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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. 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.

  3. [Effects of Sam68 gene silence on proliferation of acute T lymphoblastic leukemia cell line Jurkat].

    PubMed

    Wang, Chi-Juan; Xu, Hua; Zhang, Hai-Rui; Wang, Jian; Lin, Ya-Ni; Pang, Tian-Xiang; Li, Qing-Hua

    2014-08-01

    This study was purpose to investigate the effect of Sam68 gene silence on proliferation of human acute T lymphoblastic leukemia cell line Jurkat. The sequence of shRNA targeting the site 531-552 of Sam68 mRNA was designed and chemically synthesized, then a single-vector lentiviral, Tet-inducible shRNA-Sam68 system (pLKO-Tet-On) was constructed; next the Jurkat cells were infected with lentivirus to create stable cell clones with regulatable Sam68 gene expression. The inhibitory efficiency of Sam68 gene was assayed by Real-time PCR and Western blot; the cell activity of Jurkat cells was detected with MTT assay; the change of colony forming potential of Jurkat cells was analyzed by colony forming test; the cell cycle distribution was tested by flow cytometry. The results indicated that the expression of Sam68 in experimental cells was statistically decreased as compared with that of the control cells; the cells activity and colony forming capacity of the Jurkat cells with Sam68 gene silence were significantly inhibited; with Sam68 gene silencing, the percentage of S phase cells was significantly increased, while the percentage of G2 phase cells was significantly decreased. It is concluded that the silencing Sam68 gene using shRNA interference can effectively inhibit the proliferation of human acute T lymphoblastic leukemia cell line Jurkat.

  4. 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

  5. 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

  6. Cytotoxic effects of Fisturalin-3 and 11-Deoxyfisturalin-3 on Jurkat and U937 cell lines.

    PubMed

    Mijares, Michael Rodney; Ochoa, Mariana; Barroeta, Amairelys; Martinez, Gricelis Patricia; Suarez, Alirica Isabel; Compagnone, Reinaldo Santi; Chirinos, Perla; Avila, Ramona; De Sanctis, Juan Bautista

    2013-09-01

    Fisturalines are bromotyrosine compounds isolated from marine sponges. Previous studies have shown antineoplasic, antiviral and antibacterial effects in Vitro; however, the possible effects of these compounds in hematologic malignancies have not been assessed. In the present study, the antiproliferative and pro apoptotic effects of Fistularin-3 (F) and 11-Deoxyfistularin-3 (DF) were assessed using the MTT method and annexin V/propidium iodide by flow cytometry using the cell lines: Jurkat E6.1 and U937. In addition, the cell cycle was assessed by flow cytometry. Inhibition of the proliferative response was concentration and time dependent. The IC50 of F was 7.39 and 8.10 µM for Jurkat E6.1 and U937 respectively. At 24 and 48 h, in the U937 cell line, but not in the Jurkat cell line, both compounds induced up to 35% annexin V increase. Necrosis was not observed in any case. Compound F induced, in both cell lines, a decrease in the number of cells in the S phase and increase in the G0/G1 phase. In the Jurkat cell line only, there was an increase in the number of cells in the G2/M phase. Compound DF was not as effective as F. F is more active than DF in repressing the cell cycle and inducing apoptosis. Both compounds are potentially useful in the development of new drugs to treat hematologic malignancies.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Probing Mechanical Properties of Jurkat Cells under the Effect of ART Using Oscillating Optical Tweezers

    PubMed Central

    2015-01-01

    Acute lymphoid leukemia is a common type of blood cancer and chemotherapy is the initial treatment of choice. Quantifying the effect of a chemotherapeutic drug at the cellular level plays an important role in the process of the treatment. In this study, an oscillating optical tweezer was employed to characterize the frequency-dependent mechanical properties of Jurkat cells exposed to the chemotherapeutic agent, artesunate (ART). A motion equation for a bead bound to a cell was applied to describe the mechanical characteristics of the cell cytoskeleton. By comparing between the modeling results and experimental results from the optical tweezer, the stiffness and viscosity of the Jurkat cells before and after the ART treatment were obtained. The results demonstrate a weak power-law dependency of cell stiffness with frequency. Furthermore, the stiffness and viscosity were increased after the treatment. Therefore, the cytoskeleton cell stiffness as the well as power-law coefficient can provide a useful insight into the chemo-mechanical relationship of drug treated cancer cells and may serve as another tool for evaluating therapeutic performance quantitatively. PMID:25928073

  12. 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

  13. Effects of gravitational perturbation on the expression of genes regulating metabolism in Jurkat cells.

    PubMed

    Singh, Kanika; Cubano, Luis; Lewis, Marian

    2010-01-01

    Gravitational pertubation altered gene expression and increased glucose consumption in spaceflown Jurkat cells. The purpose of this study was to determine if the acceleration experienced during launch was responsible for these changes. In ground-based studies, cells were subjected to typical launch centrifugal acceleration (3g of force for eight minutes) and centrifugal force of 90g for five minutes (commonly used to sediment cells) in a laboratory centrifuge. Controls consisted of static cultures. Gene expression was analyzed by RT-PCR. pH and glucose concentrations were evaluated to monitor metabolic changes. Comparison with controls indicated no significant change in pH or glucose use. Gene expression of Jurkat cells subjected to 3g or 90g of force was altered for only two genes out of seven tested. This research suggests that the changes observed in Jurkat cells flown on STS-95 were not a result of launch acceleration but to other conditions experienced during space flight.

  14. Effect of Light Irradiation and Sex Hormones on Jurkat T Cells: 17β-Estradiol but Not Testosterone Enhances UVA-Induced Cytotoxicity in Jurkat Lymphocytes

    PubMed Central

    Cohly, Hari H.P.; Graham-Evans, Barbara; Ndebele, Kenneth; Jenkins, John K.; McMurray, Robert; Yan, Jian; Yu, Hongtao; Angel, Michael F

    2005-01-01

    In Eastern cultures, such as India, it is traditionally recommended that women but not men cover their heads while working in the scorching sun. The purpose of this pilot study was to determine whether there was any scientific basis for this cultural tradition. We examined the differential cytotoxic effects of ultraviolet A light (UVA) on an established T cell line treated with female and male sex hormones. CD4+ Jurkat T cells were plated in 96 well plates at 2 × 106 cells/ml and treated with 17β-estradiol (EST) or testosterone (TE). These cells were irradiated by UVA light with an irradiance of 170 J/cm2 for 15min at a distance of 6 cm from the surface of the 96-well plate. Controls included cells not treated with hormones or UVA. The effects of EST and TE were investigated between 1 and 20 ng/mL. Cytotoxicity by fluorescein-diacetate staining and COMET assay generating single strand DNA cleavage, tail length and tail moment measurements were examined. The effect of estrogen (5ng/mL) on apoptosis and its mediators was further studied using DNA laddering and western blotting for bcl-2 and p53. We found that EST alone, without UVA, enhanced Jurkat T cell survival. However, EST exhibited a dose-related cytotoxicity in the presence of UVA; up to 28% at 20 ng/ml. TE did not alter UVA-induced cytotoxicity. Since TE did not alter cell viability in the presence of UVA further damaging studies were not performed. COMET assay demonstrated the harmful effects of EST in the presence of UVA while EST without UVA had no significant effect on the nuclear damage. Apoptosis was not present as indicated by the absence of DNA laddering on agarose gel electrophoresis at 5ng/ml EST or TE ± UVA. Western blot showed that estrogen down regulated bcl-2 independently of UVA radiation while p53 was down regulated in the presence of UVA treatment. EST and TE have differential effects on UVA-induced cytotoxicity in Jurkat T-lymphocyte which suggested that women may be more susceptible to

  15. Effects Of Gravitational Perturbation On The Expression Of Genes Regulating Metabolism In Jurkat Cells

    PubMed Central

    Singh, Kanika; Cubano, Luis; Lewis, Marian

    2015-01-01

    Gravitational perturbation altered gene expression and increased glucose consumption in spaceflown Jurkat cells. The purpose of this study was to determine if the acceleration experienced during launch was responsible for these changes. In ground-based studies, cells were subjected to typical launch centrifugal acceleration (3g of force for eight minutes) and centrifugal force of 90g for five minutes (commonly used to sediment cells) in a laboratory centrifuge. Controls consisted of static cultures. Gene expression was analyzed by RT-PCR. pH and glucose concentrations were evaluated to monitor metabolic changes. Comparison with controls indicated no significant change in pH or glucose use. Gene expression of Jurkat cells subjected to 3g or 90g of force was altered for only two genes out of seven tested. This research suggests that the changes observed in Jurkat cells flown on STS-95 were not a result of launch acceleration but to other conditions experienced during space flight. PMID:23875517

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. Moxifloxacin enhances antiproliferative and apoptotic effects of etoposide but inhibits its proinflammatory effects in THP-1 and Jurkat cells

    PubMed Central

    Fabian, I; Reuveni, D; Levitov, A; Halperin, D; Priel, E; Shalit, I

    2006-01-01

    Etoposide (VP-16) is a topoisomerase II (topo II) inhibitor chemotherapeutic agent. Studies indicate that VP-16 enhances proinflammatory cytokines secretion from tumour cells, including IL-8, a chemokine associated with proangiogenic effects. Fluoroquinolones inhibit topo II activity in eukaryotic cells by a mechanism different from that of VP-16. The fluoroquinolone moxifloxacin (MXF) has pronounced anti-inflammatory effects in vitro and in vivo. We studied the effects of MXF and VP-16 on purified human topo II activity and further analysed their combined activity on proliferation, apoptosis and caspase-3 activity in THP-1 and Jurkat cells. Moxifloxacin alone slightly inhibited the activity of human topo II; however, in combination with VP-16 it led to a 73% reduction in enzyme activity. VP-16 inhibited cell proliferation in a time and dose-dependent manner. The addition of moxifloxacin for 72 h to low-dose VP-16 doubled its cytotoxic effect in THP-1 and Jurkat cells (1.8- and 2.6-fold decrease in cell proliferation, respectively) (P<0.004). Moxifloxacin given alone did not induce apoptosis but enhanced VP-16-induced apoptosis in THP-1 and Jurkat cells (1.8- and two-fold increase in annexin V positive cells and caspase-3 activity, respectively) (P<0.04). VP-16 induced the release of IL-8 in a time and dose-dependent manner from THP-1 cells. Moxifloxacin completely blocked the enhanced release of IL-8 induced by 0.5 and 1 μg ml−1 VP-16, and decreased IL-8 release from cells incubated for 72 h with 3 μg ml−1 VP-16 (P<0.001). VP-16 enhanced the release of IL-1β and TNF-α from THP-1 cells, whereas the addition of MXF prevented the enhanced cytokine secretion (P<0.001). We conclude that MXF significantly enhances VP-16 cytotoxicity in tumour-derived cells while preventing VP-16-induced proinflammatory cytokine release. This unique combination may have clinical benefits and cytotoxic drug ‘sparing effect' and should be further studied in vivo. PMID

  1. 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. Copyright 2002 Wiley-Liss, Inc.

  2. 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

  3. Synergistic Effect of TPD7 and Berberine against Leukemia Jurkat Cell Growth through Regulating Ephrin-B2 Signaling.

    PubMed

    Ma, Weina; Zhu, Man; Yang, Liu; Yang, Tianfeng; Zhang, Yanmin

    2017-09-01

    TPD7, a novel biphenyl urea taspine derivative, and berberine have presented inhibition on VEGFR2 that can be regulated by ephrin-B2 reverse signaling through interactions with the PDZ domain. The purpose of this study is to investigate the inhibitory effect of the combination of TPD7 and berberine (TAB) on T-cell acute lymphoblastic leukemia cell growth. TPD7 and berberine together synergistically inhibited the proliferation of Jurkat cells. Also, the combination of TAB induced G1 -phase cell-cycle arrest by downregulating the level of cyclin D1, cyclin E, and CDC2. Furthermore, the combination of TAB significantly enhanced apoptosis in Jurkat cells, and the apoptosis most likely resulted from the modulation of the level of Bcl-2 family members. Most importantly, the concomitant treatment simultaneously regulated the ephrin-B2 and VEGFR2 signaling, as well as modulated the MEK/ERK and PTEN/PI3K/AKT/mTOR signaling. Therefore, the combination treatment of TAB may be a promising therapeutic method in treating T-cell acute lymphoblastic leukemia. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  4. 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

  5. Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells

    PubMed Central

    Jazi, Marie Saghaeian; Mohammadi, Saeed; Yazdani, Yaghoub; Sedighi, Sima; Memarian, Ali; Aghaei, Mehrdad

    2016-01-01

    Objective(s): T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA) as a recently emerged anti-neoplastic histone deacetylase (HDAC) inhibitor and pioglitazone (PGZ) as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ) agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. Materials and Methods: Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. Results: Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after 24 hr; however, PGZ 400 μM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A) phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27) expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. Conclusion: These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies. PMID:27635203

  6. 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

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

    PubMed

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

    2010-07-01

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

  8. 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.

  9. [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.

  10. GABAA receptor plasticity in Jurkat T cells.

    PubMed

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

    2013-12-01

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

  11. 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

  12. 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.

  13. 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.

  14. Escin sodium induces apoptosis of human acute leukemia Jurkat T cells.

    PubMed

    Zhang, Zhenzhen; Gao, Jian; Cai, Xueting; Zhao, Youlong; Wang, Yafei; Lu, Wuguang; Gu, Zhenhua; Zhang, Shuangquan; Cao, Peng

    2011-12-01

    Escin sodium has been used in the clinic as an antioedematous, antiexudative and vasoprotective agent for many years and has shown excellent tolerability. However, little is known about its anticancer activity. This is a report for the first time that escin sodium exerts a cytotoxic effect on human acute leukemia Jurkat T cells via the induction of apoptosis rather than cell cycle arrest. Escin sodium activated the initiator caspase-8, -9, and the effector caspase-3, degraded poly (ADP-ribose) polymerase (PARP) and attenuated the expression of Bcl-2. In addition, escin sodium inhibited the growth of cancer cells in a selective manner with Jurkat cells most sensitive to it. Taken together, the data show that escin sodium possesses potent apoptogenic activity toward human acute leukemia Jurkat T cells. Copyright © 2011 John Wiley & Sons, Ltd.

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

    PubMed Central

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

    2015-01-01

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

  16. 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

  17. 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.

  18. Black tea extract supplementation decreases oxidative damage in Jurkat T cells.

    PubMed

    Erba, D; Riso, P; Foti, P; Frigerio, F; Criscuoli, F; Testolin, G

    2003-08-15

    The purpose of this study was to investigate the protective effect of black tea (BT) extract against induced oxidative damage in Jurkat T-cell line. Cells supplemented with 10 or 25 mg/L BT were subjected to oxidation with ferrous ions. Malondialdehyde (MDA) production as marker of lipid peroxidation, DNA single strand breaks as marker of DNA damage, and modification of the antioxidant enzyme activity, glutathione peroxidase (GPX) were measured. Results show the efficacy of BT polyphenols to decrease DNA oxidative damage and to affect GPX activity (P<0.05), while no effect was shown on MDA production. The succeeding investigation of the activity of caffeine and epigallocatechin gallate demonstrated their antioxidant potential with respect to the cellular markers evaluated. In conclusion, this study supports the protective effect of BT against ferrous ions induced oxidative damage to DNA and the ability of BT to affect the enzyme antioxidant system of Jurkat cells.

  19. RHAMNAZIN INHIBITS PROLIFERATION AND INDUCES APOPTOSIS OF HUMAN JURKAT LEUKEMIA CELLS IN VITRO.

    PubMed

    Philchenkov, A A; Zavelevych, M P

    2015-01-01

    Antiproliferative and apoptogenic effects of rhamnazin, a dimethoxylated derivative of quercetin, were studied in human acute lymphoblastic leukemia Jurkat cells. The cytotoxicity and apoptogenic activity of rhamnazin in vitro are inferior to that of quercetin. The apoptogenic activity of rhamnazin is realized via mitochondrial pathway and associated with activation of caspase-9 and -3. The additive apoptogenic effect of rhamnazin and suboptimal doses of etoposide, a DNA topoisomerase II inhibitor, is demonstrated. Therefore, methylation of quercetin modifies its biological effects considerably.

  20. Inhibition of amino acid-mTOR signaling by a leucine derivative induces G1 arrest in Jurkat cells.

    PubMed

    Hidayat, Sujuti; Yoshino, Ken-ichi; Tokunaga, Chiharu; Hara, Kenta; Matsuo, Masafumi; Yonezawa, Kazuyoshi

    2003-02-07

    We have previously demonstrated that N-acetylleucine amide, a derivative of L-leucine, inhibits leucine-induced p70(S6k) activation in a rat hepatoma cell line. In the present study, we investigated whether N-acetylleucine amide is capable of inhibiting amino acid-mTOR signaling. N-Acetylleucine amide caused cell cycle arrest at G1 stage in Jurkat cells, a human leukemia T cell line, concomitant with the inhibition of serum-induced p70(S6k) activation and p27 degradation. Treatment of Jurkat cells with this compound also exhibited dephosphorylation of retinoblastoma protein. These effects are similar to the inhibitory effects of rapamycin on amino acid-mTOR signaling pathway and suggest that N-acetylleucine amide acts as a rapamycin-like reagent to inhibit cell cycle progression in Jurkat cells.

  1. 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.

  2. 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.

  3. 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

  4. The aminopeptidase inhibitor, z-L-CMK is toxic and induced cell death in Jurkat T cells through oxidative stress.

    PubMed

    Yeo, E H; Goh, W L; Chow, S C

    2017-08-29

    The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK) was found to be toxic and readily induced cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular GSH and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of gluthatione (GSH) and accumulation ROS.

  5. 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.

  6. The Nrf2 activator, tBHQ, differentially affects early events following stimulation of Jurkat cells.

    PubMed

    Zagorski, Joseph W; Turley, Alexandra E; Dover, Heather E; VanDenBerg, Kelly R; Compton, Jacob R; Rockwell, Cheryl E

    2013-11-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is activated by cellular stresses, such as oxidative compounds. After activation, Nrf2 induces transcription of its target genes, many of which have cytoprotective functions. Previously, we have shown that activation of Nrf2 by tert-butylhydroquinone (tBHQ) skews murine CD4⁺ T-cell differentiation. Although the role of Nrf2 in murine T cells is somewhat characterized, it is largely uncharacterized in human T cells. Therefore, the aim of the current studies was to characterize the effects of the Nrf2 activator, tBHQ, on the early events of human CD4⁺ T-cell activation. Pretreatment of Jurkat T cells with tBHQ, prior to activation with anti-CD3/anti-CD28, diminished the production of interleukin-2 (IL-2) at both the transcript and protein levels. Similarly, the expression of CD25 also diminished, albeit to a lesser degree than IL-2, after pretreatment with tBHQ. The decrease in IL-2 production was not due to decreased nuclear translocation of c-fos or c-jun. Although tBHQ caused both a delay and a decrease in Ca²⁺ influx in activated Jurkat cells, no decrease in nuclear factor of activated T cells (NFAT) DNA binding or transcriptional activity was observed. In contrast to NFAT, tBHQ significantly decreased NFκB transcriptional activity. Collectively, our studies show that the Nrf2 activator, tBHQ, inhibits IL-2 and CD25 expression, which correlates with decreased NFκB transcriptional activity in activated Jurkat cells. Overall, our studies suggest that Nrf2 represents a novel mechanism for the regulation of both human and mouse T cell function.

  7. The Nrf2 Activator, tBHQ, Differentially Affects Early Events Following Stimulation of Jurkat Cells

    PubMed Central

    Rockwell, Cheryl E.

    2013-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is activated by cellular stresses, such as oxidative compounds. After activation, Nrf2 induces transcription of its target genes, many of which have cytoprotective functions. Previously, we have shown that activation of Nrf2 by tert-butylhydroquinone (tBHQ) skews murine CD4+ T-cell differentiation. Although the role of Nrf2 in murine T cells is somewhat characterized, it is largely uncharacterized in human T cells. Therefore, the aim of the current studies was to characterize the effects of the Nrf2 activator, tBHQ, on the early events of human CD4+ T-cell activation. Pretreatment of Jurkat T cells with tBHQ, prior to activation with anti-CD3/anti-CD28, diminished the production of interleukin-2 (IL-2) at both the transcript and protein levels. Similarly, the expression of CD25 also diminished, albeit to a lesser degree than IL-2, after pretreatment with tBHQ. The decrease in IL-2 production was not due to decreased nuclear translocation of c-fos or c-jun. Although tBHQ caused both a delay and a decrease in Ca2+ influx in activated Jurkat cells, no decrease in nuclear factor of activated T cells (NFAT) DNA binding or transcriptional activity was observed. In contrast to NFAT, tBHQ significantly decreased NFκB transcriptional activity. Collectively, our studies show that the Nrf2 activator, tBHQ, inhibits IL-2 and CD25 expression, which correlates with decreased NFκB transcriptional activity in activated Jurkat cells. Overall, our studies suggest that Nrf2 represents a novel mechanism for the regulation of both human and mouse T cell function. PMID:23945499

  8. 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

  9. [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.

  10. ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

    PubMed Central

    Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

    2017-01-01

    The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

  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. A synthesized nostocionone derivative potentiates programmed cell death in human T-cell leukemia Jurkat cells through mitochondria via the release of endonuclease G.

    PubMed

    Itoh, Tomohiro; Muramatsu, Yuji; Masu, Masayo; Tsuge, Ayaka; Taniguchi, Masaki; Ninomiya, Masayuki; Ando, Masashi; Tsukamasa, Yasuyuki; Koketsu, Mamoru

    2014-01-01

    Nostocionone (Nost), a compound isolated from Nostoc commune, and its synthesized derivatives (NostDs) were evaluated for in vitro cytotoxicity against human T-cell leukemia Jurkat cells. NostD3 [(1E,4E)-1-(3,4-dihydroxyphenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4-dien-3-one] inhibited cell growth more potently than Nost. To elucidate the mechanisms of NostD3-induced cell death, we examined changes in cell morphology, the loss of mitochondrial membrane potential (MMT), and DNA fragmentation. From these results, the cytotoxic effects of NostD3 were found to be mainly due to Type I programmed cell death (PCDI; i.e., apoptosis). Using caspase inhibitors, we further found that NostD-3-induced PCDI occurred through a caspase-independent pathway. Moreover, NostD3 decreased MMT and modulated multiple signaling molecules (MAPKs, Akt, Bcl-2, Bax, and c-Myc) in Jurkat cells, thereby inducing the release of endonuclease G (Endo-G) from mitochondria. The level of intracellular reactive oxygen species (ROS) in cells treated with NostD3 was elevated up to 1 h after the treatment. However, suppression of ROS by N-acetyl-l-cysteine restored Jurkat cell growth. Taken together, our data suggested that ROS production acted as a trigger in NostD3-induced PCDI in Jurkat cells through release of Endo-G from the mitochondria.

  13. 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.

  14. Butterfat fatty acids differentially regulate growth and differentiation in Jurkat T-cells.

    PubMed

    Bergamo, Paolo; Luongo, Diomira; Maurano, Francesco; Rossi, Mauro

    2005-10-01

    Synthetic Conjugated Linoleic Acid mixture (CLA; c9,t11; t10,c12-18:2) has been previously shown to inhibit growth, and enhance apoptosis and IL-2 mRNA synthesis in human lymphoblastic Jurkat T-cells. In this study, two different butterfat types were evaluated and compared for their effects on Jurkat cell viability, oxidative stress, pro-apoptotic activity, and cytokine synthesis: the conventionally produced butterfat (CBF), and organic butterfat (OBF) containing significantly higher amounts of c9,t11 (Rumenic Acid, RA), trans-vaccenic acid (VA; t11-18:1), alpha-linolenic acid (ALA), and lower levels of linoleic acid (LA). Results from cell treatment with both butterfat mixtures showed comparable oxidative stress (superoxide production, intracellular GSH depletion,and lipid peroxides yield), NADPH oxidase activation, cytotoxicity (LDH release), and IL-2 transcript level, whereas the effects of enhanced growth-inhibitory and pro-apoptotic activities were associated with OBF treatment. To then investigate each butterfat-induced effect caused by RA, VA, LA, and ALA, cells were exposed to synthetic FA concentrations similar to those from the different butterfats. Higher oxidative stress (superoxide production, intracellular GSH depletion) was induced by alpha-linolenic (ALA) and linoleic (LA) incubation (P<0.01) and superoxide production was suppressed by specific PKCalpha inhibitor (Gö 6976) and linked to increased toxicity and IL-2 synthesis inhibition. By contrast, cell treatment with RA increased apoptosis and IL-2 synthesis. These results suggest that a supply of ALA and LA is responsible for BF-induced oxidative stress via PKCalpha-NADPH oxidase pathway, and that enhanced antiproliferative effects in OBF treated cells is essentially determined by RA-induced pro-apoptotic activity. Copyright (c) 2005 Wiley-Liss, Inc.

  15. Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

    PubMed Central

    Gottlieb, R A; Nordberg, J; Skowronski, E; Babior, B M

    1996-01-01

    We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction. Images Fig. 5 PMID:8570610

  16. Photoactivated fullerene C60 induces store-operated Ca2 entry and cytochrome c release in Jurkat cells.

    PubMed

    Grebinyk, S M; Palyvoda, K O; Prylutska, S V; Grynyuk, I I; Samoylenko, A A; Drobot, L B; Matyshevska, O P

    2012-01-01

    The values of endoplasmic reticulum Ca(2+)-pool and store-operated Ca2+ entry (SOCE) were estimated in rat thymocytes and Jurkat cells loaded with indo-1 and treated with thapsigargin. It was shown that the relative value of SOCE in thymocytes was substantially lower than in Jurkat cells. Significant increase of SOCE in Jurkat cells preincubated with 10(-5) M C60 and exposed to uv/visible light irradiation was detected at 1-3 h after exposure. At this time FCCP-induced Ca(2+)-release from mitochondria was shown to be reduced, while cytochrome c level into the cytoplasm of Jurkat cells, detected by Western blot analysis, to be increased. It is supposed that Ca2+ flux remodulation induced by photoexcited fullerene C60 in Jurkat cells might be involved in the initiation of signalling events leading to cell apoptosis.

  17. 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. © 2014 Wiley Periodicals, Inc.

  18. 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.

  19. 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.

  20. Lithium Induces ER Stress and N-Glycan Modification in Galactose-Grown Jurkat Cells

    PubMed Central

    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 Ca2+ 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. PMID:23894652

  1. 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

  2. Nrf2-Dependent and -Independent Effects of tert-Butylhydroquinone, CDDO-Im, and H2O2 in Human Jurkat T Cells as Determined by CRISPR/Cas9 Gene Editing.

    PubMed

    Zagorski, Joseph W; Maser, Tyler P; Liby, Karen T; Rockwell, Cheryl E

    2017-05-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor activated by stimuli such as electrophilic compounds and other reactive xenobiotics. Previously, we have shown that the commonly used food additive and Nrf2 activator tert-butylhydroquinone (tBHQ) suppresses interleukin-2 (IL-2) production, CD25 expression, and NFκB activity in human Jurkat T cells. The purpose of the current studies was to determine whether these effects were dependent upon Nrf2 by developing a human Nrf2-null T cell model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. The current studies show that suppression of CD25 expression by tBHQ is partially dependent on Nrf2, whereas inhibition of IL-2 secretion is largely Nrf2-independent. Interestingly, tBHQ inhibited NFκB activation in an Nrf2-independent manner. This was an unexpected finding since Nrf2 inhibits NFκB activation in other models. These results led us to investigate another more potent Nrf2 activator, the synthetic triterpenoid 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Treatment of wild-type and Nrf2-null Jurkat T cells with CDDO-Im resulted in an Nrf2-dependent suppression of IL-2. Furthermore, susceptibility to reactive oxygen species was significantly enhanced in the Nrf2-null clones as determined by decreased mitochondrial membrane potential and cell viability. Importantly, this study is the first to describe the generation of a human Nrf2-null model, which is likely to have multiple applications in immunology and cancer biology. Collectively, this study demonstrates a role for Nrf2 in the effects of CDDO-Im on CD25 and IL-2 expression, whereas the effect of tBHQ on these parameters is complex and likely involves modulation of multiple stress-activated transcription factors, including NFκB and Nrf2. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  3. 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

  4. Arecoline inhibits interleukin-2 secretion in Jurkat cells by decreasing the expression of alpha7-nicotinic acetylcholine receptors and prostaglandin E2.

    PubMed

    Hwang, G S; Hu, S; Lin, Y H; Chen, S T; Tang, T K; Wang, P S; Wang, S W

    2013-10-01

    The purpose of the present study was to explore the effect of arecoline on phytohemagglutinin (PHA)-stimulated interleukin-2 (IL-2) secretion, the expression of alpha7-nicotinic acetylcholine receptors (α7-nAChRs), prostaglandin E2(PGE2) protein, and IL-2 mRNA in human lymphocyte cells (Jurkat cell line). The IL-2 and PGE2 were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of phosphorylated extracellular signal-regulated kinase (ERK) and α7-nAChRs were determined by Western blotting. The level of IL-2 mRNA was determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Arecoline, in a dose-dependent manner, significantly decreased IL-2 and PGE2 secretion by Jurkat cells incubated with 0 or 5 μg/ml 5 μg/ml PHA. PGE2 also significantly inhibited IL-2 secretion by Jurkat cells in a dose-dependent manner. In addition, reduced expression of PHA-induced ERK phosphorylation was observed in Jurkat cells treated with arecoline. PHA-enhanced IL-2 mRNA expression was also inhibited by arecoline. These results imply that arecoline inhibits the release of PGE2 and PHA-induced IL-2 secretion by Jurkat cells and that these effects seem to occur, at least in part, either through the attenuation of ERK in conjunction with a decrease of PHA-induced IL-2 mRNA expression. These results imply that arecoline inhibits the protein expression of α7-nAChRs , the release of PGE2 and PHA-induced IL-2 secretion by Jurkat cells.

  5. 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.

  6. IL-1β and IL-18 inhibition of HIV-1 replication in Jurkat cells and PBMCs.

    PubMed

    Wang, Xue; Mbondji-Wonje, Christelle; Zhao, Jiangqin; Hewlett, Indira

    2016-05-13

    HIV-1 infection-induced apoptosis is able to ensure viral replication. The death of some CD4+ T cells residing in lymphoid tissues can be induced by HIV-1 infection through caspase-1 driven pyroptosis with release of cytokine of IL-1β and IL-18. It is not well known whether IL-1β and IL-18 affect HIV-1 replication in lymphocytic cells. Using susceptible lymphocytic cell line, Jurkat cells, and primary peripheral blood mononuclear cells (PBMCs), we studied the effects of IL-1β and IL-18 on HIV-1 replication. We found that treatment with exogenous IL-1β protein (rIL-1β) and IL-18 protein (rIL-18), or expression of IL-1β and IL-18 significantly reduced HIV-1 replication. HIV-1 infection enhanced caspase-3 expression and its activation, and had no effects on caspase-1 activity. Treatment with rIL-1β and rIL-18 dramatically lowered caspase-3 activity. IL-1β and IL-18 also played roles in diminishing reactivation of viral replication from latency in J1.1 cells. These results indicate that IL-1β and IL-18 are able to inhibit HIV-1 replication, and their effects may be due to signaling through apoptosis involved in inactivation of caspase-3 activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Fructose sensitizes Jurkat cells oxidative stress-induced apoptosis via caspase-dependent and caspase-independent mechanisms.

    PubMed

    Diaz-Aguirre, Viviana; Velez-Pardo, Carlos; Jimenez-Del-Rio, Marlene

    2016-11-01

    Whether fructose (FRU), as the sole energy source, confers a metabolic advantage on cancer cells against noxious stimuli is unknown. The aim of this study was to evaluate the effects of low (11 mM), moderate (25 mM), and high (55 mM) FRU concentrations alone or in combination with rotenone (ROT) or doxorubicin (DOX) in Jurkat cells, an acute lymphoblastic leukemia cell model. Glucose (GLU) was used as a control. Using different cell analysis techniques, we demonstrated that FRU was predominantly metabolized via oxidative phosphorylation (∼95%) (i.e., lactate production was reduced >120-fold), resulting in endogenous oxidative stress-induced conditions. The cells were characterized by generation of O2(•)(-) (43%)/ H2 O2 (40%) and activation of NF-κB (∼95-fold increase, fi), c-Jun-N terminal kinase (JNK), p53 (40-fi), and c-Jun (9-fi). In addition, we observed a loss of ΔΨm (10%), activation of caspase-3 (50-fi) and apoptosis-inducing factor (AIF, 2-fi), and condensation and fragmentation of the nuclei [20% by acridine orange/ethidium bromide/Hoechst (AO/EB/H) staining, 15% by flow cytometry] compared to those of GLU 11 at 24 h. Although DOX killed Jurkat cells independent of sugar content in the culture medium, leukemic cells in low, but not high, FRU were extremely sensitive to ROT. Taken together, our findings suggest that Jurkat cells are more susceptible to cell death if forced to shift from GLU metabolism (i.e., aerobic glycolysis) to FRU metabolism (i.e., oxidative phosphorylation) after treatment with mitochondria-targeting molecules. These observations may help elucidate the cell death mechanism of leukemic cells cultured in FRU. © 2016 International Federation for Cell Biology.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Some findings of FADD knockdown in inhibition of HIV-1 replication in Jurkat cells and PBMCs.

    PubMed

    Wang, Xue; Tan, Jiying; Zhao, Jiangqin; Ragupathy, Viswannath; Haleyurgirisetty, Mohan; Hewlett, Indira

    2014-08-01

    Fas-associated protein with death domain (FADD) is a key adaptor molecule transmitting the death signal mediated by death receptors, and it is also required for T cell proliferation. A recent study indicated that FADD is able to affect HIV-1 production, but the mechanism is not known. Using the susceptible Jurkat cell line and peripheral blood mononuclear cells, we studied the effects of FADD on HIV-1 production. TaqMan RT-PCR was used to quantify HIV-1 viral RNA copies, and Western blot analysis was used to detect protein expression. FADD knockdown decreased HIV-1 replication and inactivated caspase-3 activity in the cells and blocked CD4 translocation to the lipid rafts of the plasma membrane. Reduced expression of FADD suppressed TCR signaling through downregulation of TCR, CD3, and Zap-70 in response to HIV-1 infection and blocked the trafficking of TCR, CD3, CD28, and Zap-70 to lipid rafts, leading to reduced activation of NF-κB and NFAT, which are required for HIV-1 replication. FADD knockdown diminished caspase-8 migration to lipid rafts and its expression in response to HIV-1 infection. These results indicate that FADD, as a host pro-apoptotic protein, plays important roles in regulating HIV-1 replication and production in several ways, and apoptotic pathway inhibition is able to decrease HIV-1 replication and production.

  13. 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.

  14. 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

  15. 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

  16. Modulation of stress genes expression profile by nitric oxide-releasing aspirin in Jurkat T leukemia cells.

    PubMed

    Nath, Niharika; Chattopadhyay, Mitali; Kodela, Ravinder; Tian, Song; Vlismas, Peter; Boring, Daniel; Crowell, James A; Kashfi, Khosrow

    2010-06-15

    NO-donating aspirin (NO-ASA, para isomer) has been reported to exhibit strong growth inhibitory effect in Jurkat T-acute lymphoblastic leukemia (T-ALL) cells mediated in part by beta-catenin degradation and caspase activation, but the mechanism(s) still remains unclear. In this study, DNA oligoarrays with 263 genes were used to examine the gene expression profiles relating to stress and drug metabolism, and characterize the stress responses at IC(50) and subIC(50) concentrations of p-NO-ASA (20 and 10microM, respectively) in Jurkat T cells. A total of 22 genes related to heat shock response, apoptosis signaling, detoxifiers and Phase II enzymes, and regulators of cell growth were altered in expression by array analysis based on the expression fold change criteria of > or =1.5-fold or < or =0.65-fold. Real time quantitative RT-PCR confirmed that 20microM p-NO-ASA strongly upregulated the mRNA levels of two heat shock genes HSPA1A (41.5+/-7.01-fold) and HSPA6 (100.4+/-8.11-fold), and FOS (16.2+/-3.2-fold), moderately upregulated HSPH1 (1.71+/-0.43-fold), FMO4 (4.5+/-1.67-fold), CASP9 (1.77+/-0.03-fold), DDIT3 (5.6+/-0.51-fold), and downregulated NF-kappaB1 (0.54+/-0.01-fold) and CCND1 (0.69+/-0.06-fold). Protein levels of Hsp70, the product of HSPA1A, and fos were increased in p-NO-ASA-treated Jurkat T and HT-29 colon cancer cells in a dose-dependent manner. Silencing of Hsp70 enhanced the growth inhibitory effect of p-NO-ASA at low concentrations. The altered gene expression patterns by NO-ASA in Jurkat T cells suggest mechanisms for carcinogen metabolism, anti-proliferative activity and possible chemoprotective activity in T-ALL. Copyright 2010 Elsevier Inc. All rights reserved.

  17. Reduced scytonemin isolated from Nostoc commune induces autophagic cell death in human T-lymphoid cell line Jurkat cells.

    PubMed

    Itoh, Tomohiro; Tsuzuki, Ryosuke; Tanaka, Toshiomi; Ninomiya, Masayuki; Yamaguchi, Yuji; Takenaka, Hiroyuki; Ando, Masashi; Tsukamasa, Yasuyuki; Koketsu, Mamoru

    2013-10-01

    Nostoc commune is a terrestrial benthic blue-green alga that often forms an extended mucilaginous layer on the soil, accumulates on stones and mud in aquatic environments. Reduced-scytonemin (R-scy), isolated from N. commune Vaucher, has been shown to suppress the human T-lymphoid Jurkat cell growth. To reveal the mechanisms underlying the R-scy-mediated inhibition of Jurkat cell growth, we examined cell morphology, DNA fragmentation, and microtubule-associated protein light chain 3 (LC3) modification in these cells. We observed multiple vacuoles as well as the conversion of LC3-I to LC3-II in R-scy-treated cells. These results suggest that the R-scy induced Jurkat cell growth inhibition is attributable to the induction of type II programmed cell death (PCD II; autophagic cell death or autophagy). We further examined the mechanisms underlying R-scy-induced PCDII. The cells treated with R-scy produced large amounts of reactive oxygen species (ROS), leading to the induction of mitochondrial dysfunction. However, the elimination of R-scy-induced ROS by treatment with N-acetyl-L-cysteine (NAC) markedly opposed R-scy-induced PCDII. Based on these results, we conclude that ROS formation plays a critical role in R-scy-induced PCDII. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. 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.

  19. Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

    PubMed Central

    Lee, Young Ah; Kim, Kyeong Ah; Min, Arim

    2014-01-01

    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. PMID:25246714

  20. 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.

  1. Calcium Signals Are Affected by Ciprofloxacin as a Consequence of Reduction of Mitochondrial DNA Content in Jurkat Cells

    PubMed Central

    Kozieł, Rafał; Zabłocki, Krzysztof; Duszyński, Jerzy

    2006-01-01

    The effects of ciprofloxacin on mitochondrial DNA (mtDNA) content, oxygen consumption, mitochondrial membrane potential, cellular ATP formation, and capacitative Ca2+ entry into Jurkat cells were investigated. In cells incubated for several days with 25 μg/ml ciprofloxacin, a 60% reduction of mtDNA content, inhibition of the respiratory chain, and a significant decrease in mitochondrial membrane potential were observed. These changes led to a decrease in the calcium buffering capacity of mitochondria which, in turn, resulted in a gradual inhibition of the capacitative Ca2+ entry. On days 4, 7, and 11 of incubation with ciprofloxacin, the initial rates of Ca2+ entry were reduced by 33%, 50%, and 50%, respectively. Ciprofloxacin caused a transient decrease in the cellular capability for ATP formation. In cells incubated for 15 min with glucose, pyruvate, and glutamine as exogenous fuel, ciprofloxacin reduced ATP content by 16% and 35% on days 4 and 7, respectively, of incubation with the drug. However, on day 11 of incubation with ciprofloxacin, a recovery of cellular ATP formation was observed. In conclusion, long-term exposure of Jurkat cells to ciprofloxacin at a concentration of 25 μg/ml seriously affects cellular energy metabolism and calcium homeostasis. PMID:16641433

  2. 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

  3. 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.

  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

    decreased cell viability caused by ManNAc analogs is attributable to apoptosis. DNA Fragmentation Assays—ManNAc analog-induced apo- ptosis was first...demonstrated by DNA fragmentation , which is a biochemical hallmark of apoptosis. Fig. 4A shows typical DNA fragmentation in cells undergoing apoptosis...morphological change and intranucleosomal DNA fragmentation indicative of apoptosis (47). Similar DNA frag- mentation was observed in cells treated with

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

    PubMed

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

    2012-03-16

    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. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. 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.

  7. 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.

  8. Systems biology approach to transplant tolerance: proof of concept experiments using RNA interference (RNAi) to knock down hub genes in Jurkat and HeLa cells in vitro.

    PubMed

    Lwin, Wint Wah; Park, Ken; Wauson, Matthew; Gao, Qin; Finn, Patricia W; Perkins, David; Khanna, Ajai

    2012-07-01

    Systems biology is gaining importance in studying complex systems such as the functional interconnections of human genes [1]. To investigate the molecular interactions involved in T cell immune responses, we used databases of physical gene-gene interactions to constructed molecular interaction networks (interconnections) with R language algorithms. This helped to identify highly interconnected "hub" genes AT(1)P5C1, IL6ST, PRKCZ, MYC, FOS, JUN, and MAPK1. We hypothesized that suppression of these hub genes in the gene network would result in significant phenotypic effects on T cells and examined this in vitro. The molecular interaction networks were then analyzed and visualized with Cytoscape. Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BrdU labeling, which were measured 36, 72, and 96 h after activation. Following T cell stimulation, we found a significant decrease in ATP production (P < 0.05) when the hub genes ATP5C1 and PRKCZ were knocked down using siRNA transfection, whereas no difference in ATP production was observed in siRNA transfected HeLa cells. However, HeLa cells showed a significant (P < 0.05) decrease in cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN, and FOS were knocked down. In both Jurkat and HeLa cells, targeted gene knockdown using siRNA showed decreased cell proliferation and ATP production in both Jurkat and HeLa cells. However, Jurkat T cells and HELA cells use different hub genes to regulate activation responses. This experiment provides proof of principle of applying siRNA knockdown of T cell hub genes to evaluate their proliferative capacity and ATP production. This novel concept outlines a systems biology approach to identify hub genes for targeted therapeutics. Published by Elsevier Inc.

  9. 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

  10. MUC1 (CD227) interacts with lck tyrosine kinase in Jurkat lymphoma cells and normal T cells.

    PubMed

    Mukherjee, P; Tinder, T L; Basu, G D; Gendler, S J

    2005-01-01

    MUC1 (CD227) is a large transmembrane epithelial mucin glycoprotein, which is aberrantly overexpressed in most adenocarcinomas and is a target for immune therapy for epithelial tumors. Recently, MUC1 has been detected in a variety of hematopoietic cell malignancies including T and B cell lymphomas and myelomas; however, its function in these cells is not clearly defined. Using the Jurkat T cell lymphoma cell line and normal human T cells, we demonstrate that MUC1 is not only expressed in these cells but is also phosphorylated upon T cell receptor (TCR) ligation and associates with the Src-related T cell tyrosine kinase, p56lck. Upon TCR-mediated activation of Jurkat cells, MUC1 is found in the low-density membrane fractions, where linker of T cell activation is contained. Abrogation of MUC1 expression in Jurkat cells by MUC1-specific small interfering RNA resulted in defects in TCR-mediated downstream signaling events associated with T cell activation. These include reduction in Ca2+ influx and extracellular signal-regulated kinase 1/2 phosphorylation, leading to a decrease in CD69 expression, proliferation, and interleukin-2 production. These results suggest a regulatory role of MUC1 in modulating proximal signal transduction events through its interaction with proteins of the activation complex.

  11. 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.

  12. A comprehensive characterization of the impact of mycophenolic acid on the metabolism of Jurkat T cells.

    PubMed

    Fernández-Ramos, Ana A; Marchetti-Laurent, Catherine; Poindessous, Virginie; Antonio, Samantha; Petitgas, Céline; Ceballos-Picot, Irène; Laurent-Puig, Pierre; Bortoli, Sylvie; Loriot, Marie-Anne; Pallet, Nicolas

    2017-09-05

    Metabolic reprogramming is critical for T cell fate and polarization and is regulated by metabolic checkpoints, including Myc, HIF-1α, AMPK and mTORC1. Our objective was to determine the impact of mycophenolic acid (MPA) in comparison with rapamycin (Rapa), an inhibitor of mTORC1, on the metabolism of Jurkat T cells. We identified a drug-specific transcriptome signature consisting of the key enzymes and transporters involved in glycolysis, glutaminolysis or nucleotide synthesis. MPA produced an early and transient drop in the intracellular ATP content related to the inhibition of de novo synthesis of purines, leading to the activation of the energy sensor AMPK. MPA decreases glycolytic flux, consistent with a reduction in glucose uptake, but also in the oxidation of glutamine. Additionally, both drugs reduce aerobic glycolysis. The expression of HIF-1α and Myc, promoting the activation of glycolysis and glutaminolysis, was inhibited by MPA and Rapa. In conclusion, we report that MPA profoundly impacts the cellular metabolism of Jurkat T cells by generating an energetic distress, decreasing the glycolytic and glutaminolytic fluxes and by targeting HIF-1α and Myc. These findings open interesting perspectives for novel combinatorial therapeutic strategies targeting metabolic checkpoints to block the proliferation of T cells.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. Down-Regulation of 11β-Hydroxysteroid Dehydrogenase Type 2 by Bortezomib Sensitizes Jurkat Leukemia T Cells against Glucocorticoid-Induced Apoptosis

    PubMed Central

    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. PMID:23826195

  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. A novel agonistic anti-human death receptor 5 monoclonal antibody with tumoricidal activity induces caspase- and mitochondrial-dependent apoptosis in human leukemia Jurkat cells.

    PubMed

    Du, Yao-Wu; Chen, Ju-Gao; Bai, Hui-Ling; Huang, Hong-Ying; Wang, Jing; Li, Shu-Lian; Liu, Guang-Chao; Jiang, Qi; Chai, Jing; Zhao, Yue-Ping; Ma, Yuan-Fang

    2011-04-01

    An agonistic antibody against TNF-related apoptosis-inducing ligand death receptor 5 (DR5) is a practicable candidate drug for antitumor therapy. In this study, a novel murine anti-human DR5 monoclonal antibody, mDRA-6(IgG1-κ), has been generated. This study aimed to explore the caspase-dependent and mitochondrial mechanisms of mDRA-6 in inducing apoptosis in human leukemia Jurkat cells. The apoptotic effects of mDRA-6 on Jurkat cells, which express DR5 on the cell surface, were detected by flow cytometry and western blot after exposure to different doses of mDRA-6 and at fixed doses of mDRA-6 at different times. It was demonstrated that mDRA-6 can induce Jurkat cell apoptosis via caspase- and mitochondrial-dependent pathways. These results indicate that the novel antibody mDRA-6 against DR5 has an antitumor function and may provide a new reagent for tumor therapy.

  5. DIFFERENTIAL DIELECTRIC RESPONSES OF CHONDROCYTE AND JURKAT CELLS IN ELECTROMANIPULATION BUFFERS

    PubMed Central

    Sabuncu, Ahmet C.; Asmar, Anthony J.; Stacey, Michael W.; Beskok, Ali

    2015-01-01

    Electromanipulation of cells as a label free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation, particularly dielectrophoresis (DEP), to biological cells is limited to cells suspended in buffers containing lower amounts of salts relative to the physiological buffers. One might question the use of low conductivity buffers (LCB) for DEP separation, as cells are stressed in buffers lacking physiological levels of salt. In LCB, cells leak ions and undergo volume regulation. Therefore, cells exhibit time-dependent DEP response in LCB. In this work, cellular changes in LCB are assessed by dielectric spectroscopy, cell viability assay, and gene expression of chondrocytes and Jurkats. Results indicate leakage of ions from cells, increases in cytoplasmic conductivity, membrane capacitance and conductance. Separability factor, which defines optimum conditions for DEP cell separation, for the two cell types is calculated using the cellular dielectric data. Optimum DEP separation conditions change as cellular dielectric properties evolve in LCB. Genetic analyses indicate no changes in expression of ionic channel proteins for chondrocytes suspended in LCB. Retaining cellular viability might be important during dielectrophoretic separation, especially when cells are to be biologically tested at a downstream microfluidic component. PMID:25958778

  6. Differential dielectric responses of chondrocyte and Jurkat cells in electromanipulation buffers.

    PubMed

    Sabuncu, Ahmet C; Asmar, Anthony J; Stacey, Michael W; Beskok, Ali

    2015-07-01

    Electromanipulation of cells as a label-free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation, particularly dielectrophoresis (DEP), to biological cells is limited to cells suspended in buffers containing lower amounts of salts relative to the physiological buffers. One might question the use of low conductivity buffers (LCBs) for DEP separation, as cells are stressed in buffers lacking physiological levels of salt. In LCB, cells leak ions and undergo volume regulation. Therefore, cells exhibit time-dependent DEP response in LCB. In this work, cellular changes in LCB are assessed by dielectric spectroscopy, cell viability assay, and gene expression of chondrocytes and Jurkats. Results indicate leakage of ions from cells, increases in cytoplasmic conductivity, membrane capacitance, and conductance. Separability factor, which defines optimum conditions for DEP cell separation, for the two cell types is calculated using the cellular dielectric data. Optimum DEP separation conditions change as cellular dielectric properties evolve in LCB. Genetic analyses indicate no changes in expression of ionic channel proteins for chondrocytes suspended in LCB. Retaining cellular viability might be important during dielectrophoretic separation, especially when cells are to be biologically tested at a downstream microfluidic component. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Glycosyl chains and 25-hydroxycholesterol contribute to the intracellular transport of amyloid beta (Aβ-42) in Jurkat T cells.

    PubMed

    Sharma, Neha; Baek, KeangOK; Phan, Huong T T; Shimokawa, Naofumi; Takagi, Masahiro

    2017-06-01

    Amyloid beta (Aβ) is a peptide responsible for the development of Alzheimer's disease (AD). Misfolding and accumulation of endogenous Aβ can lead to neural cell apoptosis through endoplasmic reticulum (ER) stress. Added exogenous Aβ can also result in ER stress, leading to neurotoxicity and apoptosis, which is identical to that caused by the endogenous peptide. We have speculated that the endocytic transport of Aβ causes ER stress and have previously shown that the oxysterol, in particular, 7-ketocholesterol (7-keto) induces more surface interaction between Aβ-42 and Jurkat cells than cholesterol. However, the interaction was not enough to induce intracellular transfer of the peptide. In this study, we investigated the effect of another oxysterol, 25-hydroxycholesterol (25-OH) on the membrane raft-dependent transport of Aβ-42 in Jurkat cells. Interestingly, intracellular transfer of Aβ-42 was observed in the presence of 25-OH only after the inclusion of cholera toxin B subunit (CT-B), a marker used to detect the raft domain. We speculated that 25-OH can induce intracellular movement of Aβ peptides. Furthermore, CT-B together with GM1 provided negative curvature, which resulted in the intracellular transport of Aβ-42. Notably, we used a protofibrillar species of Aβ-42 in this study. We have shown that the transport was microtubule-dependent since it could not be observed in depolymerized microtubules. These results demonstrate that oxysterols and glycosyl chains are important factors affecting intracellular transport. These compounds are also associated with aging and advanced glycation are risk factors for AD. Thus, this study should further understanding of the pathology of AD.

  8. 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.

  9. Development of a luciferase reporter Jurkat cell line under the control of endogenous interleukin-2 promoter.

    PubMed

    Liu, Jinqi; Liu, Ren; Gray, Peter; Liu, Zhenyi; Cui, Xiaoxia; Li, Guanghua; Liu, Zhong

    2017-08-26

    During new drug development, it is critical to have a cell-based reporter bioassay to measure drug-mediated physiological changes. In a conventional reporter cell line, a reporter expression construct is randomly inserted into the host cell genome with the reporter gene under control of an engineered promoter. This design ensures high signal output but may not represent the true physiological cell signaling. Here we used the CRISPR/Cas9 technology to engineer a Jurkat cell line by replacing one interleukin 2 (IL2) allele with firefly luciferase gene while keeping the other IL2 allele intact. The expression of luciferase is thus under control of endogenous IL2 promoter. We demonstrated that, in this engineered cell line, the IL-2 secretion pathway remained intact and luciferase activity significantly increased upon stimulation with phorbol ester or CD3/CD28 antibodies. We next expressed glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) in this cell line and observed dose-dependent IL-2 and luciferase responses to GITR agonist antibody. Thus we have successfully constructed a reporter cell line by engineering a reporter gene under control of an endogenous target gene promoter. This novel strategy may provide a more physiologically relevant alternative to the traditional method of reporter cell line construction. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Caspase-dependent inhibition of store-operated Ca{sup 2+} entry into apoptosis-committed Jurkat cells

    SciTech Connect

    Onopiuk, Marta; Wierzbicka, Katarzyna; Brutkowski, Wojciech; Szczepanowska, Joanna; Zablocki, Krzysztof

    2010-08-20

    Activation of T-cells triggers store-operated Ca{sup 2+} entry, which begins a signaling cascade leading to induction of appropriate gene expression and eventually lymphocyte proliferation and differentiation. The simultaneous enhancement of Fas ligand gene expression in activated cells allows the immune response to be limited by committing the activated cells to apoptosis. In apoptotic cells the store-operated calcium entry is significantly inhibited. It has been documented that moderate activation of Fas receptor may cause reversible inhibition of store-operated channels by ceramide released from hydrolyzed sphingomyelin. Here we show that activation of Fas receptor in T-cells results in caspase-dependent decrease of cellular STIM1 and Orai1 protein content. This effect may be responsible for the substantial inhibition of Ca{sup 2+} entry into Jurkat cells undergoing apoptosis. In turn, this inhibition might prevent overloading of cells with calcium and protect them against necrosis. -- Research highlights: {yields} Fas activation reduces STIM1 and Orai1 protein content in caspase dependent manner. {yields} Fas activation partially reduces mitochondrial potential in caspase dependent manner. {yields} Fas stimulation inhibits of store-operated Ca{sup 2+} entry in caspase dependent manner. {yields} Inhibition of Ca{sup 2+} entry in apoptotic cells may protect them from secondary necrosis.

  11. 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.

  12. 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.

  13. [The role of heat shock proteins 27 and 70 in redox-dependent regulation of apoptosis in Jurkat tumor cells].

    PubMed

    Nosareva, O L; Ryazantseva, N V; Stepovaya, E A; Shakhristova, E V; Stepanova, E A; Gulaya, V S

    2016-11-01

    Heat shock proteins Hsp) act as molecular chaperones, protecting enzymes and other proteins against reactive oxygen species. The objective of the study was to investigate the role of Hsp27 in maintaining the balance of the glutathione system and Hsp70 concentrations as well as in implementing Jurkat tumor cell apoptosis. Addition of the Hsp27 inhibitor KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)-isoxazol) to Jurkat cells resulted in glutathione redox imbalance (increased GSSG and increased glutathione reductase activity), a decrease in Hsp70 concentrations, and also increased cell apoptosis as compared with to the intact cell culture. The proposed selective regulation of chaperone activity is a promising direction in regulating apoptosis at the cellular level.

  14. 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.

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

    DTIC Science & Technology

    2016-05-02

    membrane pores. These “electro-pores” allow for the transfer of genetic and proteomic material , drugs and chemicals into a cell, for the purpose of inducing...calcium was likely due to several mechanisms, including the for- mation of nanopores , intracellular calcium release from internal calcium stores such...mechanisms responsible for nanoporation , we performed a microarray analysis of both U937 and Jurkat cells exposed to 100 nsEPs at a duration of 10 ns

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. Kaempferol Activates G₂-Checkpoint of the Cell Cycle Resulting in G₂-Arrest and Mitochondria-Dependent Apoptosis in Human Acute Leukemia Jurkat T Cells.

    PubMed

    Kim, Ki Yun; Jang, Won Young; Lee, Ji Young; Jun, Do Youn; Ko, Jee Youn; Yun, Young Ho; Kim, Young Ho

    2016-02-01

    The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G2-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G2- arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G1 cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G2-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G2-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G2-checkpoint activation, which caused not only G2-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondriadependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δpsim loss, and caspase cascade activation.

  6. Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in Jurkat cells.

    PubMed

    Szabo, M; Czompoly, T; Kvell, K; Talaber, G; Bartis, D; Nemeth, P; Berki, T; Boldizsar, F

    2012-02-01

    Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling.

  7. Role of intracellular labile iron, ferritin, and antioxidant defence in resistance of chronically adapted Jurkat T cells to hydrogen peroxide.

    PubMed

    Al-Qenaei, Abdullah; Yiakouvaki, Anthie; Reelfs, Olivier; Santambrogio, Paolo; Levi, Sonia; Hall, Nick D; Tyrrell, Rex M; Pourzand, Charareh

    2014-03-01

    To examine the role of intracellular labile iron pool (LIP), ferritin (Ft), and antioxidant defence in cellular resistance to oxidative stress on chronic adaptation, a new H2O2-resistant Jurkat T cell line "HJ16" was developed by gradual adaptation of parental "J16" cells to high concentrations of H2O2. Compared to J16 cells, HJ16 cells exhibited much higher resistance to H2O2-induced oxidative damage and necrotic cell death (up to 3mM) and had enhanced antioxidant defence in the form of significantly higher intracellular glutathione and mitochondrial ferritin (FtMt) levels as well as higher glutathione-peroxidase (GPx) activity. In contrast, the level of the Ft H-subunit (FtH) in the H2O2-adapted cell line was found to be 7-fold lower than in the parental J16 cell line. While H2O2 concentrations higher than 0.1mM fully depleted the glutathione content of J16 cells, in HJ16 cells the same treatments decreased the cellular glutathione content to only half of the original value. In HJ16 cells, H2O2 concentrations higher than 0.1mM increased the level of FtMt up to 4-fold of their control values but had no effect on the FtMt levels in J16 cells. Furthermore, while the basal cytosolic level of LIP was similar in both cell lines, H2O2 treatment substantially increased the cytosolic LIP levels in J16 but not in HJ16 cells. H2O2 treatment also substantially decreased the FtH levels in J16 cells (up to 70% of the control value). In contrast in HJ16 cells, FtH levels were not affected by H2O2 treatment. These results indicate that chronic adaptation of J16 cells to high concentrations of H2O2 has provoked a series of novel and specific cellular adaptive responses that contribute to higher resistance of HJ16 cells to oxidative damage and cell death. These include increased cellular antioxidant defence in the form of higher glutathione and FtMt levels, higher GPx activity, and lower FtH levels. Further adaptive responses include the significantly reduced cellular response to

  8. 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.

  9. 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

  10. 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

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

    PubMed

    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

    2008-04-11

    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. 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. 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. 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 by serum in Jurkat cells and the

  12. 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

  13. Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells

    PubMed Central

    Kaizuka, Yoshihisa; Douglass, Adam D.; Varma, Rajat; Dustin, Michael L.; Vale, Ronald D.

    2007-01-01

    T cells interacting with antigen-presenting cells (APCs) form an “immunological synapse” (IS), a bull's-eye pattern composed of a central supramolecular activation cluster enriched with T cell receptors (TCRs) surrounded by a ring of adhesion molecules (a peripheral supramolecular activation cluster). The mechanism responsible for segregating TCR and adhesion molecules remains poorly understood. Here, we show that immortalized Jurkat T cells interacting with a planar lipid bilayer (mimicking an APC) will form an IS, thereby providing an accessible model system for studying the cell biological processes underlying IS formation. We found that an actin-dependent process caused TCR and adhesion proteins to cluster at the cell periphery, but these molecules appeared to segregate from one another at the earliest stages of microdomain formation. The TCR and adhesion microdomains attached to actin and were carried centripetally by retrograde flow. However, only the TCR microdomains penetrated into the actin-depleted cell center, whereas the adhesion microdomains appeared to be unstable without an underlying actin cytoskeleton. Our results reveal that TCR and adhesion molecules spatially partition from one another well before the formation of a mature IS and that differential actin interactions help to shape and maintain the final bull's-eye pattern of the IS. PMID:18077330

  14. Caspase-6 Induces 7A6 Antigen Localization to Mitochondria During FAS-induced Apoptosis of Jurkat Cells.

    PubMed

    Suita, Hiroaki; Shinomiya, Takahisa; Nagahara, Yukitoshi

    2017-04-01

    Mitochondria are central to apoptosis. However, apoptosis progression involving mitochondria is not fully understood. A factor involved in mitochondria-mediated apoptosis is 7A6 antigen. 7A6 localizes to mitochondria from the cytosol during apoptosis, which seems to involve 'effector' caspases. In this study, we investigated the precise role of effector caspases in 7A6 localization to mitochondria during apoptosis. Human T-cell lymphoma Jurkat cells were treated with an antibody against FAS. 7A6 localization was analyzed by confocal laser scanning microscopy and flow cytometry. Caspases activation was determined by western blot analysis. 7A6 localization to mitochondria during anti-FAS-induced apoptosis was significantly reduced by the caspase-6 inhibitor, N-acetyl-Val-Glu-Ile-Asp-aldehyde, but not by the caspase-3 inhibitor, N-acetyl-Asp-Asn-Leu-Asp-aldehyde, nor caspase-7/3 inhibitor, N-acetyl-Asp-Gln-Thr-Asp-aldehyde. Moreover, caspase-6 down-regulation suppressed 7A6 localization to mitochondria. Caspase-6 regulates 7A6 localization to mitochondria during anti-FAS-induced apoptosis of Jurkat cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  15. 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.

  16. 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

  17. Increased expression of Lewis X and Y antigens on the cell surface and FUT 4 mRNA during granzyme B-induced Jurkat cell apoptosis.

    PubMed

    Azuma, Yutaro; Kurusu, Yoshikazu; Sato, Hirotaka; Higai, Koji; Matsumoto, Kojiro

    2007-04-01

    Cytotoxic T cells and natural killer cells play key roles in cell-mediated cytotoxicity and can induce apoptosis in virus-infected and malignant cells by releasing cytotoxic granules. In the current study, apoptosis was induced in Jurkat cells, a human T cell line, by delivering granzyme B into the cells using BioPORTER, a cationic lipid formulation. During granzyme B-induced apoptosis, there was an increase in the cell surface expression of Lewis X and Y antigens. To clarify the roles of initiator and executioner caspases in the expression of Lewis X and Y antigens, we treated Jurkat cells with granzyme B in the presence of caspase 3, 8, and 9 inhibitors. The results indicated that delivery of granzyme B into Jurkat cells induces apoptosis by activating caspase 3 and that caspase 3 but not caspase 8 and 9 plays a key role in enhancing the expression of Lewis X and Y antigens. Real-time PCR revealed that expression of the mRNAs for alpha1,3-fucosyltransferases FUT4 was increased at 3 h during granzyme B-induced apoptosis, while FUT9 mRNA expression gradually increased after 12 h. This increased expression of FUT4 mRNA occurred downstream of caspase 3 activation and resulted in the increased cell surface expression of Lewis X and Y antigens.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. The distinct role of guanine nucleotide exchange factor Vav1 in Bcl-2 transcription and apoptosis inhibition in Jurkat leukemia T cells

    PubMed Central

    Yin, Jie; Wan, Ya-juan; Li, Shi-yang; Du, Ming-juan; Zhang, Cui-zhu; Zhou, Xing-long; Cao, You-jia

    2011-01-01

    Aim: To investigate a novel function of proto-oncogene Vav1 in the apoptosis of human leukemia Jurkat cells. Methods: Jurkat cells, Jurkat-derived vav1-null cells (J.Vav1) and Vav1-reconstituted J.WT cells were treated with a Fas agonist antibody, IgM clone CH11. Apoptosis was determined using propidium iodide (PI) staining, Annexin-V staining, DNA fragmentation, cleavage of caspase 3/caspase 8, and poly (ADP-ribose) polymerase (PARP). Mitochondria transmembrane potential (ΔΨm) was measured using DiOC6(3) staining. Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot, respectively. Bcl-2 promoter activity was analyzed using luciferase reporter assays. Results: Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells. J.Vav1 cells lost mitochondria transmembrane potential (ΔΨm) more rapidly upon Fas induction. These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells. The expression of Vav1 increased the transcription of pro-survival Bcl-2. The guanine nucleotide exchange activity of Vav1 was required for enhancing Bcl-2 promoter activity, and the Vav1 downstream substrate, small GTPase Rac2, was likely involved in the control of Bcl-2 expression. Conclusion: Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity. PMID:21151158

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Pulse and Trapezoidal Voltage Clamp Applied To Jurkat Cells: A T- Lymphocyte Cell Line

    DTIC Science & Technology

    1993-04-01

    by the wholp cell patch technique, use a protocol of volLage pulses similar to that of Hodgkin and Huxley in their famous 1952 series of papers on...with the voltage pulses that produced them. We call the above protocol the HH protocol after Hodgkin and Huxley . Upward deflection in the current...T-cell intracellular calcium flux c-myc expression via a potassium channel. Journal of Neuroimmunology. 27: 163-171, 1990. (7) Hodgkin , A. L., and A

  11. Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells.

    PubMed

    Ulmer, Candice Z; Yost, Richard A; Chen, Jing; Mathews, Clayton E; Garrett, Timothy J

    2015-06-01

    Metabolomics is the comprehensive study of metabolism as it pertains to an organism or biological system. Lipidomics, a subset discipline of metabolomics, encompasses the study of cellular lipid functions: including pathways, networks, and interactions. The abundance of metabolites and lipids, along with their contribution to health and disease, makes metabolomics a valuable tool for biomarker research. Disease biomarker identification requires a reproducible, sensitive, and accurate analytical platform. Although transcriptomic and proteomic areas have well-established protocols for sample preparation and data processing, the metabolomics field is still developing comparable standardized conventions. Furthermore, of the few comparative LC-MS metabolomic studies that have been applied to mammalian cell cultures, most are targeted to adherent cell lines. The purpose of this work was to optimize a sample preparation workflow for the cellular metabolomic analysis of suspension-cultured mammalian cells using commercially available Jurkat T lymphocytes as a model system. The current investigation evaluated commonly used sample preparation techniques for reproducibility, accuracy, and applicability to untargeted biomarker discovery. Results show ammoniated cell rinsing solutions to be an effective means to remove extracellular components present in the media without causing ion suppression or affecting the integrity of the cellular membrane. Additionally, a novel workflow was designed to allow for the combined analysis of metabolites and lipids from mammalian suspension cells from a single cell pellet. The Folch lipid extraction protocol was coupled to an 80% MeOH metabolite isolation to ensure high extraction efficiency for phospholipids and triacylglycerides. While the workflow was tailored to cells in suspension, it could also be applied to adherent cell lines.

  12. Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells

    PubMed Central

    Ulmer, Candice Z.; Yost, Richard A.; Chen, Jing; Mathews, Clayton E.; Garrett, Timothy J.

    2015-01-01

    Metabolomics is the comprehensive study of metabolism as it pertains to an organism or biological system. Lipidomics, a subset discipline of metabolomics, encompasses the study of cellular lipid functions: including pathways, networks, and interactions. The abundance of metabolites and lipids, along with their contribution to health and disease, makes metabolomics a valuable tool for biomarker research. Disease biomarker identification requires a reproducible, sensitive, and accurate analytical platform. Although transcriptomic and proteomic areas have well-established protocols for sample preparation and data processing, the metabolomics field is still developing comparable standardized conventions. Furthermore, of the few comparative LC-MS metabolomic studies that have been applied to mammalian cell cultures, most are targeted to adherent cell lines. The purpose of this work was to optimize a sample preparation workflow for the cellular metabolomic analysis of suspension-cultured mammalian cells using commercially available Jurkat T lymphocytes as a model system. The current investigation evaluated commonly used sample preparation techniques for reproducibility, accuracy, and applicability to untargeted biomarker discovery. Results show ammoniated cell rinsing solutions to be an effective means to remove extracellular components present in the media without causing ion suppression or affecting the integrity of the cellular membrane. Additionally, a novel workflow was designed to allow for the combined analysis of metabolites and lipids from mammalian suspension cells from a single cell pellet. The Folch lipid extraction protocol was coupled to an 80% MeOH metabolite isolation to ensure high extraction efficiency for phospholipids and triacylglycerides. While the workflow was tailored to cells in suspension, it could also be applied to adherent cell lines. PMID:26401069

  13. Antitumor and immunomodulatory activities of thiosemicarbazones and 1,3-Thiazoles in Jurkat and HT-29 cells.

    PubMed

    Dos Santos, Thiago André R; da Silva, Aline Caroline; Silva, Elany Barbosa; Gomes, Paulo André Teixeira de Moraes; Espíndola, José Wanderlan Pontes; Cardoso, Marcos Veríssimo de Oliveira; Moreira, Diogo Rodrigo Magalhaes; Leite, Ana Cristina Lima; Pereira, Valéria R A

    2016-08-01

    Cancer remains a high incidence and mortality disease, causing around 8.2 million of deaths in the last year. Current chemotherapy needs to be expanded, making research for new drugs a necessary task. Immune system modulation is an emerging concept in cancer cell proliferation control. In fact, there are a number of mechanisms underlying the role immune system plays in tumor cells. In this work, we describe the structural design, synthesis, antitumor and immunomodulatory potential of 31 new 1,3-thiazole and thiosemicarbazone compounds. Cisplatin was used as anticancer drug control. Cytotoxicity against J774A.1 macrophages and antitumor activity against HT-29 and Jurkat cells was determined. These 1,3-thiazole and thiosemicarbazone compounds not only exhibited cytotoxicity in cancer cells, but were able to cause irreversible cancer cell damage by inducing necrosis and apoptosis. In addition, these compounds, especially pyridyl-thiazoles compounds, regulated immune factors such as interleukin 10 and tumor necrosis factor, possible by directing immune system in favor of modulating cancer cell proliferation. By examining their pharmacological activity, we were able to identify new potent and selective anticancer compounds. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  14. Significance of the rapid increase in GSH levels in the protective response to cadmium exposure through phosphorylated Nrf2 signaling in Jurkat T-cells.

    PubMed

    Ogasawara, Yuki; Takeda, Yuko; Takayama, Hazuki; Nishimoto, Shouichi; Ichikawa, Keisuke; Ueki, Maiko; Suzuki, Toshihiro; Ishii, Kazuyuki

    2014-04-01

    Although cadmium (Cd) is a redox system disruptor, the systematic defensive responses to Cd-induced oxidative stress remain unclear. In this study, we initially determined that when human T-cell-derived Jurkat cells were exposed to a low concentration of Cd, the glutathione (GSH) concentration rapidly increased via the transient nuclear accumulation of the transcription factor Nrf2. Therefore, we hypothesized that this increase in the GSH levels was a significant event that occurred in response to the Cd toxicity in the Jurkat T-cells. To test this hypothesis, the expression of Nrf2 in the cells was silenced using siRNA transfection. These restricted expression conditions demonstrated that the sensitivity of the Jurkat T-cells to Cd toxicity was significantly higher in the knockdown cells. Whereas we could not find differences in the metallothionein (MT) expression responses, accumulation of Nrf2 in the nuclei and the GSH increase after Cd exposure were clearly suppressed in the Nrf2 knockdown cells. These findings strongly suggest that the Cd-induced activation of GSH synthesis is initiated as an acute response for Cd detoxification. Furthermore, the Cd remaining in the Jurkat T-cells did not cause a significant inhibition of cell growth after the rapid and transient increase in the GSH concentration returned to its basal level. Additionally, we found that MT expression induced by Cd occurred much later, with the expression seen at least 12h or more after the Nrf2-dependent immediate responses were almost completed. These results indicate that the rapid increase in GSH is an essential defensive response, with the subsequent induction of MT potentially chelating the Cd retained in the cell, thereby leading to continued suppression of Cd toxicity. Copyright © 2014 Elsevier Inc. All rights reserved.

  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. Potentiation of the store-operated calcium entry (SOCE) induces phytohemagglutinin-activated Jurkat T cell apoptosis.

    PubMed

    Djillani, Alaeddine; Doignon, Isabelle; Luyten, Tomas; Lamkhioued, Bouchaib; Gangloff, Sophie C; Parys, Jan B; Nüße, Oliver; Chomienne, Christine; Dellis, Olivier

    2015-08-01

    Store-operated Ca(2+) entry (SOCE) is the main Ca(2+) entry pathway of non-excitable cells. In the past decade, the activation of this entry has been unveiled, with STIM1, a protein of the endoplasmic reticulum able to sense the intraluminal Ca(2+) content, and Orai1, the pore-forming unit of the Ca(2+) release activated Ca(2+) (CRAC) channels. When Ca(2+) ions are released from the endoplasmic reticulum, STIM1 proteins oligomerize and directly interact with Orai1 proteins, allowing the opening of the CRAC channels and a massive Ca(2+) ion influx known as SOCE. As Ca(2+) is involved in various cellular processes, the discovery of new drugs acting on the SOCE should be of interest to control the cell activity. By testing analogs of 2-aminoethyl diphenylborinate (2-APB), a well known, though not so selective effector of the SOCE, we identified methoxy diethylborinate (MDEB), a molecule able to potentiate the SOCE in three leukocyte and two breast cancer cell lines by increasing the Ca(2+) influx amplitude. Unlike 2-APB, MDEB does not affect the Ca(2+) pumps or the Ca(2+) release from the endoplasmic reticulum. MDEB could therefore represent the first member of a new group of molecules, specifically able to potentiate SOCE. Although not toxic for non-activated Jurkat T cells, it could induce the apoptosis of phytohemagglutinin-stimulated cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. 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.

  18. 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.

  19. 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

  20. Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

    SciTech Connect

    O'Connell, A.R.; Lee, B.W.; Stenson-Cox, C. . E-mail: catherine.stenson@nuigalway.ie

    2006-06-30

    Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events.

  1. Characterization of the EBV/C3d receptor on the human Jurkat T cell line: evidence for a novel transcript.

    PubMed

    Sinha, S K; Todd, S C; Hedrick, J A; Speiser, C L; Lambris, J D; Tsoukas, C D

    1993-06-15

    EBV is a human herpes virus that causes mononucleosis and is associated with various tumors. EBV infects cells via the CR2 that was previously thought to be expressed only on the surface of B cells and certain epithelial cells. Recent findings in our laboratory and those of others, however, have shown that the EBV receptor is also present on T cells. Our study shows that Jurkat human T cells have a molecule that reacts with both anti-CR2 antibodies and the third component of complement, C3. Furthermore, the data indicate that this molecule binds EBV detected by incubation with biotin-conjugated virus and streptavidin phycoerythrin. Viral binding is specific, as it is inhibited by nonconjugated virus, with anti-CR2 antibodies, and with an antibody reactive with the glycoprotein (gp350) that EBV uses to bind CR2. In addition, EBV variably infects Jurkat cells as demonstrated by the presence of transcripts of Epstein Barr nuclear Ag (EBNA-1) using the polymerase chain reaction. Immunoprecipitation experiments with anti-CR2 antibodies and SDS-PAGE analysis reveal a protein with an apparent molecular mass of 155 kDa which is higher than the one seen in B cells. The size of this molecule is reduced to 119 kDa upon endoglycosidase F treatment. Northern blot analysis of Jurkat poly(A)+ RNA shows a transcript of 4.7 kb upon probing with the B cell CR2 cDNA. This size is consistent with that of B cell CR2 mRNA. Two cDNA clones were identified upon screening of a Jurkat cell cDNA library with the B cell CR2 cDNA. One of the clones possesses an identical nucleotide sequence to the one corresponding to B cell CR2, whereas the other represents a differentially spliced transcript which lacks the exon 8b of B cell CR2. Analysis of Jurkat and Raji mRNA by PCR demonstrated the presence of this novel splice variant in both cell lines.

  2. Novel HIV-1 Knockdown Targets Identified by an Enriched Kinases/Phosphatases shRNA Library Using a Long-Term Iterative Screen in Jurkat T-Cells

    PubMed Central

    Rato, Sylvie; Maia, Sara; Brito, Paula M.; Resende, Leonor; Pereira, Carina F.; Moita, Catarina; Freitas, Rui P.; Moniz-Pereira, José; Hacohen, Nir; Moita, Luis Ferreira; Goncalves, Joao

    2010-01-01

    HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies. PMID:20174665

  3. 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

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

    PubMed Central

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

    2012-01-01

    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. PMID:22219382

  5. 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.

  6. Increase of RhoB in gamma-radiation-induced apoptosis is regulated by c-Jun N-terminal kinase in Jurkat T cells.

    PubMed

    Kim, Chun-Ho; Won, Misun; Choi, Chung-Hae; Ahn, Jiwon; Kim, Bo-Kyung; Song, Kyung-Bin; Kang, Chang-Mo; Chung, Kyung-Sook

    2010-01-08

    The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in gamma-radiation-induced apoptosis. However, it is unclear how JNK activation and gamma-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by gamma-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. gamma-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the gamma-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to gamma-radiation. Copyright 2009 Elsevier Inc. All rights reserved.

  7. Inhibitors of MAPK pathway ERK1/2 or p38 prevent the IL-1{beta}-induced up-regulation of SRP72 autoantigen in Jurkat cells.

    PubMed

    Arana-Argáez, Victor E; Delgado-Rizo, Vidal; Pizano-Martínez, Oscar E; Martínez-Garcia, Erika A; Martín-Márquez, Beatriz T; Muñoz-Gómez, Andrea; Petri, Marcelo H; Armendáriz-Borunda, Juan; Espinosa-Ramírez, Guillermo; Zúñiga-Tamayo, Diego A; Herrera-Esparza, Rafael; Vázquez-Del Mercado, Mónica

    2010-10-22

    Phosphorylation is the most important post-translational event at a cellular level that is regulated by protein kinases. MAPK is a key player in the important cellular signaling pathway. It has been hypothesized that phosphorylation might have a role in the induction of break tolerance against some autoantigens such as SRP72. The aim of this study was to explore the pathways of phosphorylation and overexpression of the SRP72 polypeptide, using an in vitro model of Jurkat cells stimulated by recombinant human (rh)IL-1β in the presence of MAPK inhibitors. We used Jurkat cells as a substrate stimulated with rhIL-1β in the presence of MAPK inhibitors at different concentrations in a time course in vitro experiment by immunoprecipitation, immunoprecipitation-Western blotting, and real time PCR. Our results showed that rhIL-1β causes up-regulation of protein expression and phosphorylation of SRP72 in Jurkat cells. Inhibitors of the MAPK pathway ERK1/2 or p38α/β down-regulate the expression of SRP72 autoantigen in Jurkat cells stimulated by rhIL-1β. Our results highlight the importance of studying the pathways of activation and overexpression of autoantigens. It will be necessary to perform careful research on various kinases pathways, including MAPK in dermatomyositis and other rheumatic diseases, to help to explain the routes of activation and inhibition of autoantigens. The understanding of this process may help to develop new therapies to prevent and control the loss of tolerance toward own normal proteins.

  8. Inhibitors of MAPK Pathway ERK1/2 or p38 Prevent the IL-1β-induced Up-regulation of SRP72 Autoantigen in Jurkat Cells*

    PubMed Central

    Arana-Argáez, Victor E.; Delgado-Rizo, Vidal; Pizano-Martínez, Oscar E.; Martínez-Garcia, Erika A.; Martín-Márquez, Beatriz T.; Muñoz-Gómez, Andrea; Petri, Marcelo H.; Armendáriz-Borunda, Juan; Espinosa-Ramírez, Guillermo; Zúñiga-Tamayo, Diego A.; Herrera-Esparza, Rafael; Vázquez-Del Mercado, Mónica

    2010-01-01

    Phosphorylation is the most important post-translational event at a cellular level that is regulated by protein kinases. MAPK is a key player in the important cellular signaling pathway. It has been hypothesized that phosphorylation might have a role in the induction of break tolerance against some autoantigens such as SRP72. The aim of this study was to explore the pathways of phosphorylation and overexpression of the SRP72 polypeptide, using an in vitro model of Jurkat cells stimulated by recombinant human (rh)IL-1β in the presence of MAPK inhibitors. We used Jurkat cells as a substrate stimulated with rhIL-1β in the presence of MAPK inhibitors at different concentrations in a time course in vitro experiment by immunoprecipitation, immunoprecipitation-Western blotting, and real time PCR. Our results showed that rhIL-1β causes up-regulation of protein expression and phosphorylation of SRP72 in Jurkat cells. Inhibitors of the MAPK pathway ERK1/2 or p38α/β down-regulate the expression of SRP72 autoantigen in Jurkat cells stimulated by rhIL-1β. Our results highlight the importance of studying the pathways of activation and overexpression of autoantigens. It will be necessary to perform careful research on various kinases pathways, including MAPK in dermatomyositis and other rheumatic diseases, to help to explain the routes of activation and inhibition of autoantigens. The understanding of this process may help to develop new therapies to prevent and control the loss of tolerance toward own normal proteins. PMID:20729213

  9. 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.

  10. 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.

  11. Enhanced expression of membrane-associated sialidase Neu3 decreases GD3 and increases GM3 on the surface of Jurkat cells during etoposide-induced apoptosis.

    PubMed

    Azuma, Yutaro; Sato, Hirotaka; Higai, Koji; Matsumoto, Kojiro

    2007-09-01

    We previously reported that, in Jurkat human T cells, the topoisomerase II inhibitor etoposide enhances sialidase activity and reduces cell surface sialic acid levels at an early stage of apoptosis and that the decreases in sialic acid are suppressed by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid [Azuma Y., et al., Glycoconj. J., 17, 301-306 (2000)]. In the current studies, we treated Jurkat cells with etoposide and examined the changes in the cell surface levels of gangliosides GM1, GM2, GM3, GD1a, and GD3 at physiological pH using anti-ganglioside antibodies. We also examined the sialidase activity on the cell surface using 4-methylumbelliferyl N-acetylneuraminic acid and measured the mRNA expression of the plasma membrane-associated sialidase Neu3 and the lysozomal Neu1 using real-time PCR. We found an increase in GM3 and a decrease in GD3 during the early stage (4 h) of etoposide-induced apoptosis that preceded the increase in cell surface exposure of phosphatidylserine (4 to 6 h). The caspase 3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde significantly suppressed changes in GM3 and GD3 and blocked the enhanced cell surface sialidase activity. Furthermore, etoposide caused a gradual up-regulation of Neu3 mRNA expression but not Neu1 mRNA expression. Enhanced Neu3 mRNA expression was suppressed in the presence of caspase 3 inhibitor. These results indicate that Neu3 is up-regulated in Jurkat cells undergoing etoposide-induced apoptosis through intracellular signaling events downstream of caspase 3 activation and that enhanced Neu3 activity is closely related to the changes of cell surface ganglioside composition.

  12. 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

  13. 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

  14. 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

  15. Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering.

    PubMed

    Zhang, Yun; Liu, Xiaomei; Zhang, Jinju; Zhang, Chun

    2016-09-01

    To develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins. AAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed "CELiD" DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with "CELiD" DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %. The AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.

  16. Epigonal Conditioned Media from Bonnethead Shark, Sphyrna tiburo, Induces Apoptosis in a T-Cell Leukemia Cell Line, Jurkat E6-1

    PubMed Central

    Walsh, Catherine J.; Luer, Carl A.; Yordy, Jennifer E.; Cantu, Theresa; Miedema, Jodi; Leggett, Stephanie R.; Leigh, Brittany; Adams, Philip; Ciesla, Marissa; Bennett, Courtney; Bodine, Ashby B.

    2013-01-01

    Representatives of Subclass Elasmobranchii are cartilaginous fish whose members include sharks, skates, and rays. Because of their unique phylogenetic position of being the most primitive group of vertebrates to possess all the components necessary for an adaptive immune system, the immune regulatory compounds they possess may represent the earliest evolutionary forms of novel compounds with the potential for innovative therapeutic applications. Conditioned medium, generated from short term culture of cells from the epigonal organ of bonnethead sharks (Sphyrna tiburo), has been shown to have potent reproducible cytotoxic activity against a variety of human tumor cell lines in vitro. Existing data suggest that epigonal conditioned medium (ECM) exerts this cytotoxic activity through induction of apoptosis in target cells. This manuscript describes apoptosis induction in a representative tumor cell line, Jurkat E6-1, in response to treatment with ECM at concentrations of 1 and 2 mg/mL. Data indicate that ECM exposure initiates the mitochondrial pathway of apoptosis through activation of caspase enzymes. Future purification of ECM components may result in the isolation of an immune-regulatory compound with potential therapeutic benefit for treatment of human cancer. PMID:24065163

  17. Human fetal retinal pigment epithelium induces apoptosis in human T-cell line Jurkat which is independent from its expression of TRAIL.

    PubMed

    Farrokh-Siar, Lili; Rezai, Kourous A; Palmer, Ellen M; van Seventer, Jean; Hamann, Kimm J; Rajadurai, Henrietta; Patel, Samir C; Ernest, J Terry; van Seventer, Gijs A

    2002-03-01

    To evaluate whether human fetal retinal pigment epithelial (HFRPE) cells express TRAIL (tumor necrosis factor related apoptosis inducing ligand). The role of TRAIL in HFRPE induced apoptosis was evaluated. Pure cultures of HFRPE cells were isolated. The expression of TRAIL protein and mRNA in non-activated and IFN-gamma activated HFRPE cells was evaluated with RT-PCR. The role of TRAIL in HFRPE induced apoptosis was assessed by incubating HFRPE cells with human T-cell leukemia line Jurkat (Jkt) in the presence or absence of neutralizing TRAIL antibodies. Cultures were pulsed with [(3)H]-thymidine to measure Jkt cell proliferation. The role of TRAIL was further examined by western blott evaluating the cleavage of caspases 8 and 10 in Jkt cells after their incubation with HFRPE cells. HFRPE cells expressed TRAIL mRNA. The expression of TRAIL mRNA and protein was up-regulated by IFN-gamma activation. However, anti-TRAIL antibodies were not able to prevent the HFRPE induced suppression of Jkt cell proliferation. The caspases 8 and 10 were also not cleaved in Jkt cells after their incubation with IFN-gamma activated HFRPE cells. Although HFRPE cells express TRAIL and its expression is upregulated by IFN-gamma activation, TRAIL is not involved in HFRPE induced apoptosis in Jkt cells. Currently the role of TRAIL in HFRPE cells is under investigation.

  18. 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

  19. The oncogenic 70Z Cbl mutation blocks the phosphotyrosine binding domain-dependent negative regulation of ZAP-70 by c-Cbl in Jurkat T cells.

    PubMed

    van Leeuwen, J E; Paik, P K; Samelson, L E

    1999-10-01

    T-cell receptor (TCR) engagement results in the activation of Src family (Lck and Fyn) and ZAP-70 protein tyrosine kinases, leading to tyrosine phosphorylation of multiple cellular substrates including the complex adapter protein c-Cbl. Moreover, Cbl is tyrosine phosphorylated upon engagement of growth factor receptors, cytokine receptors, and immunoreceptors and functions as a negative regulator of tyrosine kinase signalling pathways. Cbl associates via its phosphotyrosine binding (PTB) domain to the ZAP-70 pY292 negative regulatory phosphotyrosine. We recently demonstrated that the oncogenic Cbl mutant, 70Z Cbl, requires its PTB domain to upregulate NFAT in unstimulated Jurkat T cells. Here, we demonstrate that kinase-dead but not wild-type forms of Fyn, Lck, and ZAP-70 block 70Z Cbl-mediated NFAT activation. Moreover, 70Z Cbl does not upregulate NFAT in the ZAP-70-deficient P116 Jurkat T-cell line. The requirement for Fyn, Lck, and ZAP-70 is not due to tyrosine phosphorylation of 70Z Cbl, as mutation of all tyrosines in, or deletion of, the C-terminal region of 70Z Cbl (amino acids 655 to 906) blocks 70Z Cbl tyrosine phosphorylation but enhances 70Z Cbl-mediated NFAT activation. Further, 70Z Cbl does not cooperate with ZAP-70 Y292F to upregulate NFAT, indicating that 70Z Cbl and ZAP-70 do not activate parallel signalling pathways. Finally, the upregulation of NFAT observed upon ZAP-70 overexpression is blocked by Cbl in a PTB domain-dependent manner. We conclude that oncogenic 70Z Cbl acts as a dominant negative to block the PTB domain-dependent negative regulatory role of endogenous Cbl on ZAP-70, leading to constitutive ZAP-70 signalling and activation of transcription factors.

  20. 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.

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

    PubMed Central

    Balakrishna, Acharya; Kumar, M. Hemanth

    2015-01-01

    Ayurvedic system of medicine is using Berberis aristata and Curcuma longa herbs to treat different diseases including cancer. The study was performed to evaluate the synergetic anticancer activity of Berberine and Curcumin by estimating the inhibition of the cell proliferation by cytotoxicity assay using MTT method on specified human cell lines (A549, Hep-G2, MCF-7, Jurkat, and K562). All the cells were harvested from the culture and seeded in the 96-well assay plates at seeding density of 2.0 × 104 cells/well and were incubated for 24 hours. Test items Berberine with Curcumin (1 : 1), Curcumin 95% pure, and Berberine 95% pure were exposed at the concentrations of 1.25, 0.001, and 0.5 mg/mL, respectively, and incubated for a period of 48 hours followed by dispensing MTT solution (5 mg/mL). The cells were incubated at 37 ± 1°C for 4 hours followed by addition of DMSO for dissolving the formazan crystals and absorbance was read at 570 nm. Separate wells were prepared for positive control, controls (only medium with cells), and blank (only medium). The results had proven the synergetic anticancer activity of Berberine with Curcumin inducing cell death greater percentage of >77% when compared to pure curcumin with <54% and pure Berberine with <45% on average on all cell line models. PMID:26247019

  2. 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.

  3. FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

    PubMed

    Aguirre, Adam; Shoji, Kenji F; Sáez, Juan C; Henríquez, Mauricio; Quest, Andrew F G

    2013-02-01

    Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. Copyright © 2012 Wiley Periodicals, Inc.

  4. 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.

  5. Detailed Analysis of Apoptosis and Delayed Luminescence of Human Leukemia Jurkat T Cells after Proton Irradiation and Treatments with Oxidant Agents and Flavonoids

    PubMed Central

    Baran, Irina; Ganea, Constanta; Privitera, Simona; Scordino, Agata; Barresi, Vincenza; Musumeci, Francesco; Mocanu, Maria Magdalena; Condorelli, Daniele F.; Ursu, Ioan; Grasso, Rosaria; Gulino, Marisa; Garaiman, Alexandru; Musso, Nicolò; Cirrone, Giuseppe A. Pablo; Cuttone, Giacomo

    2012-01-01

    Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL) in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H2O2. 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H2O2 and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G2/M arrest. Quercetin reduced apoptosis and prolonged the G2/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site. PMID:22829956

  6. 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

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

    PubMed

    Zunino, Susan J; Storms, David H

    2017-08-01

    Resveratrol has been reported to inhibit or induce DNA damage, depending upon the type of cell and the experimental conditions. Dietary resveratrol is present in the body predominantly as metabolites and limited data is available concerning the activities of these metabolic products. In the present study, physiologically obtainable levels of the resveratrol metabolites resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide and resveratrol-3-O-sulfate were evaluated for their ability to protect Jurkat T cells against DNA damage induced by the topoisomerase I inhibitors camptothecin and topotecan. The cells were pretreated for 24 h with 10 µM resveratrol aglycone or each resveratrol metabolite prior to the induction of DNA damage with camptothecin or topotecan. In separate experiments, the cells were co-treated with resveratrol or its metabolites, and a topoisomerase I inhibitor. The detection of histone 2AX phosphorylation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) were used to determine DNA damage, and apoptosis was measured using an antibody against cleaved poly ADP-ribose polymerase. It was identified that pretreatment of the cells with resveratrol-3-O-glucuronide and resveratrol-4'-O-glucuronide reduced the mean fluorescence intensity of staining for DNA strand breaks following treatment with camptothecin, while the percentage of cells undergoing apoptosis was unchanged. However, pretreatment of the cells with resveratrol aglycone increased the DNA damage and apoptosis induced by the drugs. These results suggest that the glucuronide metabolites of resveratrol partially protected the cells from DNA damage, but did not influence the induction of cell death by camptothecin and topotecan. These data suggest that resveratrol aglycone treatment may be beneficial for treating types of cancer that have direct contact with resveratrol prior to its metabolism, including gastrointestinal cancers, which are routinely treated with

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

    PubMed Central

    Zunino, Susan J.; Storms, David H.

    2017-01-01

    Resveratrol has been reported to inhibit or induce DNA damage, depending upon the type of cell and the experimental conditions. Dietary resveratrol is present in the body predominantly as metabolites and limited data is available concerning the activities of these metabolic products. In the present study, physiologically obtainable levels of the resveratrol metabolites resveratrol-3-O-glucuronide, resveratrol-4′-O-glucuronide and resveratrol-3-O-sulfate were evaluated for their ability to protect Jurkat T cells against DNA damage induced by the topoisomerase I inhibitors camptothecin and topotecan. The cells were pretreated for 24 h with 10 µM resveratrol aglycone or each resveratrol metabolite prior to the induction of DNA damage with camptothecin or topotecan. In separate experiments, the cells were co-treated with resveratrol or its metabolites, and a topoisomerase I inhibitor. The detection of histone 2AX phosphorylation and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) were used to determine DNA damage, and apoptosis was measured using an antibody against cleaved poly ADP-ribose polymerase. It was identified that pretreatment of the cells with resveratrol-3-O-glucuronide and resveratrol-4′-O-glucuronide reduced the mean fluorescence intensity of staining for DNA strand breaks following treatment with camptothecin, while the percentage of cells undergoing apoptosis was unchanged. However, pretreatment of the cells with resveratrol aglycone increased the DNA damage and apoptosis induced by the drugs. These results suggest that the glucuronide metabolites of resveratrol partially protected the cells from DNA damage, but did not influence the induction of cell death by camptothecin and topotecan. These data suggest that resveratrol aglycone treatment may be beneficial for treating types of cancer that have direct contact with resveratrol prior to its metabolism, including gastrointestinal cancers, which are routinely treated with

  9. Low activity of LSD1 elicits a pro-inflammatory gene expression profile in riboflavin-deficient human T Lymphoma Jurkat cells.

    PubMed

    Liu, Dandan; Zempleni, Janos

    2014-09-01

    Mono- and dimethylation of lysine (K)-4 in histone H3 (H3K4me1, H3K4me2) create epigenetic gene activation marks that are enriched near the transcription start site of genes. Lysine-specific demethylase 1 (LSD1) is a flavin adenine dinucleotide (FAD)-dependent demethylase that catalyzes the demethylation of H3K4me1 and H3K4me2, thereby mediating gene repression. This study tested the hypothesis that LSD1 activity depends on the concentrations of the FAD precursor, riboflavin, in cell culture media, and that riboflavin deficiency causes derepression of pro-inflammatory cytokines. Human T lymphoma Jurkat cells were cultured in riboflavin-defined media, representing plasma levels of riboflavin in moderately deficient, sufficient, and supplemented humans. The expression of LSD1 mRNA and protein followed the pattern riboflavin-deficient > riboflavin-sufficient > riboflavin-supplemented cells. However, the increase in LSD1 expression was insufficient to compensate for FAD depletion, and LSD activities were more than 30 % higher in riboflavin-supplemented cells compared with the other treatment groups. The enrichment of H3K4me2 marks was 11-137 % greater in riboflavin-deficient cells compared with sufficient cells in exon 1 of genes coding for the pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor-α. Consistent with the enrichment of gene activation marks, the expression of mRNA coding for pro-inflammatory cytokines was 62-487 % higher in riboflavin-deficient cells compared with sufficient cells. These findings support the hypothesis that riboflavin deficiency contributes toward a pro-inflammatory gene expression pattern through a loss of LSD1 activity.

  10. 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.

  11. 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.

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

    PubMed

    Cubano, L A; Lewis, M L

    2001-05-01

    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.

  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. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. Anti-inflammatory properties of potato glycoalkaloids in stimulated Jurkat and Raw 264.7 mouse macrophages.

    PubMed

    Kenny, Olivia M; McCarthy, Catherine M; Brunton, Nigel P; Hossain, Mohammad B; Rai, Dilip K; Collins, Stuart G; Jones, Peter W; Maguire, Anita R; O'Brien, Nora M

    2013-04-19

    The potato glycoalkaloids, α-chaconine, α-solanine and solanidine, along with potato peel extracts were investigated for potential anti-inflammatory effects in vitro. Their potential to reduce two biomarkers of inflammation, cytokine and nitric oxide (NO) productions, were assessed in the stimulated Jurkat and macrophage models, respectively. Cytokine and nitric oxide productions were stimulated in Jurkat and Raw 264.7 macrophages with Concanavalin A (Con A; 25 μg/ml) and lipopolysaccaride (LPS; 1 μg/ml), respectively. Selective concentrations of glycoalkaloids and potato peel extracts were added simultaneously with Con A or LPS for 24h to investigate their potential to reduce inflammatory activity. α-Chaconine and solanidine significantly reduced interleukin-2 (IL-2) and interleukin-8 (IL-8) productions in Con A-induced Jurkat cells. The potato peel extracts did not influence cytokine production. In LPS-stimulated Raw macrophages, α-solanine, solanidine and two potato peel extracts significantly reduced induced NO production. Our findings suggest that sub-cytotoxic concentrations of potato glycoalkaloids and potato peel extracts possess anti-inflammatory effects in vitro and with further investigation may be useful in the prevention of anti-inflammatory diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. 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.

  5. Myristica fragrans Houtt. methanolic extract induces apoptosis in a human leukemia cell line through SIRT1 mRNA downregulation.

    PubMed

    Chirathaworn, Chintana; Kongcharoensuntorn, Wisatre; Dechdoungchan, Thitiporn; Lowanitchapat, Alisa; Sa-nguanmoo, Pattaratida; Poovorawan, Yong

    2007-11-01

    Myristica fragrans Houtt. (nutmeg) contains antibacterial, antiviral and anti-cancer activities. However the mechanisms underlying those activities have not been clearly explained. To study the effect of Myristica fragrans Houtt. methanolic extract on Jurkat human leukemia T cell line. Methanol extract of Myristica fragrans Houtt. (Myristicaceae) was used to study the effect on Jurkat cell metabolic activity using an MTT assay and on apoptosis using annexin V staining. Expression of SIRT1 gene was determined by RT-PCR. At the concentrations 50 and 100 ig/mL, the methanol extract of Myristica fragrans Houtt significantly inhibited Jurkat cell proliferation and induced apoptosis as detected by annexin V staining. Downregulation of SIRT1 mRNA expression in Jurkat cells was observed even when the amount of methanol extract was 10 microg/mL. Methanol extract of Myristica fragrans Houtt induced apoptosis of Jurkat leukemia T cell line in a mechanisms involving SIRTI mRNA downregulation.

  6. Dual-purpose sample trap for on-line strong cation-exchange chromatography/reversed-phase liquid chromatography/tandem mass spectrometry for shotgun proteomics. Application to the human Jurkat T-cell proteome.

    PubMed

    Kang, Dukjin; Nam, Hyungwook; Kim, Yu-Sam; Moon, Myeong Hee

    2005-04-08

    A dual-purpose sample-trapping column is introduced for the capacity enhancement of proteome analysis in on-line two-dimensional nanoflow liquid chromatography (strong cation-exchange chromatography followed by reversed-phase liquid chromatography) and tandem mass spectrometry. A home-made dual trap is prepared by sequentially packing C18 reversed-phase (RP) particles and SCX resin in a silica capillary tubing (1.5 cm x 200 microm I.D. for SCX, 0.7 cm x 200 microm for RP) ended with a home-made frit and is connected to a nanoflow column having a pulled tip treated with an end frit. Without having a separate fraction collection and concentration process, digested peptide mixtures were loaded directly in the SCX part of the dual trap, and the SCX separation of peptides was performed with a salt step elution initiated by injecting only 8 microL of NH4HCO3 solution from the autosampler to the dual trap. The fractionated peptides at each salt step were directly transferred to the RP trap packed right next to the SCX part for desalting, and a nanoflow LC-MS-MS run was followed. During the sample loading-SCX fractionation-desalting, flow direction was set to bypass the analytical column to prevent contamination. The entire 2D-LC separation and MS-MS analysis were automated. Evaluation of the technique was made with an injection of 15 microg peptide mixtures from human Jurkat T-cell proteome, and the total seven salt step cycles followed by each RPLC run resulted in an identification of 681 proteins.

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Membrane chloride conductance and capacitance in Jurkat T lymphocytes during osmotic swelling.

    PubMed Central

    Ross, P E; Garber, S S; Cahalan, M D

    1994-01-01

    Video microscopy and whole-cell patch-clamp recording were used to monitor changes in relative cell volume (V/Vo), chloride conductance (gCl), and membrane capacitance (Cm) during osmotically induced swelling in Jurkat T lymphocytes. Cellular swelling was initiated with hyperosmotic pipette solutions. Simultaneous evaluation of V/Vo and gCl revealed a 59-s delay between the inception of swelling and the activation of outwardly rectifying, ATP-dependent Cl- channels. Following the delay, increases in V/Vo and gCl progressed in parallel. In contrast, Cm, a measure of cell surface area, fell gradually at a rate of approximately 150 fF/min after whole-cell access was achieved. The decline in Cm lasted 200 s and was followed by a rapid rise (approximately 750 fF/min). The rise in Cm coincided with a variable increase in "leak" current, gCl increased at a slower rate and reached lower peak values in experiments performed without ATP; ATP had no effect on the biphasic Cm time course. The temporal separation of conductance and capacitance during swelling suggests that gCl and Cm vary independently, supporting the hypothesis that a large portion, if not all, of the whole-cell Cl- conductance activated during swelling is provided by volume-sensitive Cl- channels preexisting in the plasma membrane. Images FIGURE 6 PMID:8130336

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

    SciTech Connect

    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 (IC{sub 50}) of 2- to 3-fold lower than HCPT as a control. In particular, 2–47 inhibited the proliferation of Jurkat cells with an IC{sub 50} 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 CHCl{sub 3} 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. - Highlights: • Compound 2–47 showed a wide inhibitory effect on the tested tumor cell lines with an IC{sub 50} of 3 times lower than that of HCPT in general. • Compound 2–47 inhibited the proliferation of the human leukemia cell Jurkat at an IC{sub 50} of as low as 40 nM. • As compared to HCPT, compound 2–47 showed much reduced cytotoxicity on normal human cells. • As compared to others, compound 2–47 showed a hundreds-fold higher solubility in non-polar organic solution.

  14. In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL-2 production in human T-cells

    PubMed Central

    Midgett, Kristin; Peden-Adams, Margie M.; Gilkeson, Gary S.; Kamen, Diane L.

    2014-01-01

    Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)-2 production in the human Jurkat T-cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD-3/anti CD-28, or anti CD-3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 μg ml−1 PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μg ml−1 PFOA. Jurkat cells stimulated with PHA/PMA or anti CD-3 exhibited decreased IL-2 production beginning at 50 μg PFOS ml−1 and 5 μg PFOS ml−1 respectively, but stimulation with anti-CD3/anti-CD28 resulted in no changes compared with the control. Addition of the PPAR-alpha antagonist GW6471 to PFOS-dosed cells stimulated with PHA/PMA resulted in decreases in IL-2 production starting at 50 μg PFOS ml−1, which suggests PFOS affected T-cell IL-2 production via PPAR-alpha-independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL-2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL-2 production, but PFOS suppressed IL-2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL-2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. PMID:25056757

  15. In vitro evaluation of the effects of perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) on IL-2 production in human T-cells.

    PubMed

    Midgett, Kristin; Peden-Adams, Margie M; Gilkeson, Gary S; Kamen, Diane L

    2015-05-01

    Perfluorinated compounds, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been shown to alter various immune functions suggesting they are immunotoxic. This study assessed the effects of PFOS and PFOA on interleukin (IL)-2 production in the human Jurkat T-cell line and PFOS in healthy human primary T cells. Jurkat cells were stimulated with phytohemagglutinin (PHA)/phorbol myristate acetate (PMA), anti CD-3/anti CD-28, or anti CD-3, and dosed with 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 75, or 100 µg ml(-1) PFOS or 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 µg ml(-1) PFOA. Jurkat cells stimulated with PHA/PMA or anti CD-3 exhibited decreased IL-2 production beginning at 50 µg PFOS ml(-1) and 5 µg PFOS ml(-1) respectively, but stimulation with anti-CD3/anti-CD28 resulted in no changes compared with the control. Addition of the PPAR-alpha antagonist GW6471 to PFOS-dosed cells stimulated with PHA/PMA resulted in decreases in IL-2 production starting at 50 µg PFOS ml(-1), which suggests PFOS affected T-cell IL-2 production via PPAR-alpha-independent mechanisms. Exposure to PFOA, PFOA + GW6471, or PFOS + PFOA in Jurkat cells resulted in no significant differences in IL-2 production. In vitro dosing studies using healthy primary human CD4+ T cells were consistent with the Jurkat results. These data demonstrated that PFOA did not impact IL-2 production, but PFOS suppressed IL-2 production in both a human cell line and human primary cells at dose levels within the high end of the human exposure range. A decrease in IL-2 production is characteristic of autoimmune diseases such as systemic lupus erythematosus and should be further investigated. Copyright © 2014 John Wiley & Sons, Ltd.

  16. 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.

  17. 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.

  18. The effects of ursolic acid on cytokine production via the MAPK pathways in leukemic T-cells

    PubMed Central

    Kaewthawee, Narawan; Brimson, Sirikalaya

    2013-01-01

    Ursolic acid (UA) is a pentacyclic triterpenoid carboxylic acid that is found in plants and herbal products. It is one of the chemopreventive agents, which can suppress cancer cell proliferation and induce apoptosis. UA possesses various biological activities including anticancer, anti-inflammatory, anti-oxidative and hepatoprotective activity. We investigated the effect of UA on cytokine production via the MAPK pathways in Jurkat leukemic T-cells, showing that UA inhibited cell growth and proliferation of Jurkat cells, as well as suppressing PMA/PHA induced IL-2 and TNF-α production in a concentration and time dependent manner. The inhibition of IL-2 and TNF-α production by UA involved the c-Jun N-terminal kinase (JNK) pathway, but not the extracellular-signal regulated protein kinase (ERK) pathway. Future utilization of UA as a chemopreventive or therapeutic agent may provide an alternative option for leukemia treatments. PMID:26417220

  19. Sulindac and its metabolites: sulindac sulfide and sulindac sulfone enhance cytotoxic effects of arsenic trioxide on leukemic cell lines.

    PubMed

    Stępnik, Maciej; Ferlińska, Magdalena; Smok-Pieniążek, Anna; Gradecka-Meesters, Dobrosława; Arkusz, Joanna; Stańczyk, Małgorzata

    2011-08-01

    The effects of arsenic trioxide (ATO) in combination with sulindac (SUL), sulindac sulfide (SS) or sulindac sulfone (SF) on human (Jurkat, HL-60, K562 and HPB-ALL) and mouse (EL-4) leukemic cell lines were investigated. The cells showed different sensitivity to sulindacs (2.5-200 μM) with SS being the most cytotoxic (72 h WST-1 reduction test). The cytotoxicity of ATO was enhanced by combination with sulindacs. The combination of ATO (1 μM) with SS or SF at concentrations over 50 μM induced considerable cytotoxicity in all cell lines. Normal human lymphocytes exposed for 48 h to the combinations showed smaller decrease in viability. Measurements of Jurkat, HL-60 and K562 cells exposed to ATO (1 μM) and sulindacs (100 μM or 200 μM for K562 cells) indicated apoptosis as the main cell death mechanism. The mitochondrial membrane potential measurements (JC-1 probe) indicated an active involvement of mitochondria in the process. The results did not indicate involvement of an inhibitory effect of the combinations on NF-κB activity in Jurkat, HL-60 and K562 cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. 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.

  1. Anti-tumor effects of anti-T-cell globulin.

    PubMed

    Westphal, Sabine; Brinkmann, Hanna; Kalupa, Martina; Wilke, Andrea; Seitz-Merwald, Isabell; Penack, Olaf

    2014-10-01

    In vivo T-cell depletion using anti-T-cell antibodies is a standard procedure during allogeneic hematopoietic stem cell transplantation (allo-HSCT). Clinical data demonstrate that in vivo T-cell depletion with the anti-CD52 monoclonal antibody Alemtuzumab is associated with increased relapse rates of hematologic malignancies after allo-HSCT, underlining the importance of donor T cells for graft versus tumor activity. In contrast, recent results suggest that in vivo T-cell depletion with rabbit anti-T-cell globulin (ATG) Fresenius is not associated with tumor relapse after allo-HSCT, raising the possibility that ATG mediates antitumor effects. However, data on ATG's ability to bind to tumor cells and on its effect on the induction of antibody-dependent cellular cytotoxicity (ADCC) are lacking. We used ATG Fresenius, which contains polyclonal rabbit IgG directed against the human T-lymphoma cell line Jurkat, to study relevant mechanisms of ATG-mediated antitumor effects, including ADCC, complement-dependent cytotoxicity, and induction of apoptosis. Based on the knowledge that Jurkat cells aberrantly express myeloid markers and B-cell markers, we hypothesized that rabbit ATG Fresenius binds to a variety of hematologic malignancies. We found that ATG specifically binds to a variety of hematologic malignancies including acute myeloid leukemia and B-cell lymphoma in a concentration-dependent manner. We demonstrate that ATG mediates antitumor activity, including induction of ADCC, complement-dependent cytotoxicity, and apoptosis, toward different hematologic malignancies. Our results contribute to a better understanding of the effects of ATG on posttransplant immunology in patients undergoing allo-HSCT. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  2. Effect of steviol, steviol glycosides and stevia extract on glucocorticoid receptor signaling in normal and cancer blood cells.

    PubMed

    Panagiotou, Christina; Mihailidou, Chrysovalantou; Brauhli, George; Katsarou, Olga; Moutsatsou, Paraskevi

    2017-07-25

    The use of steviol glycosides as non-caloric sweeteners has proven to be beneficial for patients with type 2 diabetes mellitus (T2D), obesity, and metabolic syndrome. However, recent data demonstrate that steviol and stevioside might act as glucocorticoid receptor (GR) agonists and thus correlate with adverse effects on metabolism. Herein, we evaluated the impact of steviol, steviol glycosides, and a Greek-derived stevia extract on a number of key steps of GR signaling cascade in peripheral blood mononuclear cells (PBMCs) and in Jurkat leukemia cells. Our results revealed that none of the tested compounds altered the expression of primary GR-target genes (GILZ, FKPB5), GR protein levels or GR subcellular localization in PBMCs; those compounds increased GILZ and FKPB5 mRNA levels as well as GRE-mediated luciferase activity, inducing in parallel GR nuclear translocation in Jurkat cells. The GR-modulatory activity demonstrated by stevia-compounds in Jurkat cells but not in PBMCs may be due to a cell-type specific effect. Copyright © 2017. Published by Elsevier B.V.

  3. Inducible T cell tyrosine kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor.

    PubMed

    Grasis, Juris A; Browne, Cecille D; Tsoukas, Constantine D

    2003-04-15

    The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.

  4. [Effects of (Pyr(1))apelin-13 on the proliferation and activation of Jarkat T lymphocyte].

    PubMed

    Wei, Ming; Gan, Lu; Hou, Jin; Chen, Lihong; Liu, Yantong; Ren, Ligang

    2016-12-01

    Objective To investigate the effect of (Pyr(1))apelin-13 on the proliferation and activation of Jurkat T lymphocytes. Methods Jurkat T cells were treated by 0, 10, 50 nmol/L (Pyr(1))apelin-13 for 24 hours and cell proliferation ability was detected by CCK-8 assay. The effect of (Pyr(1))apelin-13 on the activation of Jurkat T lymphocytes with anti-CD3 and anti-CD28 antibodies was observed and the molecular mechanism was investigated. Quantitative real-time PCR was applied to detect the expressions of CD69, CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) mRNAs. Results The proliferation of Jurkat T cells was not affected by (Pyr(1)) apelin-13. However, (Pyr(1))apelin-13 increased the expressions of CD69 and CD25 and then activated Jurkat T lymphocytes. Furthermore, the expressions of CTLA-4 and PD-1 had no difference between activation group and (Pyr(1))apelin-13 group. Conclusion (Pyr(1))apelin-13 promotes Jurkat T lymphocyte activation by increasing the expressions of CD69 and CD25.

  5. 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.

  6. 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

  7. 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).

  8. 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.

  9. Interactions between endothelial cells and T cells modulate responses to mixed neutron/gamma radiation.

    PubMed

    Cary, Lynnette H; Noutai, Daniel; Salber, Rudolph E; Williams, Margaret S; Ngudiankama, Barbara F; Whitnall, Mark H

    2014-06-01

    Detonation of an improvised nuclear device near a population center would cause significant casualties from the acute radiation syndrome (ARS) due to exposure to mixed neutron/gamma fields (MF). The pathophysiology of ARS involves inflammation, microvascular damage and alterations in immune function. Interactions between endothelial cells (EC) and hematopoietic cells are important not only for regulating immune cell traffic and function, but also for providing the microenvironment that controls survival, differentiation and migration of hematopoietic stem and progenitor cells in blood-forming tissues. Endothelial cells/leukocyte interactions also influence tumor progression and the results of anticancer therapies. In this study, we hypothesized that irradiation of endothelial cells would modulate their effects on hematopoietic cells and vice versa. Human umbilical vein endothelial cells (HUVEC) and immortalized T lymphocytes (Jurkat cells) were cultured individually and in co-culture after exposure to mixed fields. Effects of nonirradiated cells were compared to effects of irradiated cells and alterations in signaling pathways were determined. Mitogen-activated protein kinases (MAPKs) p38 and p44/42 (ERK1/2) in HUVEC exhibited higher levels of phosphorylated protein after exposure to mixed field radiation. IL-6, IL-8, G-CSF, platelet derived growth factor (PDGF) and angiopoietin 2 (ANG2) protein expression were upregulated in HUVEC by exposure to mixed field radiation. PCR arrays using HUVEC mRNA revealed alterations in gene expression after exposure to mixed fields and/or co-culture with Jurkat cells. The presence of HUVEC also influenced the function of Jurkat cells. Nonirradiated Jurkat cells showed an increase in proliferation when co-cultured with nonirradiated HUVEC, and a decrease in proliferation when co-cultured with irradiated HUVEC. Additionally, nonirradiated Jurkat cells incubated in media from irradiated HUVEC exhibited upregulation of activated

  10. 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

  11. Irradiation of light emitting diode at 850nm inhibits T cell-induced cytokine expression.

    PubMed

    Cheong, Kyung Ah; Kim, Chang-Hyun; Choi, Yunseok; Park, Chan Do; Lee, Ai-Young

    2012-01-01

    An anti-inflammatory effect of light obtained from light-emitting diodes (LEDs) has been discovered, however, limited ranges of wavelengths have been used and the action mechanism has been rarely demonstrated. We sought to analyze the immunomodulatory effect of LED on Jurkat T cells and human T cells. Jurkat T cells with/without stimulation were irradiated once or five times using seven ranges of LED wavelengths, from 415nm to 940nm. Cytotoxic effects were examined by an MTT assay. Changes in T cell-induced cytokines, including IL-2, IL-4, IL-10, IL-12 and IFN-gamma, and their upstream signaling molecules, ZAP-70 and PKCθ, were examined by real-time PCR, ELISA, and Western blot analysis. The effect of the LED wavelength, whose effect was identified on Jurkat T cells, was also examined in human CD3+ T cells with/without stimulation and in Dermatophagoides farinae-induced atopic dermatitis (AD) NC/Nga mice. Lower doses of LED irradiation at 850nm inhibited T cell-derived cytokines without inducing cell death in both Jurkat T cells and human T cells. Repeated exposure resulted in a greater increase of inhibitory effects than that observed with a single exposure, and these effects were identified in the NC/Nga AD model. Although more remains to be clarified, these results may support the clinical application of LED for immune regulation. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  12. 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.

  13. 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.

  14. Mechanisms of Dihydroartemisinin and Dihydroartemisinin/Holotransferrin Cytotoxicity in T-Cell Lymphoma Cells

    PubMed Central

    Zhao, Xindong; Zhao, Chunting; Zhao, Hongguo; Huo, Lanfen

    2015-01-01

    The validated therapeutic effects of dihydroartemisinin (DHA) in solid tumors have encouraged us to explore its potential in treating T-cell lymphoma. We found that Jurkat cells (a T-cell lyphoma cell line) were sensitive to DHA treatment with a IC50 of dihydroartemisinin. The cytotoxic effect of DHA in Jurkat cells showed a dose- and time- dependent manner. Interestingly, the cytotoxic effect of DHA was further enhanced by holotransferrin (HTF) due to the high expression of transferrin receptors in T-cell lymphoma. Mechanistically, DHA significantly increased the production of intracellular reactive oxygen species, which led to cell cycle arrest and apoptosis. The DHA treatment also inhibited the expression of protumorgenic factors including VEGF and telomerase catalytic subunit. Our results have proved the therapeutic effect of DHA in T-cell lymphoma. Especially in combination with HTF, DHA may provide a novel efficient approach in combating the deadly disease. PMID:26502166

  15. 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.

  16. 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.

  17. 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.

  18. Effect of substrate mechanical properties on T cell activation

    NASA Astrophysics Data System (ADS)

    Hui, King; Upadhyaya, Arpita

    2013-03-01

    T cell activation is a key process in cell-mediated immunity, and engagement of T cell receptors by peptides on antigen presenting cells leads to activation of signaling cascades as well as cytoskeletal reorganization and large scale membrane deformations. While significant advances have been made in understanding the biochemical signaling pathways, the effects imposed by the physical environment and the role of mechanical forces on cell activation are not well understood. In this study, we have used anti-CD3 coated elastic polyacrylamide gels as stimulatory substrates to enable the spreading of Jurkat T cells and the measurement of cellular traction forces. We have investigated the effect of substrate stiffness on the dynamics of T cell spreading and cellular force generation. We found that T cells display more active and sustained edge dynamics on softer gels and that they exert increased traction stresses with increasing gel stiffness. A dynamic actin cytoskeleton was required to maintain the forces generated during activation, as inferred from small molecule inhibition experiments. Our results indicate an important role for physical properties of the antigen presenting cell as well as cytoskeleton-driven forces in signaling activation.

  19. Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line

    PubMed Central

    Rahman, Heshu Sulaiman; Rasedee, Abdullah; Abdul, Ahmad Bustamam; Zeenathul, Nazariah Allaudin; Othman, Hemn Hassan; Yeap, Swee Keong; How, Chee Wun; Hafiza, Wan Abd Ghani Wan Nor

    2014-01-01

    This investigation evaluated the antileukemia properties of a zerumbone (ZER)-loaded nanostructured lipid carrier (NLC) prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat) cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans. PMID:24549090

  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. Induction of apoptosis in a leukemia cell line by triterpene saponins from Albizia adianthifolia.

    PubMed

    Haddad, Mohamed; Laurens, Véronique; Lacaille-Dubois, Marie-Aleth

    2004-09-01

    Triterpenoid saponins, which are present in plants and some marine animals, exert various important pharmacological effects. The present study examines the effects of adianthifoliosides A, B, and D (AdA, AdB, and AdD) together with two prosapogenins (Pro1 and Pro2) obtained from Albizia adianthifolia (Mimosaceae) on human leukemia T-cells (Jurkat cells) and on splenocytes. AdA, AdB, and AdD were found to exhibit a cytotoxic effect on Jurkat cells, whereas the prosapogenins were found to exert a lymphoproliferative effect on this cell type. Furthermore, all tested compounds were found to exert a synergistic lymphoproliferative activity with concanavalin A (ConA) on splenocytes. The concentrations where the saponins were found to be cytotoxic on Jurkat cells are far below the concentration of hemolysis. These results indicate that another mechanism than membrane permeabilization formation is responsible of the cell cytotoxicity. Thus, we demonstrated that at 5 microM for AdA and at 1 microM for AdD, these compounds induce apoptosis in Jurkat cells. Early apoptotic events were detected by flow cytometry analysis by using a double annexin-V-FITC and propidium iodide staining. In addition, a disrupted mitochondrial membrane potential was observed in cells treated with AdA, AdB, and AdD. Furthermore, a DNA ladder was observed when Jurkat cells were incubated with 1 microM AdD for 24h. By comparison between the biological activities of the native compounds with the prosapogenins, we show in this work the important role of the acylation and esterification by different moieties at C-21 and C-28 of the aglycone (acacic acid) in the apoptosis-inducing capacity. Particularly, the monoterpene-quinovosyl moiety is shown to be important for the induction of apoptosis.

  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. Freezing and post-thaw apoptotic behaviour of cells in the presence of palmitoyl nanogold particles

    NASA Astrophysics Data System (ADS)

    Thirumala, Sreedhar; Forman, Julianne M.; Monroe, W. Todd; Devireddy, Ram V.

    2007-05-01

    The aim of this study was to evaluate the freezing response of HeLa and Jurkat cells in the presence of commercially available nanoparticles, NPs (Palmitoyl Nanogold®, Nanoprobes). The cells were incubated with NPs for either 5 min or 3 h, and a calorimeter technique was then used to generate the volumetric shrinkage response during freezing at 20 °C min-1. Concomitantly, we also examined the effect of a commonly used cryoprotectant, dimethylsulfoxide, DMSO (10% v/v ratio) on the freezing response of HeLa and Jurkat cells. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the reference hydraulic conductivity, Lpg, (μm/min-atm) and activation energy, ELp, (kcal mol-1) were obtained. For HeLa cells, the values of Lpg ranged from 0.08 to 0.23 µm/min-atm, while ELp ranged from 10.9 to 37.4 kcal mol-1. For Jurkat cells these parameter values ranged from 0.05 to 0.16 µm/min-atm and 9.5 to 35.9 kcal mol-1. A generic optimal cooling rate equation was then used to predict the optimal rates of freezing HeLa and Jurkat cells in the presence and absence of DMSO and NPs. The post-thaw viability and apoptotic response of HeLa and Jurkat cells was further investigated by cooling cells at three rates in the presence and absence of DMSO and NPs using a commercially available controlled rate freezer. Jurkat cells treated in this manner demonstrated an increase in their adhesive properties after 18 h incubation and adhered strongly to the bottom of the culture plate. This observation prevented further analysis of Jurkat apoptotic and necrotic post-thaw responses. There was no significant effect of NPs or DMSO alone on HeLa cell viability prior to freezing. The post-thaw results from HeLa cells show that the NPs increased the measured post-freeze apoptotic response when cooled at 1 °C min-1, suggesting a possible therapeutic use of NPs in cryodestructive procedures.

  7. 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.

  8. Protective effect of ginsenoside Rg1 on lidocaine-induced apoptosis

    PubMed Central

    LI, HUI; XU, JUNMEI; WANG, XIN; YUAN, GUIXIU

    2014-01-01

    Lidocaine, as an anesthetic substance, is often used for surface and spinal anesthesia. However, studies have shown that lidocaine may induce transient neurological symptoms and cauda equina syndrome. In the present study the effects of the ginsenoside Rg1 (Rg1) on lidocaine-induced apoptosis were assessed in Jurkat cells using flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The data showed that incubation with Rg1 provides protection against lidocaine-induced apoptosis in cultured Jurkat cells. In order to investigate the effect of Rg1 on the apoptosis pathway, caspase 3 gene expression was determined. The results suggested that the protective effect of Rg1 on lidocaine-induced apoptosis is mediated by altering the level of B-cell lymphoma-2 (BCL-2) family proteins and downregulating caspase-3 expression. In conclusion, the present study demonstrated that incubation with Rg1 provides protection against lidocaine-induced apoptosis in cultured Jurkat cells. In addition, the study demonstrated that Rg1 is a notable antiapoptotic molecule that is capable of blocking the caspase-dependent signaling cascade in Jurkat cells, and that the protective effect of Rg1 on lidocaine-induced apoptosis is mediated by altering levels of BCL-2 family proteins and downregulating caspase-3 expression. The present study provides the basis for understanding and evaluating the effect of Rg1 in the in vivo treatment of lidocaine-induced transient neurological symptoms and cauda equina syndrome by lidocaine. PMID:24270314

  9. In vivo and in vitro phosphorylation of annexin II in T cells: potential regulation by annexin V.

    PubMed Central

    Dubois, T; Oudinet, J P; Russo-Marie, F; Rothhut, B

    1995-01-01

    In order to understand how signal transduction occurs during T cell activation, it is necessary to identify the key regulatory molecules whose function is influenced by phosphorylation. Annexins II (A-II) and V (A-V) belong to a large family of Ca(2+)-dependent phospholipid-binding proteins. Among many putative functions, annexins may be involved in signal transduction during cellular proliferation and differentiation. In the present study we show that A-II is phosphorylated in vivo in the Jurkat human T cell line. Indeed, A-II is phosphorylated after stimulation by phorbol myristate acetate and on serine residues after T cell antigen receptor (TcR) stimulation. In cytosol from Jurkat cells, A-II is phosphorylated only by Ca2+/phospholipid-stimulated kinases such as Ca(2+)-dependent protein kinases C (cPKCs). A-V inhibits the phosphorylation of A-II and other substrates of cPKCs and has no effect on kinases activated only by phospholipids. In conclusion, A-II is phosphorylated both in vitro and in vivo in Jurkat cells, and may play a role as a substrate during signal transduction in lymphocytes via the TcR through the PKC pathway. On the other hand, A-V could act as a potent modulator of cPKCs in Jurkat cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7646452

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. K(bg) and Kv1.3 channels mediate potassium efflux in the early phase of apoptosis in Jurkat T lymphocytes.

    PubMed

    Valencia-Cruz, Georgina; Shabala, Lana; Delgado-Enciso, Ivan; Shabala, Sergey; Bonales-Alatorre, Edgar; Pottosin, Igor I; Dobrovinskaya, Oxana R

    2009-12-01

    Microelectrode ion flux estimation (MIFE) and patch-clamp techniques were combined for noninvasive K(+) flux measurements and recording of activities of the dominant K(+) channels in the early phases of apoptosis in Jurkat cells. Staurosporine (STS, 1 microM) evoked rapid (peaking around 15 min) transient K(+) efflux, which then gradually decreased. This transient K(+) efflux occurred concurrently with the transient increase of the K(+) background (K(bg)) TWIK-related spinal cord K(+) channel-like current density, followed by a drastic decrease and concomitant membrane depolarization. The Kv1.3 current density remained almost constant. Kv1.3 activation was not altered by STS, whereas the inactivation was shifted to more positive potentials. Contribution of K(bg) and Kv1.3 channels to the transient and posttransient STS-induced K(+) efflux components, respectively, was confirmed by the effects of bupivacaine, predominantly blocking K(bg) current, and the Kv1.3-specific blocker margatoxin. Channel-mediated K(+) efflux provoked a substantial cellular shrinkage and affected the activation of caspases.

  16. 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

  17. 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

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

    PubMed

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

    2010-06-01

    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-kappaB-, 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.

  19. 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.

  20. 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.

  1. IMPACT OF LASER THERAPY ON THE PROLIFERATION OF VARIOUS CULTURED CELLS.

    PubMed

    Gabunia, T; Turabelidze, S; Machavariani, M; Enukidze, M; Kipiani, Nina V; Sharashenidze, G; Sanikidze, T

    2016-10-01

    The purpose of our study was to establish the laser effects on the epithelial tissue and immune metabolism. The research was conducted on human leukemic mature T cells (Jurkat cells) (DSMZ-Deutshe Sammulung von Mikroorganismen und Zellkulturen (Germany)) and MDCK cell line (Lugar Laboratory, Tbilisi, Georgia). Cells were radiated by Laser device "ОПТОДАН"- АЛСТ-01 (power 5 W) 3 -7 days (4 minutes per day). With the aim to model oxidative stress-induced apoptosis, 30% hydrogen peroxide (H2O2) (Sigma) is added to Jurkat cells, in doses 25 and 50μM [4, 5]; and MDCK cells, in doses 400 and 800 μM [19] added to incubation suspension with subsequent incubation for 24, 48 and 72 hrs. Control group is represented by intact Jurkat and MDCK cells. MTT test was used to assess the cells' proliferation activity (viability). Statistical analyses of the obtained results were performed by SPSS (version 10.0) program package. Our research results show that effects of laser therapy on proliferation of cell cultures depend on the type of cells and incubation conditions. Laser irradiation revealed equal efficacy in both types of the intact cells and increased their viability in time-dependent manner. Jurkat cells turned out to be more susceptible to oxidative stress. Laser therapy only slightly improved their viability at moderate intensity of oxidative stress and proved to be ineffective in strong oxidative stress conditions. The MDCK cells appeared to be more sustainable to oxidative stress; significant changes in these cells viability were observed only when high doses of hydrogen peroxide were added to their incubation medium. Thus, laser therapy was effective for these cells incubated in both regimens of oxidative stress. Our research results prove the efficacy of laser therapy use during periodontitis with the aim to recover epithelium in the oral cavity and to modulate immune metabolism in the patient's body.

  2. 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.

  3. [Mutation of palmitoylation site of linker for activation of T cells inhibits signal transduction mediated by glycosyl phosphatidyl inositol-anchored CD59 in T cells].

    PubMed

    Gao, Meihua; Wang, Lina; Wang, Bing; Cong, Beibei; Zhang, Shuchao

    2015-08-01

    To construct the lentivirus carrying the mutated palmitoylation site of the linker for activation of T cells (LAT) and infect Jurkat cells with it to establish stable cell line, and to investigate the effect of LAT palmitoylation mutation on T cell signaling induced by CD59. Negative control (neg-EGFP) and LAT-M-EGFP fusion protein gene vectors were respectively constructed and then packaged using lentivirus. Subsequently, Jurkat cells were infected with them to establish stable cell lines. Confocal laser scanning microscopy was used to observe the infection efficiency and the distribution of fusion proteins in Jurkat cells. CCK-8 assay was used to detect the change of cell proliferation activity after CD59 mAb supplementation. Flow cytometry was used to determine the apoptosis rate. Western blotting was used to examine the levels of phospholipase C-γ1 (PLC-γ1) and lymphocyte-specific protein tyrosine kinase (LCK). Confocal laser scanning microscopy revealed that LAT molecules of LAT-M group scattered on cell membrane, and there was no obvious clustered region after cross linkage with CD59 mAb. Compared with the negative control group, the cell proliferation activity of LAT-M group significantly decreased, and the quantity of middle-late apoptotic cells significantly increased; Western blotting showed that the expression levels of PLC-γ1 and LCK in LAT-M group was roughly the same with those in negative control group, and after CD59 mAb stimulation, there was no obvious change in LAT-M group, while the levels in negative control group were reduced. LAT-M-EGFP fusion protein could not locate on lipid rafts of Jurkat cells infected with LAT palmitoylation mutation. In addition, the growth of the cells carrying the LAT-M-EGFP was inhibited. The palmitoylation mutation of LAT attenuated the signal transduction induced by glycosylphosphatidylinositol-anchored CD59 in T cells.

  4. Bioactive properties of wood knot extracts on cultured human cells.

    PubMed

    Phelan, Martha; Aherne, S Aisling; Wong, Alfred; O'Brien, Nora M

    2009-12-01

    Not all felled wood is converted to timber or pulp, with the remaining material being a rich source of relatively unexplored and unexploited potentially novel bioactive compounds. Therefore the potential bioactive effects of two softwood knot (the part of the branch encased in the tree stem) extracts--namely, Pinus banksiana Lamb. (Jack pine) and Picea sitchensis (Bong.) Carr. (Sitka spruce)--were investigated by (1) determining their effects on the viability and antioxidant status of human Jurkat T cells, (2) investigating potential cytoprotective and genoprotective effects against oxidative stress in cultured cells, and (3) assessing their effects on concanavalin A (ConA)-induced interleukin-2 (IL-2) production. Initially, both Jack pine knot and Sitka spruce knot extracts were shown to possess strong antioxidant activity as determined by the ferric reducing antioxidant power assay. When added to Jurkat cells, Jack pine knot extract was more toxic compared with Sitka spruce knot extract, with concentrations that resulted in 50% cell death of 153.0 microg/mL and 376.1 microg/mL, respectively. Supplementation of Jurkat cells with wood knot extracts did not affect their glutathione content or catalase activity. Pretreatment of Jurkat cells with Sitka spruce or Jack pine knot extracts protected against H(2)O(2)-induced cell injury. However, none of the extracts protected against H(2)O(2)-induced DNA damage. Jack pine knots, at a concentration of 30 microg/mL, significantly suppressed ConA-induced IL-2 production. Although total phenol content did not differ between the two extracts, gas chromatography analysis did show variation in the types of constituents present. Further research is warranted to elucidate the selective bioactive properties of these softwood knot extracts.

  5. Timed, sequential administration of paclitaxel improves its cytotoxic effectiveness in a cell culture model.

    PubMed

    Fisi, Viktória; Kátai, Emese; Bogner, Péter; Miseta, Attila; Nagy, Tamás

    2016-05-02

    Paclitaxel (taxol) is a chemotherapeutic agent frequently used in combination with other anti-neoplastic drugs. It is most effective during the M phase of the cell-cycle and tends to cause synchronization in malignant cells lines. In this study, we investigated whether timed, sequential treatment based on the cell-cycle characteristics could be exploited to enhance the cytotoxic effect of paclitaxel. We characterized the cell-cycle properties of a rapidly multiplying cell line (Sp2, mouse myeloma cells) by propidium-iodide DNA staining such as the lengths of various cell cycle phases and population duplication time. Based on this we designed a paclitaxel treatment protocol that comprised a primary and a secondary, timed treatment. We found that the first paclitaxel treatment synchronized the cells at the G2/M phase but releasing the block by stopping the treatment allowed a large number of cells to enter the next cell-cycle by a synchronized manner. The second treatment was most effective during the time when these cells approached the next G2/M phase and was least effective when it occurred after the peak time of this next G2/M phase. Moreover, we found that after mixing Sp2 cells with another, significantly slower multiplying cell type (Jurkat human T-cell leukemia) at an initial ratio of 1:1, the ratio of the two different cell types could be influenced by timed sequential paclitaxel treatment at will. Our results demonstrate that knowledge of the cell-cycle parameters of a specific malignant cell type could improve the effectivity of the chemotherapy. Implementing timed chemotherapeutic treatments could increase the cytotoxicity on the malignant cells but also decrease the side-effects since other, non-malignant cell types will have different cell-cycle characteristic and be out of synch during the treatment.

  6. 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

  7. 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

  8. Pyrvinium selectively induces apoptosis of lymphoma cells through impairing mitochondrial functions and JAK2/STAT5.

    PubMed

    Xiao, Meifang; Zhang, Liming; Zhou, Yizheng; Rajoria, Pasupati; Wang, Changfu

    2016-01-15

    Targeting mitochondrial respiration has emerged as an attractive therapeutic strategy in blood cancer due to their unique metabolic dependencies. In this study, we show that pyrvinium, a FDA-approved anthelmintic drug, selectively targets lymphoma T-cells though inhibition of mitochondrial functions and JAK2/STAT5. Pyrvinium induces apoptosis of malignant T-cell line Jurkat and primary T-cells from lymphoma patients while sparing T-cells from healthy donors. Increased level of active caspase-3 and decreased levels of Bcl-2 and Mcl-1 were also observed in Jurkat and lymphoma T-cells but not normal T-cells treated with pyrvinium. In addition, pyrvinium impairs mitochondrial functions by inhibit mitochondrial respiration, suppressing mitochondrial respiratory complex I activity, increasing ROS and decreasing ATP levels. However, the effects of pyrvinium were abolished in mitochondrial respiration-deficient Jurkat ρ(0) cells, confirming that pyrvinium acts on lymphoma T-cells via targeting mitochondrial respiration. We further show that lymphoma T-cells derived from patients depend more on mitochondrial respiration than normal T-cells, and this explains the selective toxicity of pyrvinium in lymphoma versus normal T-cells. Finally, we demonstrate that pyrvinium also suppresses JAK2/STAT5 signaling pathway in Jurkat cells. Our study suggests that pyrvinium is a useful addition to T-cell lymphoma treatment, and emphasizes the potential therapeutic value of the differences in the mitochondrial characteristics between malignant and normal T-cells in blood cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Effect of oleic acid on store-operated calcium entry in immune-competent cells.

    PubMed

    Carrillo, Celia; Giraldo, María; Cavia, M Mar; Alonso-Torre, Sara R

    2017-04-01

    To study the mechanism by which oleic acid (OA) (C18:1) exerts its beneficial effects on immune-competent cells. Since store-operated Ca(2+) entry (SOCE) is a Ca(2+) influx pathway involved in the control of multiple physiological processes including cell proliferation, we studied the effect of OA in Ca(2+) signals of Jurkat T cells and THP-1 monocytes, paying particular attention to SOCE. Changes in [Ca(2+)]i were measured using the Fura-2 fluorescence dye. Mn(2+) uptake was monitored as a rate of quenching of Fura-2 fluorescence measured at the Ca(2+)-insensitive wavelengths. Thapsigargin was used to induce SOCE in Fura-2-loaded cells. We showed a clear dose-dependent SOCE-inhibitory effect of OA in both cell lines. Such an inhibitory effect was PKC independent and totally restored by albumin, suggesting that OA exerts its effect somewhere in the membrane. We also demonstrated that OA induces increases in [Ca(2+)]i partly mediated by an extracellular Ca(2+) influx through econazole-insensitive channels. Finally, we compared the effect of OA with stearic acid (C18:0), assuming the emerged evidence concerning the link between saturated fats and inflammation disorders. Stearic acid failed to inhibit SOCE, independently on the concentration tested, thus intensifying the physiological relevance of our findings. We suggest a physiological pathway for the beneficial effects of OA in inflammation.

  10. 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.

  11. Downregulation of Mcl-1 through inhibition of translation contributes to benzyl isothiocyanate-induced cell cycle arrest and apoptosis in human leukemia cells.

    PubMed

    Zhou, T; Li, G; Cao, B; Liu, L; Cheng, Q; Kong, H; Shan, C; Huang, X; Chen, J; Gao, N

    2013-02-28

    Benzyl isothiocyanate (BITC) is one of the compounds of ITCs' family that has attracted a great deal of interest because of its ability to exhibit anticancer activity. In this study, we investigated the effects of BITC on cell cycle arrest and apoptosis in human leukemia cell lines, primary leukemia cells, and nude mice Jurkat xenograft. Exposure of Jurkat cells to BITC resulted in dose- and time-dependent increase in apoptosis, caspase activation, cytochrome c release, nuclear apoptosis-inducing factor (AIF) accumulation, Bcl2-associated X protein (Bax) translocation, and myeloid cell leukemia-1 (Mcl-1) downregulation. Treatment with these cells also resulted in cell cycle arrest at the G2/M phase. The G2/M-arrested cells are more sensitive to undergoing Mcl-1 downregulation and apoptosis mediated by BITC. BITC downregulates Mcl-1 expression through inhibition of translation, rather than through a transcriptional, post-translational, or caspase-dependent mechanism. Dephosphorylation of eukaryotic initiation factor 4G could contribute to the inhibition of Mcl-1 translation mediated by BITC. Furthermore, ectopic expression of Mcl-1 substantially attenuates BITC-mediated lethality in these cells, whereas knockdown of Mcl-1 through small interfering RNA significantly enhances BITC-mediated lethality. Finally, administration of BITC markedly inhibited tumor growth and induced apoptosis in Jurkat xenograft model in association with the downregulation of Mcl-1. Taken together, these findings represent a novel mechanism by which agents targeting Mcl-1 potentiate BITC lethality in transformed and primary human leukemia cells and inhibitory activity of tumor growth of Jurkat xenograft model.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. 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.

  18. 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.

  19. 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

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

    PubMed

    Clerc, Isabelle; Laverdure, Sylvain; Torresilla, Cynthia; Landry, Sébastien; Borel, Sophie; Vargas, Amandine; Arpin-André, Charlotte; Gay, Bernard; Briant, Laurence; Gross, Antoine; Barbeau, Benoît; Mesnard, Jean-Michel

    2011-09-19

    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. 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. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. Borrelidin has limited anti-cancer effects in bcl-2 overexpressing breast cancer and leukemia cells and reveals toxicity in non-malignant breast epithelial cells.

    PubMed

    Gafiuc, Diana; Weiß, Marlene; Mylonas, Ioannis; Brüning, Ansgar

    2014-10-01

    Clinically effective anti-cancer drugs have to tread a narrow line between selective cytotoxicity on tumor cells and tolerable adverse effects against healthy tissues. This causes the failure of many potential cancer drugs in advanced clinical trials, hence signifying the importance of a comprehensive initial estimate of the cytotoxicity of prospective anti-cancer drugs in preclinical studies. In this study, the cytotoxicity of borrelidin, a macrolide antibiotic with a high cytotoxic selectivity for proliferating endothelial cells and leukemia cells, was tested on malignant and non-malignant breast cells. Highly metastatic breast cancer cell lines (MDA-MB-231 and MDA-MB-435) showed promising results and exhibited good sensitivity to borrelidin at low nanomolar concentrations, but borrelidin was cytotoxic to a non-malignant breast epithelial cell line (MCF10A) as well. Furthermore, although a high sensitivity of endothelial cells (human umbilical vein endothelial cells; HUVEC) and individual leukemia cell lines (Jurkat and IM9) to borrelidin was confirmed in this study, another leukemia cell line (HL60) and an immortalized endothelial cell line (EA.hy926) displayed a significantly decreased sensitivity. Reduced sensitivity to borrelidin was associated with elevated bcl-2 expression in these cell lines. In conclusion, the results presented show that borrelidin displays high and selective cytotoxicity against subgroups of cancer cells and endothelial cells, but, owing to its non-specific toxicity to non-malignant cells, its clinical application might be restricted because of likely adverse effects and limited efficacy in bcl2-overexpressing cancer cells. Copyright © 2013 John Wiley & Sons, Ltd.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics

    PubMed Central

    Evangelisti, Cecilia; Evangelisti, Camilla; Teti, Gabriella; Chiarini, Francesca; Falconi, Mirella; Melchionda, Fraia; Pession, Andrea; Bertaina, Alice; Locatelli, Franco; McCubrey, James A.; Beak, Dong Jae; Bittman, Robert; Pyne, Susan; Pyne, Nigel J.; Martelli, Alberto M.

    2014-01-01

    Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL. PMID:25226616

  13. 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.

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

    PubMed Central

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

    1997-01-01

    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. PMID:9224625

  15. 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.

  16. 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.

  17. 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

  18. Molecular effects of the phosphatidylinositol-3-kinase inhibitor NVP-BKM120 on T and B-cell acute lymphoblastic leukaemia.

    PubMed

    Pereira, João Kleber Novais; Machado-Neto, João Agostinho; Lopes, Matheus Rodrigues; Morini, Beatriz Corey; Traina, Fabiola; Costa, Fernando Ferreira; Saad, Sara Teresinha Olalla; Favaro, Patricia

    2015-09-01

    Constitutive activation of the PI3K pathway in T cell acute lymphoblastic leukaemia (T-ALL) has been reported and in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. We investigated the effects of NVP-BKM120, a potent pan-class I PI3K inhibitor, in lymphoblastic leukaemia cell lines. Effects of NVP-BKM120 on cell viability, clonogenicity, apoptosis, cell cycle, cell signalling and autophagy were assessed in vitro on T-ALL (Jurkat and MOLT-4) and BL (Daudi and NAMALWA) cell lines. NVP-BKM120 treatment decreased cell viability and clonogenic growth in all tested cells. Moreover, the drug arrested cell cycling in association with a decrease in Cyclin B1 protein levels, and increased apoptosis. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, mTOR, P70S6K and 4EBP1, with stable total protein levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, in association with augmented BAX:BCL2 ratio. Quantification of autophagy showed a dose-dependent increase in acidic vesicular organelles in all cells tested. In summary, our present study establishes that NVP-BKM120 presents an effective antitumour activity against T-ALL and BL cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. 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.

  20. Cytotoxic effect of a novel naphthylchalcone against multiple cancer cells focusing on hematologic malignancies.

    PubMed

    Maioral, Mariana Franzoni; Bodack, Camila do Nascimento; Stefanes, Natália Marceli; Bigolin, Álisson; Mascarello, Alessandra; Chiaradia-Delatorre, Louise Domeneghini; Yunes, Rosendo Augusto; Nunes, Ricardo José; Santos-Silva, Maria Cláudia

    2017-09-01

    Chalcones are natural compounds described in the literature by its several properties including cytotoxic activity against several tumor types. Considering that the search for new chemotherapeutic agents is still necessary, the aim of this study was to investigate the cytotoxic mechanisms involved in cell death induced by a synthetic chalcone (A23) on different tumor cells. Chalcone A23 reduced the cell viability of twelve tumor cell lines in a concentration and time dependent manner and it was more cytotoxic against acute leukemia cells. Interestingly, the compound was non cytotoxic to normal cells and non-hemolytic to normal red blood cells. Chalcone A23 decreased the expression of cell proliferation marker KI-67 and blocked the G2/M phase in both K562 and Jurkat cell lines. Cells treated with A23 showed morphological features suggestive of apoptosis, the "latter pattern" in agarose gel, the externalization of phosphatidylserine and caspase-3 and PARP cleavage. Chalcone A23 significantly reduced the mitochondrial membrane potential, decreased the expression of anti-apoptotic proteins Bcl-2 and survivin and increased the expression of pro-apoptotic protein Bax, confirming the involvement of the intrinsic pathway. The increased mitochondrial permeability resulted in the release of AIF, cytochrome c and endonuclease G from the mitochondria to the cytosol. In addition, chalcone A23 increased the expression of FasR and induced Bid cleavage, showing the involvement of the extrinsic pathway. Finally, chalcone A23 seems to have a synergic effect with the chemotherapy drugs cytarabine and vincristine. These results suggest that A23 is an interesting compound with strong and selective anti-tumor activity. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. 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.

  2. 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.

  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

    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.

  5. Lck Inhibits Heat Shock Protein 65-Mediated Reverse Cholesterol Transport in T Cells.

    PubMed

    Luo, Tiantian; Hu, Jing; Xi, Dan; Xiong, Haowei; He, Wenshuai; Liu, Jichen; Li, Menghao; Lu, Hao; Zhao, Jinzhen; Lai, Wenyan; Guo, Zhigang

    2016-11-15

    Previously, we reported that heat shock protein (HSP)65 impairs the effects of high-density lipoprotein on macrophages. We also showed that immune response activation adversely affects reverse cholesterol transport (RCT). In this study, we investigated the effects of the Src family kinase lymphocyte-specific protein tyrosine kinase (Lck) and elucidated the mechanism underlying HSP65-regulated cholesterol efflux in T cells. We evaluated cell proliferation, Lck expression, and inflammatory cytokine production in Jurkat cells and CD4(+) T cells. HSP65-mediated inhibition of RCT was assessed by evaluating ABCA1, ABCG1, SR-BI, PPAR-γ, and liver X receptor-α expression. A dose-dependent relationship was found between the levels of these proteins and the suppression of cholesterol efflux. Stimulation of Lck-silenced T cells with ionomycin resulted in a decrease in intracellular calcium levels. Treatment of Jurkat cells with PP2, an inhibitor of Src family kinase, inhibited calcium-induced, but not PMA-induced, ERK phosphorylation. NF-κB activation in response to PMA was minimally inhibited in cells stimulated with PP2. HSP65 failed to trigger downstream ERK or JNK phosphorylation or to activate NF-κB or protein kinase C-γ in Lck-silenced cells. Additionally, elevation of intracellular calcium was also impaired. However, HSP65 significantly enhanced cholesterol efflux and decreased cellular cholesterol content by inducing the expression of cholesterol transport proteins in Lck-silenced cells. The treatment of Jurkat cells with PP2 also inhibited cell proliferation and promoted RCT. In conclusion, Lck is a key molecule in the TCR signaling cascade that inhibits cholesterol efflux and upregulates intracellular cholesterol ester content in T cells. Our results demonstrate that the immune response plays a previously unrecognized role in RCT. Copyright © 2016 by The American Association of Immunologists, Inc.

  6. Kefir induces cell-cycle arrest and apoptosis in HTLV-1-negative malignant T-lymphocytes

    PubMed Central

    Maalouf, Katia; Baydoun, Elias; Rizk, Sandra

    2011-01-01

    Background: Adult lymphoblastic leukemia (ALL) is a malignancy that occurs in white blood cells. The overall cure rate in children is 85%, whereas it is only 40% in adults. Kefir is an important probiotic that contains many bioactive ingredients, which give it unique health benefits. It has been shown to control several cellular types of cancer. Purpose: The present study investigates the effect of a cell-free fraction of kefir on CEM and Jurkat cells, which are human T-lymphotropic virus type I (HTLV-1)-negative malignant T-lymphocytes. Methods: Cells were incubated with different kefir concentrations. The cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir on the proliferation of CEM and Jurkat cells was then assessed. The levels of transforming growth factor-alpha (TGF-α), transforming growth factor- beta1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), and MMP-9 mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, the growth inhibitory effects of kefir on cell-cycle progression/apoptosis were assessed by Cell Death Detection (ELISA) and flow cytometry. Results: The maximum cytotoxicity recorded after 48-hours treatment with 80 μg/μL kefir was only 42% and 39% in CEM and Jurkat cells, respectively. The percent reduction in proliferation was very significant, and was dose-, and time-dependent. In both cell lines, kefir exhibited its antiproliferative effect by downregulating TGF-α and upregulating TGF-β1 mRNA expression. Upon kefir treatment, a marked increase in cell-cycle distribution was noted in the preG1 phase of CEM and Jurkat cells, indicating the proapoptotic effect of kefir, which was further confirmed by Cell Death Detection ELISA. However, kefir did not affect the mRNA expression of metalloproteinases needed for the invasion of leukemic cell lines. Conclusion: In conclusion, kefir is

  7. Kefir induces cell-cycle arrest and apoptosis in HTLV-1-negative malignant T-lymphocytes.

    PubMed

    Maalouf, Katia; Baydoun, Elias; Rizk, Sandra

    2011-02-14

    Adult lymphoblastic leukemia (ALL) is a malignancy that occurs in white blood cells. The overall cure rate in children is 85%, whereas it is only 40% in adults. Kefir is an important probiotic that contains many bioactive ingredients, which give it unique health benefits. It has been shown to control several cellular types of cancer. The present study investigates the effect of a cell-free fraction of kefir on CEM and Jurkat cells, which are human T-lymphotropic virus type I (HTLV-1)-negative malignant T-lymphocytes. Cells were incubated with different kefir concentrations. The cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir on the proliferation of CEM and Jurkat cells was then assessed. The levels of transforming growth factor-alpha (TGF-α), transforming growth factor- beta1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), and MMP-9 mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, the growth inhibitory effects of kefir on cell-cycle progression/apoptosis were assessed by Cell Death Detection (ELISA) and flow cytometry. The maximum cytotoxicity recorded after 48-hours treatment with 80 μg/μL kefir was only 42% and 39% in CEM and Jurkat cells, respectively. The percent reduction in proliferation was very significant, and was dose-, and time-dependent. In both cell lines, kefir exhibited its antiproliferative effect by downregulating TGF-α and upregulating TGF-β1 mRNA expression. Upon kefir treatment, a marked increase in cell-cycle distribution was noted in the preG(1) phase of CEM and Jurkat cells, indicating the proapoptotic effect of kefir, which was further confirmed by Cell Death Detection ELISA. However, kefir did not affect the mRNA expression of metalloproteinases needed for the invasion of leukemic cell lines. In conclusion, kefir is effective in inhibiting proliferation and inducing

  8. 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

  9. Comparative cytotoxicity of chelidonine and homochelidonine, the dimethoxy analogues isolated from Chelidonium majus L. (Papaveraceae), against human leukemic and lung carcinoma cells.

    PubMed

    Havelek, Radim; Seifrtova, Martina; Kralovec, Karel; Krocova, Eliska; Tejkalova, Veronika; Novotny, Ivan; Cahlikova, Lucie; Safratova, Marcela; Opletal, Lubomir; Bilkova, Zuzana; Vavrova, Jirina; Rezacova, Martina

    2016-03-15

    The search for new anticancer compounds is a crucial element of natural products research. In this study the effects of naturally occurring homochelidonine in comparison to chelidonine on cell cycle progression and cell death in leukemic T-cells with different p53 status are described. The mechanism of cytotoxic, antiproliferative, apoptosis-inducing effects and the effect on expressions of cell cycle regulatory proteins was investigated using XTT assay, Trypan blue exclusion assay, flow cytometry, Western blot analysis, xCELLigence, epi-fluorescence and 3D super resolution microscopy. A549 cells were used for xCELLigence, clonogenic assay and for monitoring microtubule stability. We found that homochelidonine and chelidonine displayed significant cytotoxicity in examined blood cancer cells with the exception of HEL 92.1.7 and U-937 exposed to homochelidonine. Unexpectedly, homochelidonine and chelidonine-induced cytotoxicity was more pronounced in Jurkat cells contrary to MOLT-4 cells. Homochelidonine showed an antiproliferative effect on A549 cells but it was less effective compared to chelidonine. Biphasic dose-depended G1 and G2/M cell cycle arrest along with the population of sub-G1 was found after treatment with homochelidonine in MOLT-4 cells. In variance thereto, an increase in G2/M cells was detected after treatment with homochelidonine in Jurkat cells. Treatment with chelidonine induced cell cycle arrest in the G2/M cell cycle in both MOLT-4 and Jurkat cells. MOLT-4 and Jurkat cells treated with homochelidonine and chelidonine showed features of apoptosis such as phosphatidylserine exposure, a loss of mitochondrial membrane potential and an increase in the caspases -3/7, -8 and -9. Western blots indicate that homochelidonine and chelidonine exposure activates Chk1 and Chk2. Studies conducted with fluorescence microscopy demonstrated that chelidonine and homochelidonine inhibit tubulin polymerization in A549 cells. Collectively, the data indicate that

  10. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. The novel organic arsenical darinaparsin induces MAPK-mediated and SHP1-dependent cell death in T-cell lymphoma and Hodgkin lymphoma cells and human xenograft models.

    PubMed

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

    2014-12-01

    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 about its mechanism of action. 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. Biologic effects of darinaparsin on the MAPK pathway were investigated using pharmacologic inhibitors, RNAi and transient transfection for overexpression for SHP1 and MEK. Darinaparsin treatment resulted in time- and dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines. In addition, 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 coimmunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, whereas cotreatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) and MEK constitutive activation decreased darinaparsin-related cell death. Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms. ©2014 American Association for Cancer Research.

  12. 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.

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

    PubMed Central

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

    2004-01-01

    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)-γ 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-γ 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. PMID:15270849

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Significant differences in genotoxicity induced by retrovirus integration in human T cells and induced pluripotent stem cells.

    PubMed

    Zheng, Weiyan; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

    2013-04-25

    Retrovirus is frequently used in the genetic modification of mammalian cells and the establishment of induced pluripotent stem cells (iPSCs) via cell reprogramming. Vector-induced genotoxicity could induce profound effect on the physiology and function of these stem cells and their differentiated progeny. We analyzed retrovirus-induced genotoxicity in somatic cell Jurkat and two iPSC lines. In Jurkat cells, retrovirus frequently activated host gene expression and gene activation was not dependent on the distance between the integration site and the transcription start site of the host gene. In contrast, retrovirus frequently down-regulated host gene expression in iPSCs, possibly due to the action of chromatin silencing that spreads from the provirus to the nearby host gene promoter. Our data raises the issue that some of the phenotypic variability observed among iPSC clones derived from the same parental cell line may be caused by retrovirus-induced gene expression changes rather than by the reprogramming process itself. It also underscores the importance of characterizing retrovirus integration and carrying out risk assessment of iPSCs before they can be applied in basic research and clinics. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Alternative medicine remedies might stimulate viability of leukemic cells.

    PubMed

    Styczynski, Jan; Wysocki, Mariusz

    2006-01-01

    Ex vivo activity of three products of alternative therapy against leukemic and normal cells was analyzed. Extracts of Viscum album, Uncaria tomentosa, and Croton lechleri were used for the study. Leukemic cells of 53 children with acute leukemias and four cell lines, Jurkat, CCRF-CEM, HL-60, and K-562 were tested for sensitivity to alternative medicine remedies by the MTT assay, cell-cycle analysis and annexin-V binding assay. Leukemic cells showed high resistance to tested three compounds of alternative medicine in all performed assays. Additionally, tested remedies stimulated survival of leukemic cells in 45%, 96%, and 83% cases, respectively; while no effect was observed in normal lymphocytes. In combination studies, Viscum album extract did not increase prednisolone and cytarabine cytotoxicity in leukemic cells. We conclude that some alternative medicine remedies might stimulate the viability of childhood leukemic cells.

  11. 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.

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

    DTIC Science & Technology

    2011-01-01

    about specific USEP- induced cellular effects that are unlikely to be true for all cell types. We also investigated the mechanism responsible for the...Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines Bennett L. Ibey1*, Caleb...HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD50 lower than the ED50 for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism

  13. 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.

  14. MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1.

    PubMed

    Qian, Lu; Zhang, Wanggang; Lei, Bo; He, Aili; Ye, Lianhong; Li, Xingzhou; Dong, Xin

    2016-11-01

    The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention.

  15. Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1

    PubMed Central

    1994-01-01

    We have established long-term cultures of several cell lines stably and uniformly expressing human immunodeficiency virus type 1 (HIV-1) in order to (a) identify naturally processed HIV-1 peptides recognized by cytotoxic T lymphocytes (CTL) from HIV-1-seropositive individuals and (b) consider the hypothesis that naturally occurring epitope densities on HIV-infected cells may limit their lysis by CTL. Each of two A2- restricted CD8+ CTL specific for HIV-1 gag or reverse transcriptase (RT) recognized a single naturally processed HIV-1 peptide in trifluoroacetic acid (TFA) extracts of infected cells: gag 77-85 (SLYNTVATL) or RT 476-484 (ILKEPVHGV). Both processed peptides match the synthetic peptides that are optimally active in cytotoxicity assays and have the consensus motif described for A2-associated peptides. Their abundances were approximately 400 and approximately 12 molecules per infected Jurkat-A2 cell, respectively. Other synthetic HIV-1 peptides active at subnanomolar concentrations were not present in infected cells. Except for the antigen processing mutant line T2, HIV- infected HLA-A2+ cell lines were specifically lysed by both A2- restricted CTL, although infected Jurkat-A2 cells were lysed more poorly by RT-specific CTL than by gag-specific CTL, suggesting that low cell surface density of a natural peptide may limit the effectiveness of some HIV-specific CTL despite their vigorous activity against synthetic peptide-treated target cells. PMID:7523570

  16. Arsenic, cadmium, and manganese levels in shellfish from Map Ta Phut, an industrial area in Thailand, and the potential toxic effects on human cells.

    PubMed

    Rangkadilok, Nuchanart; Siripriwon, Pantaree; Nookabkaew, Sumontha; Suriyo, Tawit; Satayavivad, Jutamaad

    2015-01-01

    Map Ta Phut Industrial Estate is a major industrial area in Thailand for both petrochemical and heavy industries. The release of hazardous wastes and other pollutants from these industries increases the potential for contamination in foods in the surrounding area, especially farmed shellfish. This study determined the arsenic (As), cadmium (Cd), and manganese (Mn) concentrations in the edible flesh of farmed shellfish, including Perna viridis, Meretrix meretrix, and Scapharca inaequivalvis, around the Map Ta Phut area using inductively coupled plasma mass spectrometry. The results showed that shellfish samples contained high levels of total As [1.84-6.42 mg kg(-1) wet weight (ww)]. High Mn concentrations were found in P. viridis and M. meretrix, whereas S. inaequivalis contained the highest Cd. Arsenobetaine (AsB) was found to be the major As species in shellfish (>45% of total As). The in vitro cytotoxicity of these elements was evaluated using human cancer cells (T47D, A549, and Jurkat cells). An observed decrease in cell viability in T47D and Jurkat cells was mainly caused by exposure to inorganic As (iAs) or Mn but not to AsB or Cd. The combined elements (AsB+Mn+Cd) at concentrations predicted to result from the estimated daily intake of shellfish flesh by the local people showed significant cytotoxicity in T47D and Jurkat cells.

  17. Selective targeting of leukemic cell growth in vivo and in vitro using a gene silencing approach to diminish S-adenosylmethionine synthesis.

    PubMed

    Attia, Ramy R; Gardner, Lidia A; Mahrous, Engy; Taxman, Debra J; Legros, Leighton; Rowe, Sarah; Ting, Jenny P-Y; Geller, Arthur; Kotb, Malak

    2008-11-07

    We exploited the fact that leukemic cells utilize significantly higher levels of S-adenosylmethionine (SAMe) than normal lymphocytes and developed tools that selectively diminished their survival under physiologic conditions. Using RNA interference gene silencing technology, we modulated the kinetics of methionine adenosyltransferase-II (MAT-II), which catalyzes SAMe synthesis from ATP and l-Met. Specifically, we silenced the expression of the regulatory MAT-IIbeta subunit in Jurkat cells and accordingly shifted the K(m L-Met) of the enzyme 10-15-fold above the physiologic levels of l-Met, thereby reducing enzyme activity and SAMe pools, inducing excessive apoptosis and diminishing leukemic cell growth in vitro and in vivo. These effects were reversed at unphysiologically high l-Met (>50 microm), indicating that diminished leukemic cell growth at physiologic l-Met levels was a direct result of the increase in MAT-II K(m L-Met) due to MAT-IIbeta ablation and the consequent reduction in SAMe synthesis. In our NOD/Scid IL-2Rgamma(null) humanized mouse model of leukemia, control shRNA-transduced Jurkat cells exhibited heightened engraftment, whereas cells lacking MAT-IIbeta failed to engraft for up to 5 weeks post-transplant. These stark differences in malignant cell survival, effected by MAT-IIbeta ablation, suggest that it may be possible to use this approach to disadvantage leukemic cell survival in vivo with little to no harm to normal cells.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. PRR7 Is a Transmembrane Adaptor Protein Expressed in Activated T Cells Involved in Regulation of T Cell Receptor Signaling and Apoptosis*

    PubMed Central

    Hrdinka, Matouš; Dráber, Peter; Štěpánek, Ondřej; Ormsby, Tereza; Otáhal, Pavel; Angelisová, Pavla; Brdička, Tomáš; Pačes, Jan; Hořejší, Václav; Drbal, Karel

    2011-01-01

    Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells. PMID:21460222

  6. 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

  7. 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

  8. 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.

  9. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines.

    PubMed

    Varamini, P; Soltani, M; Ghaderi, A

    2009-01-01

    There are reports on the presence of various compounds exerting different biological activities in Ruta graveolens, a plant of Rutaceae family. The aim of the present study was to evaluate in vitro cytotoxicity of the total extract of R. graveolens against tumor cell lines of different origin. Aerial parts of the plant was extracted with 70% ethanol by sonication method and cytotoxic activity was examined on RAJI, RAMOS, RPMI8866, U937, Jurkat, MDA-MB-453, MCF-7, LNCap-FGC-10, 5637, HeLa, SK-OV-3, A549, Mehr-80 and also peripheral blood mononuclear cells (PBMC) by the use of WST-1 assay. Results were expressed as IC(50) values. R. graveolens extract showed high cytotoxic activity against RAJI and RAMOS, two Burkitt's lymphoma cell lines, with an IC(50) equal to 24.3 microg/ml and 35.2 microg/ml respectively and LNCap-FGC-10, a prostate adenocarcinoma cell line with an IC(50) equal to 27.6 microg/ml as well as Mehr-80, a newly established Large Cell Lung Carcinoma (IC(50)=46.2 microg/ml). No significant anti-proliferative activity was observed on other cell lines including MCF-7, MDA-MB-453, SK-OV-3, HeLa, 5637, JURKAT and RPMI8866. Adverse cytotoxic effect of R. graveolens was investigated against PBMCs and a significantly lower effect of this extract (IC(50)=104 microg/ml) was seen on normal cells compared with RAJI and RAMOS, two haematopoietic cell lines.

  10. HIV Type 1 Nef Is Released from Infected Cells in CD45+ Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

    PubMed Central

    Raymond, A.D.; Campbell-Sims, T.C.; Khan, M.; Lang, M.; Huang, M.B.; Bond, V.C.

    2011-01-01

    Abstract HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10 ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIVMN. Interestingly, plasma mvNef levels in HIV+ patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIV-infected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS. PMID:20964480

  11. 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

  12. 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.

  13. Altered Expression of Tyrosine Kinases of the Src and Syk Families in Human T-Cell Leukemia Virus Type 1-Infected T-Cell Lines

    PubMed Central

    Weil, Robert; Levraud, Jean-Pierre; Dodon, Madeleine Duc; Bessia, Christine; Hazan, Uriel; Kourilsky, Philippe; Israël, Alain

    1999-01-01

    During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells. PMID:10196263

  14. 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

  15. Leukemia cell microvesicles promote survival in umbilical cord blood hematopoietic stem cells.

    PubMed

    Razmkhah, Farnaz; Soleimani, Masoud; Mehrabani, Davood; Karimi, Mohammad Hossein; Kafi-Abad, Sedigheh Amini

    2015-01-01

    Microvesicles can transfer their contents, proteins and RNA, to target cells and thereby transform them. This may induce apoptosis or survival depending on cell origin and the target cell. In this study, we investigate the effect of leukemic cell microvesicles on umbilical cord blood hematopoietic stem cells to seek evidence of apoptosis or cell survival. Microvesicles were isolated from both healthy donor bone marrow samples and Jurkat cells by ultra-centrifugation and were added to hematopoietic stem cells sorted from umbilical cord blood samples by magnetic associated cell sorting (MACS) technique. After 7 days, cell count, cell viability, flow cytometry analysis for hematopoietic stem cell markers and qPCR for P53 gene expression were performed. The results showed higher cell number, higher cell viability rate and lower P53 gene expression in leukemia group in comparison with normal and control groups. Also, CD34 expression as the most important hematopoietic stem cell marker, did not change during the treatment and lineage differentiation was not observed. In conclusion, this study showed anti-apoptotic effect of leukemia cell derived microvesicles on umbilical cord blood hematopoietic stem cells.

  16. Leukemia cell microvesicles promote survival in umbilical cord blood hematopoietic stem cells

    PubMed Central

    Razmkhah, Farnaz; Soleimani, Masoud; Mehrabani, Davood; Karimi, Mohammad Hossein; Kafi-abad, Sedigheh Amini

    2015-01-01

    Microvesicles can transfer their contents, proteins and RNA, to target cells and thereby transform them. This may induce apoptosis or survival depending on cell origin and the target cell. In this study, we investigate the effect of leukemic cell microvesicles on umbilical cord blood hematopoietic stem cells to seek evidence of apoptosis or cell survival. Microvesicles were isolated from both healthy donor bone marrow samples and Jurkat cells by ultra-centrifugation and were added to hematopoietic stem cells sorted from umbilical cord blood samples by magnetic associated cell sorting (MACS) technique. After 7 days, cell count, cell viability, flow cytometry analysis for hematopoietic stem cell markers and qPCR for P53 gene expression were performed. The results showed higher cell number, higher cell viability rate and lower P53 gene expression in leukemia group in comparison with normal and control groups. Also, CD34 expression as the most important hematopoietic stem cell marker, did not change during the treatment and lineage differentiation was not observed. In conclusion, this study showed anti-apoptotic effect of leukemia cell derived microvesicles on umbilical cord blood hematopoietic stem cells. PMID:26862318

  17. Synthesis of ZnPc loaded poly(methyl methacrylate) nanoparticles via miniemulsion polymerization for photodynamic therapy in leukemic cells.

    PubMed

    Feuser, Paulo Emilio; Gaspar, Pamela Cristina; Jacques, Amanda Virtuoso; Tedesco, Antônio Claudio; Santos Silva, Maria Claudia Dos; Ricci-Júnior, Eduardo; Sayer, Claudia; de Araújo, Pedro Henrique Hermes

    2016-03-01

    The goal of this work was to synthesize and characterize ZnPc loaded poly(methyl methacrylate) (PMMA) nanoparticles (NPs) by miniemulsion polymerization. Biocompatibility assays were performed in murine fibroblast (L929) cells and human peripheral blood lymphocytes (HPBL). Finally, photobiological assays were performed in two leukemic cells: chronic myeloid leukemia in blast crisis (K562) and acute lymphoblastic leukemia (Jurkat). ZnPc loaded PMMA NPs presented an average diameter of 97±2.5 nm with a low polydispersity index and negative surface charge. The encapsulation efficiency (EE %) of ZnPc PMMA NPs was 87%±2.12. The release of ZnPc from PMMA NPs was slow and sustained without the presence of burst effect, indicating homogeneous drug distribution in the polymeric matrix. NP biocompatibility was observed on the treatment of peripheral blood lymphocytes and L929 fibroblast cells. Phototoxicity assays showed that the ZnPc loaded in PMMA NPs was more phototoxic than ZnPc after activation with visible light at 675 nm, using a low light dose of 2J/cm(2) in both leukemic cells (Jurkat and K562). The results from fluorescence microscopy (EB/OA) and DNA fragmentation suggest that the ZnPc loaded PMMA NPs induced cell death by apoptosis. Based on presented results, our study suggests that PDT combined with the use of polymeric NPs, may be an excellent alternative for leukemia treatment. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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. PMID:28400837

  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. The microfluidic multitrap nanophysiometer for hematologic cancer cell characterization reveals temporal sensitivity of the calcein-AM efflux assay.

    PubMed

    Byrd, Thomas F; Hoang, Loi T; Kim, Eric G; Pfister, Matthew E; Werner, Erik M; Arndt, Stephen E; Chamberlain, Jeffrey W; Hughey, Jacob J; Nguyen, Bao A; Schneibel, Erik J; Wertz, Laura L; Whitfield, Jonathan S; Wikswo, John P; Seale, Kevin T

    2014-05-30

    Cytometric studies utilizing flow cytometry or multi-well culture plate fluorometry are often limited by a deficit in temporal resolution and a lack of single cell consideration. Unfortunately, many cellular processes, including signaling, motility, and molecular transport, occur transiently over relatively short periods of time and at different magnitudes between cells. Here we demonstrate the multitrap nanophysiometer (MTNP), a low-volume microfluidic platform housing an array of cell traps, as an effective tool that can be used to study individual unattached cells over time with precise control over the intercellular microenvironment. We show how the MTNP platform can be used for hematologic cancer cell characterization by measuring single T cell levels of CRAC channel modulation, non-translational motility, and ABC-transporter inhibition via a calcein-AM efflux assay. The transporter data indicate that Jurkat T cells exposed to indomethacin continue to accumulate fluorescent calcein for over 60 minutes after calcein-AM is removed from the extracellular space.

  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. Anti-inflammatory effects of green soybean extract irradiated with visible light.

    PubMed

    Tanaka, Keiko; Ohgo, Yasushi; Katayanagi, Yuki; Yasui, Kensuke; Hiramoto, Shigeru; Ikemoto, Hiroyuki; Nakata, Yumi; Miyoshi, Noriyuki; Isemura, Mamoru; Ohashi, Norio; Imai, Shinjiro

    2014-04-22

    We conducted a preliminary investigation of the effects of visible light irradiation on plant extracts, and we observed a strong suppressive effect on interleukin (IL) 2 expression with the inhibition of c-Jun amino-terminal kinase (JNK) phosphorylation in Jurkat cells by visible light irradiation to ethanol extract from green soybeans (LIEGS). This effect was produced only by extracts from green soybeans (Glycine max) and not other-color soybeans. LIEGS suppressed the lipopolysaccharide-induced IL-6, IL-12 and TNF-α expression levels in human monocyte THP-1 cells in a concentration-dependent manner. LIEGS was applied for 8 weeks to NC/Nga mice. LIEGS suppressed the development of atopic dermatitis (AD)-like skin lesions and reduced the dermatitis scores of the mice. The light irradiation changed the various types of small-molecule compounds in extracts. Visible light irradiation to daidzein with chlorophyll b induced a novel oxidative product of daidzein. This product suppressed IL-2 expression in Jurkat cells.

  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. 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

  11. 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.

  12. 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

  13. 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.

  14. Enhanced cellular respiration in cells exposed to doxorubicin.

    PubMed

    Souid, Abdul-Kader; Penefsky, Harvey S; Sadowitz, Peter D; Toms, Bonnie

    2006-01-01

    Doxorubicin executes topoisomerase II mediated apoptosis, a process known to result in mitochondrial dysfunction, such as the leakage of cytochrome c and the opening of mitochondrial permeability transition pores (PTP). To further define the effects of doxorubicin on cell metabolism, we measured cellular respiration, cellular ATP, DNA fragmentation, and cytochrome c leakage in Jurkat (supersensitive), human leukemia-60 (HL-60, sensitive), and HL-60/MX2 (resistant) cells following exposure to 1.0 microM doxorubicin for 30 min. The measurements were made after 24 h of exposure to the drug. In Jurkat and HL-60 cells, doxorubicin treatment increased cellular mitochondrial oxygen consumption and ATP content by 2-3-fold. The increment in oxygen consumption was blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk) and by the PTP inhibitor cyclosporin A. In HL-60/MX2 cells, which are resistant because of a reduced topoisomerase II activity, doxorubicin treatment was without effect on either respiration or ATP content, suggesting that topoisomerase II was essential for induction of apoptosis and stimulation of respiration and ATP content. The conclusion that both of the latter processes were products of oxidations in the mitochondrial respiratory chain was supported by the further observation that rotenone and sodium cyanide inhibited oxygen consumption and substantially lowered ATP content in the treated and untreated cells. Thus, oxidative phosphorylation is enhanced in cells briefly incubated with doxorubicin for as long as 24 h post drug exposure despite apoptosis-associated mitochondrial insults caused by the drug.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cells.

    PubMed

    Nzounza, Patrycja; Chazal, Maxime; Guedj, Chloé; Schmitt, Alain; Massé, Jean-Marc; Randriamampita, Clotilde; Pique, Claudine; Ramirez, Bertha Cecilia

    2012-01-01

    Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA. Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry.

  4. 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.

  5. 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.

  6. 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

  7. Flow cytometry analysis of cell population dynamics and cell cycle during HIV-1 envelope-mediated formation of syncytia in vitro.

    PubMed

    Torres-Castro, Israel; Cortés-Rubio, César N; Sandoval, Guadalupe; Lamoyi, Edmundo; Larralde, Carlos; Huerta, Leonor

    2014-01-01

    Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell-cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4(+) cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4(+) cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env(+) cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6 h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4(+) cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell-cell fusion can be performed by flow cytometry.

  8. 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.

  9. 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.

  10. Aronia melanocarpa Juice Induces a Redox-Sensitive p73-Related Caspase 3-Dependent Apoptosis in Human Leukemia Cells

    PubMed Central

    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 G2/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. PMID:22412883

  11. 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.

  12. 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.

  13. Hybrid cell sorters for on-chip cell separation by hydrodynamics and magnetophoresis

    NASA Astrophysics Data System (ADS)

    Seo, Hye-Kyoung; Kim, Yong-Ho; Kim, Hyun-Ok; Kim, Yong-Jun

    2010-09-01

    The classification of cells is important for medical diagnosis and medicine research. A micro-scale analysis system for blood allows immediate diagnosis, regardless of time or location. Rapid handling of biological cells is required for the development of lab-on-a-chip and point-of-care technologies. In previous papers, the reported sample solution flow rates for hydrodynamic cell separation ranged from 0.1 to tens of μl min-1. This paper proposes a hybrid cell sorter that combines hydrodynamics and magnetophoresis. This cell sorter is proposed for improving the classification efficiency of a virtual impactor-based cell sorter and for permitting the hydrodynamic cell separation with high throughput. In addition, its performance has been evaluated in the classification of Jurkat cells and blood cells. In the Jurkat cell experiment, the classification efficiency of Jurkat cells at the major outlet decreased from 70 to 50.8% with the proposed hybrid scheme. The flow rate for cell separation was 1 ml min-1. The classification efficiency of red blood cells (RBCs) increased from 75.2 to 86.8% with the application of a magnetic field. Also, the classification efficiency of white blood cells (WBCs) decreased from 83.8 to 70.9% with an applied magnetic field. Experimental results demonstrated that the classification efficiency of blood cells can be modulated and enhanced by magnetophoresis and that the hybrid cell sorter has potential for lab-on-a-chip applications.

  14. 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

  15. 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

  16. 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

  17. Assessment of the involvement of oxidative stress and Mitogen-Activated Protein Kinase signaling pathways in the cytotoxic effects of arsenic trioxide and its combination with sulindac or its metabolites: sulindac sulfide and sulindac sulfone on human leukemic cell lines.

    PubMed

    Stępnik, M; Ferlińska, M; Smok-Pieniążek, A; Gradecka-Meesters, D; Arkusz, J; Stańczyk, M

    2012-06-01

    The purpose of the study was to characterize the involvement of reactive oxygen species (ROS) in mediating the cytotoxic effects of arsenic trioxide (ATO) in combination with sulindac or its metabolites: sulfide (SS) and sulfone (SF) on human leukemic cell lines. Jurkat, HL-60, K562, and HPB-ALL cells were exposed to the drugs alone or in combinations. Cell viability was measured using WST-1 or XTT reduction tests and ROS production by dichlorodihydrofluorescein diacetate staining (flow cytometry). Modulation of (a) intracellular glutathione (GSH) level was done by using L: -buthionine sulfoximine (BSO) or diethylmaleate (DEM), (b) NADPH oxidase by using diphenyleneiodonium (DPI), and (c) MAP kinases by using SB202190 (p38), SP600125 (JNK), and U0126 (ERK) inhibitors. ATO cytotoxicity (0.5 or 1 μM) was enhanced by sulindacs, with higher activity showed by the metabolites. Strong cytotoxic effects appeared at SS and SF concentrations starting from 50 μM. The induction of ROS production seemed not to be the major mechanism responsible for the cytotoxicity of the combinations. A strong potentiating effect of BSO on ATO cytotoxicity was demonstrated; DEM (10-300 μM) and DPI (0.0025-0.1 μM; 72 h) did not influence the effects of ATO. Some significant decreases in the viability of the cells exposed to ATO in the presence of MAPK inhibitors comparing with the cells exposed to ATO alone were observed; however, the effects likely resulted from a simple additive cytotoxicity of the drugs. The combinations of ATO with sulindacs offer potential therapeutic usefulness.

  18. Effects of Nonequilibrium Plasmas on Eukaryotic Cells

    DTIC Science & Technology

    2009-05-01

    on Eukaryotic Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER FA9550-06-1-0004 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Dr. Mounir Laroussi, P.I., and...plume in room air. is a dielectric barrier discharge riptions we first present the effects types of eukaryotic microalgae. e cells are presented...preliminary results on the effects 15. SUBJECT TERMS Glow discharge, Atmospheric pressure, air plasma, eukaryote , cell , non-equilibrium 19a. NAME

  19. Tumor necrosis factor alpha inhibitors have no effect on a human T-lymphotropic virus type-I (HTLV-I)-infected cell line from patients with HTLV-I-associated myelopathy.

    PubMed

    Fukui, Shoichi; Nakamura, Hideki; Takahashi, Yoshiko; Iwamoto, Naoki; Hasegawa, Hiroo; Yanagihara, Katsunori; Nakamura, Tatsufumi; Okayama, Akihiko; Kawakami, Atsushi

    2017-02-03

    While tumor necrosis factor alpha (TNF-α) inhibitors (TNFi) and other biologics are very effective against autoimmune diseases, they can also cause infectious diseases. Therefore, it is important to clarify whether the TNFi sometimes used to treat patients with rheumatoid arthritis (RA) complicated with human T-lymphotropic virus type-I (HTLV-I) infection have the unintended side effect of promoting HTLV-I proliferation. We used the HTLV-I-infected cell line HCT-5, derived from spinal fluid cells of a patient with HTLV-I associated myelopathy, to evaluate the production of cytokines and chemokines, TNF-α receptor (TNFR), the expression of HTLV-I associated genes, the HTLV-I proviral load (PVL), the expression of HTLV-I structural protein, and apoptosis. We used Jurkat cells as a control. Supernatants of HCT-5 showed time-dependent elevations of IL-6, RANTES and ICAM-1. HCT-5 supernatants treated with infliximab, adalimumab, etanercept (ETN), golimumab and certolizumab pegol showed no significant differences in the levels of these molecules compared to the control. Neither TNFR1 nor TNFR2 expression was altered by any TNFi treatment, relative to phosphate-buffered saline (PBS) treatment, with the exception that TNFR2 was significantly decreased and internalized in HCT-5 cells by ETN treatment. The HTLV-I associated genes Tax and HBZ and the PVL levels were not significantly changed. Immunofluorescence staining of HCT-5 for an HTLV-I-associated protein, GAG, was also not significantly different between any of the TNFi treatments and the PBS treatment. DNA ladders as an index of apoptosis were not detected. Apoptotic cells were not increased by the addition of any TNFi. In vitro, TNFi did not affect the cytokine profiles, expression of associated genes and proteins, proviral load or apoptosis of HCT-5 cells. The results suggested that TNFi treatment of RA patients complicated with HTLV-I might have no effect on HTLV-I infection.

  20. 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.

  1. 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.