell cycle profiles determined by flow cytometry show, that the percentage of the cells in the G0/G1 phase after 24 and 96 h of RPM-simulated microgravity is significantly increased as compared to the control group. However, no significant difference is observed after 120 h of RPM-simulated microgravity. In regard to S phase, the percentage of cells is significantly decreased after 24 and 96 h of RPM, respectively; whereas 120 h later, the number of S-phase cells is comparable to the control group. Thus, we show that simulated microgravity inhibits cell cycle progression of human EA.hy 926 cells from the G0/G1 phase to the S phase. We observe an effect of a hibernation-like state, when the growth of the cells in the RPM group slows down, but does not stop. Our results further show that simulated microgravity can affect adhesion of endothelial cells, and alpha-tubulin expression, as most cells begin to detach from the surface of OptiCell unit after 24 h, form aggregates after 48 h, and exhibit accumulation of alpha-tubulin around the nucleus after 48 h of exposure to simulated microgravity conditions. Our results demonstrate a chance in the cell cycle in a low gravitational field.

  19. Human mesenchymal stem cells enhance the systemic effects of radiotherapy.

    PubMed

    de Araújo Farias, Virgínea; O'Valle, Francisco; Lerma, Borja Alonso; Ruiz de Almodóvar, Carmen; López-Peñalver, Jesús J; Nieto, Ana; Santos, Ana; Fernández, Beatriz Irene; Guerra-Librero, Ana; Ruiz-Ruiz, María Carmen; Guirado, Damián; Schmidt, Thomas; Oliver, Francisco Javier; Ruiz de Almodóvar, José Mariano

    2015-10-13

    The outcome of radiotherapy treatment might be further improved by a better understanding of individual variations in tumor radiosensitivity and normal tissue reactions, including the bystander effect. For many tumors, however, a definitive cure cannot be achieved, despite the availablity of more and more effective cancer treatments. Therefore, any improvement in the efficacy of radiotherapy will undoubtedly benefit a significant number of patients. Many experimental studies measure a bystander component of tumor cell death after radiotherapy, which highlights the importance of confirming these observations in a preclinical situation. Mesenchymal stem cells (MSCs) have been investigated for use in the treatment of cancers as they are able to both preferentially home onto tumors and become incorporated into their stroma. This process increases after radiation therapy. In our study we show that in vitro MSCs, when activated with a low dose of radiation, are a source of anti-tumor cytokines that decrease the proliferative activity of tumor cells, producing a potent cytotoxic synergistic effect on tumor cells. In vivo administration of unirradiated mesenchymal cells together with radiation leads to an increased efficacy of radiotherapy, thus leading to an enhancement of short and long range bystander effects on primary-irradiated tumors and distant-non-irradiated tumors. Our experiments indicate an increased cell loss rate and the decrease in the tumor cell proliferation activity as the major mechanisms underlying the delayed tumor growth and are a strong indicator of the synergistic effect between RT and MSC when they are applied together for tumor treatment in this model.

  20. Inhibitory Effect of Baicalin and Baicalein on Ovarian Cancer Cells

    PubMed Central

    Chen, Jianchu; Li, Zhaoliang; Chen, Allen Y.; Ye, Xingqian; Luo, Haitao; Rankin, Gary O.; Chen, Yi Charlie

    2013-01-01

    Ovarian cancer is one of the primary causes of death for women all through the Western world. Baicalin and baicalein are naturally occurring flavonoids that are found in the roots and leaves of some Chinese medicinal plants and are thought to have antioxidant activity and possible anti-angiogenic, anti-cancer, anxiolytic, anti-inflammatory and neuroprotective activities. Two kinds of ovarian cancer (OVCAR-3 and CP-70) cell lines and a normal ovarian cell line (IOSE-364) were selected to be investigated in the inhibitory effect of baicalin and baicalein on cancer cells. Largely, baicalin and baicalein inhibited ovarian cancer cell viability in both ovarian cancer cell lines with LD50 values in the range of 45–55 μM for baicalin and 25–40 μM for baicalein. On the other hand, both compounds had fewer inhibitory effects on normal ovarian cells viability with LD50 values of 177 μM for baicalin and 68 μM for baicalein. Baicalin decreased expression of VEGF (20 μM), cMyc (80 μM), and NFkB (20 μM); baicalein decreased expression of VEGF (10 μM), HIF-1α (20 μM), cMyc (20 μM), and NFkB (40 μM). Therefore baicalein is more effective in inhibiting cancer cell viability and expression of VEGF, HIF-1α, cMyc, and NFκB in both ovarian cancer cell lines. It seems that baicalein inhibited cancer cell viability through the inhibition of cancer promoting genes expression including VEGF, HIF-1α, cMyc, and NFκB. Overall, this study showed that baicalein and baicalin significantly inhibited the viability of ovarian cancer cells, while generally exerting less of an effect on normal cells. They have potential for chemoprevention and treatment of ovarian cancers. PMID:23502466

  1. Introduction of a CD40L genomic fragment via a human artificial chromosome vector permits cell-type-specific gene expression and induces immunoglobulin secretion.

    PubMed

    Yamada, Hidetoshi; Li, Yanze C; Nishikawa, Mitsuo; Oshimura, Mitsuo; Inoue, Toshiaki

    2008-01-01

    Gene therapy using cDNA driven by an exogenous promoter is not suited for genetic disorders that require intrinsic expression of a transgene, such as hyperimmunoglobulin (Ig)M syndrome (HIGM), which is caused by mutations in the CD40L gene. The human artificial chromosome (HAC) vector has the potential to solve this problem, because it can be used to transfer large genomic fragments containing their own regulatory elements. In this study, we examined whether introduction of a genomic fragment of CD40L via the HAC vector permits intrinsic expression of the transgene and has an effect on immunoglobulin secretion. We constructed an HAC vector carrying the mouse CD40L genomic fragment (mCD40L-HAC) in Chinese hamster ovary (CHO) cells and transferred the mCD40L-HAC vector into a human CD4-positive active T-cell line (Jurkat) and a human myeloid cell line (U937) via microcell-mediated chromosome transfer (MMCT). The mCD40L-HAC vector permits mCD40L expression in human active T cells but not in human myeloid cells. The mCD40L-HAC also functions to stimulate mouse B cells derived from CD40L(-/-) mice, inducing secretion of IgG. This study may be an initial step toward the therapeutic application of HAC vectors for intrinsic expression of genes, a potential new direction for genome-based gene therapy.

  2. Neutron exposures in human cells: bystander effect and relative biological effectiveness.

    PubMed

    Seth, Isheeta; Schwartz, Jeffrey L; Stewart, Robert D; Emery, Robert; Joiner, Michael C; Tucker, James D

    2014-01-01

    Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p<0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0 ± 0.13 for micronuclei and 5.8 ± 2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety.

  3. Potentiation of Anticancer Drugs: Effects of Pentoxifylline on Neoplastic Cells

    PubMed Central

    Barancik, Miroslav; Bohacova, Viera; Gibalova, Lenka; Sedlak, Jan; Sulova, Zdena; Breier, Albert

    2012-01-01

    The drug efflux activity of P-glycoprotein (P-gp, a product of the mdr1 gene, ABCB1 member of ABC transporter family) represents a mechanism by which tumor cells escape death induced by chemotherapeutics. In this study, we investigated the mechanisms involved in the effects of pentoxifylline (PTX) on P-gp-mediated multidrug resistance (MDR) in mouse leukemia L1210/VCR cells. Parental sensitive mouse leukemia cells L1210, and multidrug-resistant cells, L1210/VCR, which are characterized by the overexpression of P-gp, were used as experimental models. The cells were exposed to 100 μmol/L PTX in the presence or absence of 1.2 μmol/L vincristine (VCR). Western blot analysis indicated a downregulation of P-gp protein expression when multidrug-resistant L1210/VCR cells were exposed to PTX. The effects of PTX on the sensitization of L1210/VCR cells to VCR correlate with the stimulation of apoptosis detected by Annexin V/propidium iodide apoptosis necrosis kit and proteolytic activation of both caspase-3 and caspase-9 monitored by Western blot analysis. Higher release of matrix metalloproteinases (MMPs), especially MMP-2, which could be attenuated by PTX, was found in L1210/VCR than in L1210 cells by gelatin zymography in electrophoretic gel. Exposure of resistant cells to PTX increased the content of phosphorylated Akt kinase. In contrast, the presence of VCR eliminated the effects of PTX on Akt kinase phosphorylation. Taken together, we conclude that PTX induces the sensitization of multidrug-resistant cells to VCR via downregulation of P-gp, stimulation of apoptosis and reduction of MMPs released from drug-resistant L1210/VCR cells. These facts bring new insights into the mechanisms of PTX action on cancer cells. PMID:22312258

  4. Inhibitory effects of megakaryocytic cells in prostate cancer skeletal metastasis.

    PubMed

    Li, Xin; Koh, Amy J; Wang, Zhengyan; Soki, Fabiana N; Park, Serk In; Pienta, Kenneth J; McCauley, Laurie K

    2011-01-01

    Prostate cancer cells commonly spread through the circulation, but few successfully generate metastatic foci in bone. Osteoclastic cellular activity has been proposed as an initiating event for skeletal metastasis. Megakaryocytes (MKs) inhibit osteoclastogenesis, which could have an impact on tumor establishment in bone. Given the location of mature MKs at vascular sinusoids, they may be the first cells to physically encounter cancer cells as they enter the bone marrow. Identification of the interaction between MKs and prostate cancer cells was the focus of this study. K562 (human MK precursors) and primary MKs derived from mouse bone marrow hematopoietic precursor cells potently suppressed prostate carcinoma PC-3 cells in coculture. The inhibitory effects were specific to prostate carcinoma cells and were enhanced by direct cell-cell contact. Flow cytometry for propidium iodide (PI) and annexin V supported a proapoptotic role for K562 cells in limiting PC-3 cells. Gene expression analysis revealed reduced mRNA levels for cyclin D1, whereas mRNA levels of apoptosis-associated specklike protein containing a CARD (ASC) and death-associated protein kinase 1 (DAPK1) were increased in PC-3 cells after coculture with K562 cells. Recombinant thrombopoietin (TPO) was used to expand MKs in the marrow and resulted in decreased skeletal lesion development after intracardiac tumor inoculation. These novel findings suggest a potent inhibitory role of MKs in prostate carcinoma cell growth in vitro and in vivo. This new finding, of an interaction of metastatic tumors and hematopoietic cells during tumor colonization in bone, ultimately will lead to improved therapeutic interventions for prostate cancer patients.

  5. Effects of stratospheric radiations on human glioblastoma cells.

    PubMed

    Cerù, Maria Paola; Amicarelli, Fernanda; Cristiano, Loredana; Colafarina, Sabrina; Aimola, Pierpaolo; Falone, Stefano; Cinque, Benedetta; Ursini, Ornella; Moscardelli, Roberto; Ragni, Pietro

    2005-01-01

    The aim of this work was to evaluate the effect of stratospheric radiations on neural tumour cells. ADF human glioblastoma cells were hosted on a stratospheric balloon within the 2002 biological experiment campaign of the Italian Space Agency. The flight at an average height of 37 km lasted about 24 hrs. Cell morphology, number and viability, cell cycle and apoptosis, some antioxidant enzymes and proteins involved in cell cycle regulation, DNA repair and gene expression were studied. Stratospheric radiations caused a significant decrease in cell number, as well as a block of proliferation, but not apoptosis or necrosis. Radiations also induced activation and induction of some antioxidant enzymes, increase in DNA repair-related proteins (p53 and Proliferating Cell Nuclear Antigen) and variations of the transcription factors Peroxisome Proliferator-Activated Receptors. Morphologically, test cells exhibited more electron dense cytoplasm and less condensed chromatin than controls and modification of their surfaces. Our results indicate that glioblastoma cells, exposed to continuous stratospheric radiations for 24 hrs, show activation of cell cycle check point, decrease of cell number, variations of Peroxisome Proliferator-Activated Receptors and increase of Reactive Oxygen Species-scavenging enzymes.

  6. Gravity effect on lymphocyte deformation through cell shape change.

    PubMed

    Hung, R J; Tsao, Y D; Spauling, G F

    1995-01-01

    The effects on human cells (lymphocyte) immersed in a culture liquid under microgravity environment has been investigated. The study was based on the numerical simulation of the Morphology of human cells affected by the time dependent variation of gravity acceleration ranging from 10(-3) to 2 g(o) (g(o) = 9.81 m/s2) in 15 s. Both the free floating cells and the cells which came into contact with the upper and lower inclined walls imposed by the time-dependent reduced gravity acceleration were considered in this study. The results show that, when the gravity acceleration increased, the cell morphology changed from spherical to horizontally elongated ellipsoid for both the free floating cells and the stationary cells on the lower inclined wall while the cell morphology varied from spherical to vertically-elongated ellipsoid for the cells hanging on the upper inclined wall. A test of the deformation of human cells exposed to the variation of gravity levels, carried out in the KC-135 free fall aircraft, show that the results of experimental observations agree exactly with the theoretical model computation described in this paper. These results will be useful for study of the behavior and morphology of cells in space.

  7. Transforming growth factor-β promotes ‘death by neglect’ in post-activated human T cells

    PubMed Central

    Sillett, H K; Cruickshank, S M; Southgate, J; Trejdosiewicz, L K

    2001-01-01

    Transforming growth factor-β (TGF-β) is central to the wound repair processes that follow local trauma and inflammation. In order to mimic the early events of wound-healing, we studied the effects of TGF-β on mitogen-stimulated peripheral blood cells. TGF-β added at the initiation of mitogenesis did not significantly alter T-cell activation, proliferation, CD45 isoform switching, or activation-induced cell death. By contrast, TGF-β added 72 hr post-activation (or later) enhanced the cumulative increase in apoptotic T cells. TGF-β had no effect on mitogen-induced up-regulation of Fas (CD95) or Fas ligand and did not enhance killing of the Fas-sensitive Jurkat cell line by activated T cells. Furthermore, TGF-β had no direct effect on levels of mRNA for members of the bcl family (bcl-X, bfl-1, bik, bak, bax, bcl-2 and mcl-1). These findings suggest that TGF-β does not directly induce apoptosis via the Fas system or by direct effects on bcl proteins. However, interleukin-2, which can ‘rescue’ lymphocytes from spontaneous apoptosis due to cytokine deprivation, abolished the pro-apoptotic effects of TGF-β on post-activated T cells, thus demonstrating that TGF-β increases the cytokine-dependence of T cells for survival. We propose a novel role for TGF-β in the suppression of inflammation by promoting the elimination of post-activated T cells once the initiating stimulus has been resolved. PMID:11298829

  8. The influence of 50-Hz magnetic fields on cytoplasmic Ca2+ oscillations in human leukemia T-cells.

    PubMed

    Galvanovskis, J; Sandblom, J; Bergqvist, B; Galt, S; Hamnerius, Y

    1996-02-02

    We have studied the effects of 50-Hz 100-microT rms magnetic fields on intracellular Ca2+ concentration in the Jurkat T lymphocyte variant E6.1 using fluorescent probes Indo-1 and Fura-2. We found, however, that the pattern of intracellular Ca2+ fluctuations also depended on the agent used for cell attachment, in our case the polypeptide poly-L-lysine. In order to isolate possible effects of magnetic field exposure from those of poly-L-lysine, the action of polypeptide on cytosolic Ca2+ was studied as well. It was found that a 10(-7)% concentration of polypeptide trigg