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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. [Effect of phenylhexyl isothiocyanate on Wnt/beta-catenin signaling pathway in Jurkat cell line].

    PubMed

    Lin, Juan; Huang, Yi-Qun; Ma, Xu-Dong

    2013-04-01

    This study was purposed to investigate the effect of phenylhexyl isothiocyanate (PHI) on Wnt/β-catenin signaling pathway, histone acetylation, histone methylation and cell apoptosis in Jurkat cell line. The viability of Jurkat cells after treatment with PHI was tested by MTT. Apoptotic rate of Jurkat cells was measured by flow cytometry. The levels of Wnt/β-catenin related proteins including β-catenin, TCF, c-myc, and cyclinD1, histone acetylated H3 and H4, histone methylated H3K9 and H3K4 were detected by Western blot. The results showed that PHI inhibited the cell growth and induced apoptosis in Jurkat cells in time-and dose-dependent manners. Its IC50 at 48 h was about 20 µmol/L. Expression of histone acetylated H3, H4 and histone methylated H3k4 increased after exposure to PHI for 3 h, while histone methylated H3K9 decreased. Expression of β-catenin was not changed after exposure to PHI for 3 h, but expression of β-catenin, and its cell cycle-related genes such as TCF, c-myc and cyclinD1 decreased after exposure to PHI for 7 h. It is concluded that PHI regulates acetylation and methylation of histone, inhibits Wnt/β-catenin signal pathway, and is able to induce apoptosis and inhibits growth of Jurkat cells. PMID:23628033

  3. 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

  4. Cytotoxic effect of inositol hexaphosphate and its Ni(II) complex on human acute leukemia Jurkat T cells.

    PubMed

    de Lima, Eliane May; Kanunfre, Carla Cristine; de Andrade, Lucas Ferrari; Granato, Daniel; Rosso, Neiva Deliberali

    2015-12-01

    Inositol hexaphosphate (InsP6) is present in cereals, legumes, nuts and seed oils and is biologically active against some tumor and cancer cells. Herein, this study aimed at evaluating the cellular toxicity, antiproliferative activity and effects on cell cycle progression of free InsP6 and InsP6-Ni(II) of leukemic T (Jurkat) and normal human cells. Treatments with InsP6 at concentrations between 1.0 and 4.0mM significantly decreased the viability of Jurkat cells, but showed no cytotoxic effect on normal human lymphocytes. Treatment with InsP6-Ni(II) complex at concentrations between 0.05 and 0.30 mM showed an anti-proliferative dose and a time-dependent effect, with significantly reduced cell viability of Jurkat cells but showed no cytotoxic effect on normal human lymphocytes as compared to the control. Ni(II) free ion was toxic to normal cells while InsP6-Ni(II) had no cytotoxic effect. The InsP6-Ni(II) complex potentiated (up to 10×) the antiproliferative effect of free InsP6 on Jurkat cells. The cytometric flow assay showed that InsP6 led to an accumulation of cells in the G0/G1 phase of the cell cycle, accompanied by a decrease in the number of cells in S and G2/M phases, whereas InsP6-Ni(II) has led to an accumulation of cells in the S and G2/M phases. Our findings showed that InsP6-Ni(II) potentiates cytotoxic effects of InsP6 on Jurkat cells and may be a potential adjuvant in the treatment of cancer. PMID:26335902

  5. 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

    2013-11-01

    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. PMID:24188375

  6. 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

    2013-11-01

    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.

  7. 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.

  8. In vitro study of AFB1 and AFM1 effects on human lymphoblastoid Jurkat T-cell model.

    PubMed

    Luongo, D; Russo, R; Balestrieri, A; Marzocco, S; Bergamo, P; Severino, L

    2014-10-01

    Aflatoxin B(1) (AFB(1)) is a mycotoxin produced by Aspergillus spp. that can occur as a natural contaminant in foods and feeds of vegetable origin. Post-ingestion, AFB(1) can be metabolized in the liver of mammals into hydroxylated aflatoxin M(1) (AFM(1)) that is excreted with milk. Although several studies have been carried out to evaluate effects of AFB(1) on the immune system, studies regarding AFM(1) are moreover lacking. The aim of the current study was to investigate effects of AFB(1) and AFM(1) on immune function using a lymphoblastoid Jurkat T-cell line as an experimental model. Both AFB(1) and AFM(1) produced significant decreases in Jurkat cell proliferation, whereas only minor effects were noted on interleukin (IL)-2 and interferon (IFN)-γ cytokines mRNA expression in stimulated cells that had been pre-incubated with AFB(1) and AFM(1). Particularly, AFB(1), but not AFM(1), at the highest concentration (50 µM) induced a marked increase in IL-8 mRNA expression. The results of the current study suggested the existence of a concentration threshold for AFB(1) and AFM(1) needed to exert biological activity on cell viability and innate immunity.

  9. 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.

  10. 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

  11. The synergistic repressive effect of NF-κB and JNK inhibitor on the clonogenic capacity of Jurkat leukemia cells.

    PubMed

    Liu, Xinli; Zhang, Jun; Li, Jing; Volk, Andrew; Breslin, Peter; Zhang, Jiwang; Zhang, Zhou

    2014-01-01

    Deregulation of Nuclear Transcription Factor-κB (NF-κB) and Jun N-terminal kinase (JNK) signaling is commonly detected in leukemia, suggesting an important role for these two signaling pathways in the pathogenesis of leukemia. In this study, using Jurkat cells, an acute T-lymphoblastic leukemia (T-ALL) cell line, we evaluated the effects of an NF-κB inhibitor and a JNK inhibitor individually and in combination on the proliferation, survival and clonogenic capacity of leukemic cells. We found that leukemic stem/progenitor cells (LSPCs) were more sensitive to NF-κB inhibitor treatment than were healthy hematopoietic stem/progenitor cells (HSPCs), as shown by a reduction in the clonogenic capacity of the former. Inactivation of NF-κB leads to the activation of JNK signaling in both leukemic cells and healthy HSPCs. Interestingly, JNK inhibitor treatment enhanced the repressive effects of NF-κB inhibitor on LSPCs but prevented such repression in HSPCs. Our data suggest that JNK signaling stimulates proliferation/survival in LSPCs but is a death signal in HSPCs. The combination of NF-κB inhibitor and JNK inhibitor might provide a better treatment for T-ALL leukemia by synergistically killing LSPCs while simultaneously preventing the death of normal HPCs. PMID:25526629

  12. The Synergistic Repressive Effect of NF-κB and JNK Inhibitor on the Clonogenic Capacity of Jurkat Leukemia Cells

    PubMed Central

    Liu, Xinli; Zhang, Jun; Li, Jing; Volk, Andrew; Breslin, Peter; Zhang, Jiwang; Zhang, Zhou

    2014-01-01

    Deregulation of Nuclear Transcription Factor-κB (NF-κB) and Jun N-terminal kinase (JNK) signaling is commonly detected in leukemia, suggesting an important role for these two signaling pathways in the pathogenesis of leukemia. In this study, using Jurkat cells, an acute T-lymphoblastic leukemia (T-ALL) cell line, we evaluated the effects of an NF-κB inhibitor and a JNK inhibitor individually and in combination on the proliferation, survival and clonogenic capacity of leukemic cells. We found that leukemic stem/progenitor cells (LSPCs) were more sensitive to NF-κB inhibitor treatment than were healthy hematopoietic stem/progenitor cells (HSPCs), as shown by a reduction in the clonogenic capacity of the former. Inactivation of NF-κB leads to the activation of JNK signaling in both leukemic cells and healthy HSPCs. Interestingly, JNK inhibitor treatment enhanced the repressive effects of NF-κB inhibitor on LSPCs but prevented such repression in HSPCs. Our data suggest that JNK signaling stimulates proliferation/survival in LSPCs but is a death signal in HSPCs. The combination of NF-κB inhibitor and JNK inhibitor might provide a better treatment for T-ALL leukemia by synergistically killing LSPCs while simultaneously preventing the death of normal HPCs. PMID:25526629

  13. The synergistic repressive effect of NF-κB and JNK inhibitor on the clonogenic capacity of Jurkat leukemia cells.

    PubMed

    Liu, Xinli; Zhang, Jun; Li, Jing; Volk, Andrew; Breslin, Peter; Zhang, Jiwang; Zhang, Zhou

    2014-01-01

    Deregulation of Nuclear Transcription Factor-κB (NF-κB) and Jun N-terminal kinase (JNK) signaling is commonly detected in leukemia, suggesting an important role for these two signaling pathways in the pathogenesis of leukemia. In this study, using Jurkat cells, an acute T-lymphoblastic leukemia (T-ALL) cell line, we evaluated the effects of an NF-κB inhibitor and a JNK inhibitor individually and in combination on the proliferation, survival and clonogenic capacity of leukemic cells. We found that leukemic stem/progenitor cells (LSPCs) were more sensitive to NF-κB inhibitor treatment than were healthy hematopoietic stem/progenitor cells (HSPCs), as shown by a reduction in the clonogenic capacity of the former. Inactivation of NF-κB leads to the activation of JNK signaling in both leukemic cells and healthy HSPCs. Interestingly, JNK inhibitor treatment enhanced the repressive effects of NF-κB inhibitor on LSPCs but prevented such repression in HSPCs. Our data suggest that JNK signaling stimulates proliferation/survival in LSPCs but is a death signal in HSPCs. The combination of NF-κB inhibitor and JNK inhibitor might provide a better treatment for T-ALL leukemia by synergistically killing LSPCs while simultaneously preventing the death of normal HPCs.

  14. 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.

  15. 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.

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

    PubMed

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

    2011-07-01

    Naegleria fowleri, a free-living amoeba, is the causative pathogen of primary amoebic meningoencephalitis in humans and experimental mice. N. fowleri is capable of destroying tissues and host cells through lytic necrosis. However, the mechanism by which N. fowleri induces host cell death is unknown. Electron microscopy indicated that incubation of Jurkat T cells with N. fowleri trophozoites induced necrotic morphology of the Jurkat T cells. N. fowleri also induced cytoskeletal protein cleavage, extensive poly (ADP-ribose) polymerase hydrolysis and lactate dehydrogenase (LDH) release. Although no activation of caspase-3 was observed in Jurkat T cells co-incubated with amoebae, intracellular reactive oxygen species (ROS) were strongly generated by NADPH oxidase (NOX). Pretreating cells with necroptosis inhibitor necrostatin-1 or NOX inhibitor diphenyleneiodonium chloride (DPI) strongly inhibited amoeba-induced ROS generation and Jurkat cell death, whereas pan-caspase inhibitor z-VAD-fmk did not. N. fowleri-derived secretory products (NfSP) strongly induced intracellular ROS generation and cell death. Necroptotic effects of NfSP were effectively inhibited by pretreating NfSP with proteinase K. Moreover, NfSP-induced LDH release and intracellular ROS accumulation were inhibited by pretreating Jurkat T cells with DPI or necrostatin-1. These results suggest that N. fowleri induces ROS-dependent necroptosis in Jurkat T cells. PMID:21535020

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

    PubMed

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

    2011-07-01

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

  19. 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.

  20. 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. PMID:27433287

  1. 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

  2. Umbelliprenin from Ferula szowitsiana Activates both Intrinsic and Extrinsic Pathways of Apoptosis in Jurkat T-CLL cell line.

    PubMed

    Gholami, Omid; Jeddi-Tehrani, Mahmood; Iranshahi, Mehrdad; Zarnani, Amir Hassan; Ziai, Seyed Ali

    2013-01-01

    Umbelliprenin is a prenylated compound, which belongs to the class of sesquiterpene coumarins. It is extracted from dried roots of Ferula szwitsiana collected from the mountains of Golestan forest (Golestan Province, north of Iran). Induction of apoptosis in Jurkat T-CLL cells has been previously shown. In this study, effect of umbelliprenin on proapoptotic caspases (caspase-8 and -9) and antiapoptotic Bcl-2 family protein was studied. Jurkat cells were incubated with umbelliprenin. Cells were then lysed and activation of proteins was studied by Western blot analysis. In this study, we showed that umbelliprenin activates intrinsic and extrinsic pathways of apoptosis by the activation of caspase-8 and -9 respectively. Inhibition of Bcl-2 was also shown. In conclusion, umbelliprenin induced apoptosis in Jurkat cells through caspase-dependent apoptosis pathway.

  3. Umbelliprenin from Ferula szowitsiana Activates both Intrinsic and Extrinsic Pathways of Apoptosis in Jurkat T-CLL cell line

    PubMed Central

    Gholami, Omid; Jeddi-Tehrani, Mahmood; Iranshahi, Mehrdad; Zarnani, Amir Hassan; Ziai, Seyed Ali

    2013-01-01

    Umbelliprenin is a prenylated compound, which belongs to the class of sesquiterpene coumarins. It is extracted from dried roots of Ferula szwitsiana collected from the mountains of Golestan forest (Golestan Province, north of Iran). Induction of apoptosis in Jurkat T-CLL cells has been previously shown. In this study, effect of umbelliprenin on proapoptotic caspases (caspase-8 and -9) and antiapoptotic Bcl-2 family protein was studied. Jurkat cells were incubated with umbelliprenin. Cells were then lysed and activation of proteins was studied by Western blot analysis. In this study, we showed that umbelliprenin activates intrinsic and extrinsic pathways of apoptosis by the activation of caspase-8 and -9 respectively. Inhibition of Bcl-2 was also shown. In conclusion, umbelliprenin induced apoptosis in Jurkat cells through caspase-dependent apoptosis pathway. PMID:24250644

  4. Baicalein suppresses the proliferation of acute T-lymphoblastic leukemia Jurkat cells by inhibiting the Wnt/β-catenin signaling.

    PubMed

    Liu, Xiaoping; Liu, Shengcai; Chen, Jiarui; He, Li; Meng, Xiangyu; Liu, Shangqin

    2016-10-01

    Although the response rates of chemotherapy in patients with acute T-lymphoblastic leukemia (T-ALL) have improved significantly, the outcome of these patients is still poor. Previous studies suggested that baicalein could inhibit the growth of several cancers, while its effect on T-ALL cells remains unclear. We used Jurkat cells as an in vitro model of T-ALL. Cell counting kit-8 assay and cytometric analysis with Annexin V-FITC/PI double staining were used to investigate the proliferation and apoptosis of Jurkat cells treated with increasing concentration of baicalein for indicated time. RT-PCR and western blotting was used to test the expression of Wnt/β-catenin associated genes and proteins. In cell viability assay, baicalein could inhibit the proliferation of Jurkat cells both in dose- and time-dependent manners. In cell apoptosis assay, baicalein could stimulate apoptosis of Jurkat cells both in dose- and time-dependent manners. Moreover, we demonstrated that baicalein could down-regulated the mRNA and protein levels of β-catenin and its widely accepted downstream targets (c-Myc, cyclin D1, and Axin2) in dose-dependent manners. These results proved that baicalein might be a potential choice for the treatment of T-ALL.

  5. Deficiency of ADAP/Fyb/SLAP-130 destabilizes SKAP55 in Jurkat T cells.

    PubMed

    Huang, Yanping; Norton, Darrell D; Precht, Patricia; Martindale, Jennifer L; Burkhardt, Janis K; Wange, Ronald L

    2005-06-24

    ADAP (adhesion and degranulation-promoting adaptor protein) and SKAP55 (Src kinase-associated phosphoprotein of 55 kDa) are T cell adaptors that mediate inside-out signaling from the T cell antigen receptor to integrins, giving rise to increased integrin affinity/avidity and formation of the immunological synapse between the T cell and the antigen-presenting cell. These two proteins are tightly and constitutively associated with one another, and their ability to interact is required for inside-out signaling. Here we show in an ADAP-deficient Jurkat T cell line that the co-dependence of ADAP and SKAP55 extends beyond their functional and physical interactions and show that SKAP55 protein is unstable in the absence of ADAP. Restoration of ADAP to the ADAP-deficient Jurkat T cell line restores SKAP55 expression by causing a 5-fold decrease in the rate of SKAP55 proteolysis. Inactivation of the Src homology 3 domain of SKAP55, which mediates the association between SKAP55 with ADAP, blocks the protective effect of ADAP. The half-life of SKAP55, in the absence of ADAP, is approximately 15-20 min, increasing to 90 min in the presence of ADAP. This is a remarkably rapid rate of turnover for a signaling protein and suggests the possibility that stimuli that signal for the stabilization of SKAP55 may play an important role in T cell adhesion and conjugate formation.

  6. Damage of neuroblastoma cell SH-SY5Y mediated by MPP+ inhibits proliferation of T-cell leukemia Jurkat by co-culture system.

    PubMed

    Wang, Fuli; Awan, Umer Farooq; Wang, Yuanyuan; Wang, Luna; Qing, Hong; Ma, Hong; Deng, Yulin

    2014-01-01

    The adaptive immune system has implications in pathology of Parkinson's disease (PD). Research data demonstrated that the peripheral CD4+ T-cell population decreased in pathogenesis of PD. The effect of damaged dopaminergic neurons on peripheral T cells of PD is still unknown. In this study, we constructed a neuronal and glial cells co-culture model by using human neuroblastoma cells SH-SY5Y and gliomas cells U87. After the co-culture cells were treated with neurotoxin 1-methyl-4-phenylpyridinium (MPP+) for 24 h, the conditioned media was harvested and used to cultivate T-cell leukemia Jurkat cells for another 24 h. We then analyzed the cell proliferation, cell cycle and necrosis effect of Jurkat cells. The results showed that co-culture medium of SH-SY5Y and U87 cells with MPP+ treatment inhibited the proliferation of Jurkat cells compared to control medium without MPP+, even though the same concentration of MPP+ had very little toxicity to the Jurkat cell. Furthermore, co-culture medium with low concentration of MPP+ (100 µM) arrested Jurkat cells cycle in G2/M phase through increasing cell cycle division 2 (CDC2) and CyclinB1 expression level, whereas co-culture medium with high concentration of MPP+ (500 µM) induced Jurkat cell necrosis through cellular swelling and membrane breakage. Our data implies that damaged dopamine neurons with glial cells can lead to the reduced number or inhibited proliferation activity of peripheral T cells. PMID:24933638

  7. Influence of microgravity on mitogen binding and cytoskeleton in Jurkat cells

    NASA Astrophysics Data System (ADS)

    Sciola, L.; Cogoli-Greuter, M.; Cogoli, A.; Spano, A.; Pippia, P.

    1999-01-01

    The effects of microgravity on Jurkat cells — a T-lymphoid cell line — was studied on a sounding rocket flight. An automated pre-programmed instrument permitted the injection of fluorescent labelled concanavalin A (Con A), culture medium and/or fixative at given times. An in-flight 1 g centrifuge allowed the comparison of the data obtained in microgravity with a 1 g control having the same history related to launch and re-entry. After flight, the cells fixed either at the onset of microgravity or after a 12 minute incubation time with fluorescent concanavalin A were labelled for vimentin and actin and analysed by fluorescence microscopy. Binding of Con A to Jurkat cells is not influenced by microgravity, whereas patching of the Con A receptors is significantly lower. A significant higher number of cells show changes in the structure of vimentin in microgravity. Most evident is the appearance of large bundles, significantly increased in the microgravity samples. No changes are found in the structure of actin and in the colocalisation of actin on the inner side of the cell membrane with the Con A receptors after binding of the mitogen.

  8. [MIP-1α promotes the migration ability of Jurkat cell through human brain microvascular endothelial cell monolayer].

    PubMed

    Ma, Yi-Ran; Zhang, Shuang; Sun, Ying; Liu, Yi-Yang; Song, Qian; Hao, Yi-Wen

    2014-02-01

    This study was purposed to explore the mechanism of central nervous system (CNS) leukemia resulting from brain metastasis of human acute T-cell leukemia (T-ALL) cells and the role of MIP-1α in migration of Jurkat cells through human brain microvascular endothelial cells (HBMEC). The real-time PCR, siRNA test, transendothelial migration test, endothelial permeability assay and cell adhesion assay were used to detect MIP-1α expression, penetration and migration ability as well as adhesion capability respectively. The results showed that the MIP-1α expression in Jurkat cells was higher than that in normal T cells and CCRF-HSB2, CCRF-CEM , SUP-T1 cells. The MIP-1α secreted from Jurkat cells enhanced the ability of Jurkat cells to penetrate through HBMEC, the ability of Jurkat cells treated by MIP-1α siRNA to adhere to HBMEC and to migrate trans endothelial cells decreased. It is concluded that the MIP-1α secreted from Jurkat cells participates in process of penetrating the Jurkat cells through HBMEC monolayer.

  9. 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

  10. Differential Response of Mono Mac 6, BEAS-2B, and Jurkat Cells to Indoor Dust

    PubMed Central

    Riechelmann, Herbert; Deutschle, Tom; Grabow, Ariane; Heinzow, Birger; Butte, Werner; Reiter, Rudolf

    2007-01-01

    Background Airway toxicity of indoor dust is not sufficiently understood. Objectives Our goal in this study was to describe the effects of indoor dust on human monocyte, epithelial, and lymphocyte cell lines. We aimed to a) obtain a comprehensive and intelligible outline of the transcriptional response; b) correlate differential transcription with cellular protein secretion; c) identify cell line–specific features; and d) search for indoor dust–specific responses. Methods Settled dust was sampled in 42 German households, and various contaminants were characterized. We exposed Mono Mac 6, BEAS-2B, and Jurkat cells to 500 μg/mL indoor dust for 6 hr. Outcome parameters included the transcriptional profile of an oligonucleotide microarray covering 1,232 genes. Significantly enriched Gene Ontology themes were calculated. Supernatant protein levels of 24 inflammatory response proteins served to confirm transcriptional results. Results An intraclass correlation coefficient of 0.8 indicated reasonable microarray reproducibility. The transcriptional profile was characterized by enhancement of detoxification and a danger and defense response. Differential gene regulation correlated with protein secretion (Goodman and Kruskal’s gamma coefficient: 0.72; p < 0.01). Mono Mac 6 cells revealed the highest fraction of differentially expressed genes, dominated by up-regulation of various cytokines and chemokines. BEAS-2B cells revealed weaker changes in a limited set of inflammatory response proteins. No significant changes were observed in Jurkat cells. Conclusions Monocytes are particularly responsive to indoor dust. We observed a classical T-helper 1-dominated immune response, which suggested that bioorganic contaminants are relevant effectors in indoor dust. PMID:17805423

  11. 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.

  12. Osmocenyl-tamoxifen derivatives target the thioredoxin system leading to a redox imbalance in Jurkat cells.

    PubMed

    Scalcon, Valeria; Top, Siden; Lee, Hui Zhi Shirley; Citta, Anna; Folda, Alessandra; Bindoli, Alberto; Leong, Weng Kee; Salmain, Michèle; Vessières, Anne; Jaouen, Gérard; Rigobello, Maria Pia

    2016-07-01

    The synthesis and the biological effects of two ferrocifen analogs in the osmium series, namely the monophenolic complex 1, the tamoxifen-like complex 2 and their oxidized quinone methide (QM) derivatives, 1-QM and 2-QM, are reported. Inhibition of purified thioredoxin reductase (TrxR) is observed with 1 and 2 only after their enzymatic oxidation by the hydrogen peroxide/horseradish peroxidase (H2O2/HRP) system with IC50 of 2.4 and 1.2μM respectively. However, this inhibition is larger than that obtained with the corresponding quinone methides (IC50=5.4μM for 1-QM and 3.6μM for 2-QM). The UV-Vis spectra of 1 or 2 incubated in the presence of H2O2/HRP show that the species generated is not a quinone methide, but probably the corresponding cation. In Jurkat cells, 2 shows high toxicity (IC50=7.4μM), while 1 is less effective (IC50=42μM). Interestingly, a significant inhibition of TrxR activity is observed in cells incubated with 2 (about 70% inhibition with 15μM) while the inhibition induced by 1 is much weaker (about 30% inhibition with 50μM). This strong inhibition of TrxR by 2 leads to accumulation of thioredoxin and peroxiredoxin 3 in oxidized form and to a decrease of the mitochondrial membrane potential (MMP). These results show that cytotoxicity of the osmocifens depends on their oxidation within the cell and that inhibition of thioredoxin reductase by oxidized species is a key factor in rationalizing the cytotoxicity of these complexes on Jurkat cells. PMID:27130146

  13. 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.

  14. Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells).

    PubMed

    Zhang, Min; Zhu, Qi; Shi, Ming; Liu, Yang; Ma, Lei; Yang, Yining; Feng, Dongyun; Dai, Wen; Zhang, Lin; Kang, Tao; Chen, Ping; He, Ying; Liu, Tingting; Zhao, Qing; Wang, Wenjing; Zhi, Jin; Feng, Guodong; Zhao, Gang

    2015-08-01

    This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis.

  15. 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

  16. 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

  17. 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.

  18. CEACAM1 regulates Fas-mediated apoptosis in Jurkat T-cells via its interaction with β-catenin.

    PubMed

    Li, Yun; Shively, John E

    2013-05-01

    CEACAM1 (Carcinoembryonic Antigen Cell Adhesion molecule 1), an activation induced cell surface marker of T-cells, modulates the T-cell immune response by inhibition of the T-cell and IL-2 receptors. Since T-cells undergo activation induced cell death via Fas activation, it was of interest to determine if this pathway was also affected by CEACAM1. Previously, we identified a novel biochemical interaction between CEACAM1 and the armadillo repeats of β-catenin in Jurkat cells, in which two critical residues, H469 and K470 of the cytoplasmic domain of CEACAM1-4L played an essential role; while in other studies, β-catenin was shown to regulate Fas-mediated apoptosis in Jurkat cells. CEACAM1 expression in Jurkat cells leads to the re-distribution of β-catenin to the actin cytoskeleton as well as inhibition of β-catenin tyrosine phosphorylation and its degradation after Fas stimulation. As a result, Fas-mediated apoptosis in these cells was inhibited. The K470A mutation of CEACAM1 partially rescued the inhibitory effect, in agreement with the prediction that a CEACAM1-β-catenin interaction pathway is involved. Although CEACAM1 has two ITIMs, they were not tyrosine-phosphorylated upon Fas ligation, indicating an ITIM independent mechanism; however, mutation of the critical residue S508, located between the ITIMs, to aspartic acid and a prerequisite for ITIM activation, abrogates the inhibitory activity of CEACAM1 to Fas-mediated apoptosis. Since Fas-mediated apoptosis is a major form of activation-induced cell death, our finding supports the idea that CEACAM1 is a general inhibitory molecule for T-cell activation utilizing a variety of pathways.

  19. Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells.

    PubMed

    Balasubramanyam, M; Gardner, J P

    1995-12-01

    Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca2+i) extrusion and 45Ca2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca2+i extrusion were further explored. PMA significantly increased the rate of Ca2+ extrusion in TG-treated cells from 0.28 +/- 0.02 to 0.35 +/- 0.03 s-1 (mean +/- SEM) and stimulated the initial rate of 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i

  20. Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells.

    PubMed

    Hage-Sleiman, Rouba; Hamze, Asmaa B; El-Hed, Aimée F; Attieh, Randa; Kozhaya, Lina; Kabbani, Sarah; Dbaibo, Ghassan

    2016-08-01

    Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase's translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation-where PKCθ plays a critical role. PMID:26798039

  1. Ceramide inhibits PKCθ by regulating its phosphorylation and translocation to lipid rafts in Jurkat cells.

    PubMed

    Hage-Sleiman, Rouba; Hamze, Asmaa B; El-Hed, Aimée F; Attieh, Randa; Kozhaya, Lina; Kabbani, Sarah; Dbaibo, Ghassan

    2016-08-01

    Protein kinase C theta (PKCθ) is a novel, calcium-independent member of the PKC family of kinases that was identified as a central player in T cell signaling and proliferation. Upon T cell activation by antigen-presenting cells, PKCθ gets phosphorylated and activated prior to its translocation to the immunological synapse where it couples with downstream effectors. PKCθ may be regulated by ceramide, a crucial sphingolipid that is known to promote differentiation, growth arrest, and apoptosis. To further investigate the mechanism, we stimulated human Jurkat T cells with either PMA or anti-CD3/anti-CD28 antibodies following induction of ceramide accumulation by adding exogenous ceramide, bacterial sphingomyelinase, or Fas ligation. Our results suggest that ceramide regulates the PKCθ pathway through preventing its critical threonine 538 (Thr538) phosphorylation and subsequent activation, thereby inhibiting the kinase's translocation to lipid rafts. Moreover, this inhibition is not likely to be a generic effect of ceramide on membrane reorganization. Other lipids, namely dihydroceramide, palmitate, and sphingosine, did not produce similar effects on PKCθ. Addition of the phosphatase inhibitors okadaic acid and calyculin A reversed the inhibition exerted by ceramide, and this suggests involvement of a ceramide-activated protein phosphatase. Such previously undescribed mechanism of regulation of PKCθ raises the possibility that ceramide, or one of its derivatives, and may prove valuable in novel therapeutic approaches for disorders involving autoimmunity or excessive inflammation-where PKCθ plays a critical role.

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

    SciTech Connect

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

    2009-03-15

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

  3. 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. PMID:26794080

  4. Type I interferons activate apoptosis in a Jurkat cell variant by caspase-dependent and independent mechanisms

    PubMed Central

    Gamero, Ana M.; Potla, Ramesh; Sakamoto, Shuji; Baker, Darren P.; Abraham, Robert; Larner, Andrew C.

    2007-01-01

    Although the antiviral actions of interferons (IFNs) are observed in most types of cells, the antiproliferative effects of IFNα/β are variable as are the mechanisms of growth inhibition that may or may not be due to the induction of apoptosis. To understand more about the mechanisms that are responsible for IFNα/β -stimulated apoptosis, we have characterized a new human Jurkat T cell variant named H123 where IFNα activates programmed cell death (PCD). No differences in IFNα -stimulated, Stat-dependent gene expression were detected between H123 cells and the parental Jurkat cells, which are growth inhibited, but do not undergo apoptosis with IFNα. Although IFNα stimulates the activity of both caspase 3 and 9 in H123 cells, the general caspase inhibitor Z-VAD only partially reverses the apoptotic actions of IFNα. Induction of apoptosis by IFNα occurs through a mitochondrial-dependent pathway in H123 cells, as demonstrated by the release of cytochrome C from the mitochondria. Furthermore, IFNα treatment of H123 cells stimulates the release of the serine protease HtrA2/Omi from the mitochondria, suggesting that it plays a role in the apoptotic actions of this cytokine. These results provide evidence for a novel type 1 IFN-mediated pathway that regulates apoptosis of T cells through a mitochondrial-dependent and caspase-dependent and independent pathway. PMID:16337360

  5. 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.

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

    PubMed

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

    2014-08-01

    The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis. PMID:25246714

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

    NASA Astrophysics Data System (ADS)

    Grundt, Jessica E.; Roth, Caleb C.; Rivest, Benjamin D.; Doroski, Michael L.; Payne, Jason; Ibey, Bennett L.; Wilmink, Gerald J.

    2011-03-01

    Terahertz (THz) radiation sources are now being used in a host of military, defense, and medical applications. Widespread employment of these applications has prompted concerns regarding the health effects associated with THz radiation. In this study, we examined the gene expression profile of mammalian cells exposed to THz radiation. We hypothesized that if THz radiation couples directly to cellular constituents, then exposed cells may express a specific gene expression profile indicative of ensuing damage. To test this hypothesis, Jurkat cells were irradiated with a molecular gas THz laser (2.52 THz, 636 mWcm-2, durations: 5, 10, 20, 30, 40, or 50 minutes). Viability was assessed 24 h post-exposure using MTT assays, and gene expression profiles were evaluated 4 h post-exposure using mRNA microarrays. Comparable analyses were also performed for hyperthermic positive controls (44°C for 40 minutes). We found that cellular temperatures increased by ~6 °C during THz exposures. We also found that cell death increased with exposure duration, and the median lethal dose (LD50) was calculated to be ~44 minutes. The microarray data showed that THz radiation induced the transcriptional activation of genes associated with cellular proliferation, differentiation, transcriptional activation, chaperone protein stabilization, and apoptosis. For most genes, we found that the magnitude of differential expression was comparable for both the THz and thermal exposure groups; however, several genes were specifically activated by the THz exposure. These results suggest that THz radiation may elicit effects that are not exclusively due to the temperature rise created during THz exposures (i.e. thermal effects). In future work, we plan to verify the results of our microarray experiments using qPCR techniques.

  8. 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.

  9. 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

  10. Plasma membrane charging of Jurkat cells by nanosecond pulsed electric fields.

    PubMed

    White, Jody A; Pliquett, Uwe; Blackmore, Peter F; Joshi, Ravindra P; Schoenbach, Karl H; Kolb, Juergen F

    2011-08-01

    The initial effect of nanosecond pulsed electric fields (nsPEFs) on cells is a change of charge distributions along membranes. This first response is observed as a sudden shift in the plasma transmembrane potential that is faster than can be attributed to any physiological event. These immediate, yet transient, effects are only measurable if the diagnostic is faster than the exposure, i.e., on a nanosecond time scale. In this study, we monitored changes in the plasma transmembrane potential of Jurkat cells exposed to nsPEFs of 60 ns and amplitudes from 5 to 90 kV/cm with a temporal resolution of 5 ns by means of the fast voltage-sensitive dye Annine-6. The measurements suggest the contribution of both dipole effects and asymmetric conduction currents across opposite sides of the cell to the charging. With the application of higher field strengths the membrane charges until a threshold voltage value of 1.4-1.6 V is attained at the anodic pole. This indicates when the ion exchange rates exceed charging currents, thus providing strong evidence for pore formation. Prior to reaching this threshold, the time for the charging of the membrane by conductive currents is qualitatively in agreement with accepted models of membrane charging, which predict longer charging times for lower field strengths. The comparison of the data with previous studies suggests that the sub-physiological induced ionic imbalances may trigger other intracellular signaling events leading to dramatic outcomes, such as apoptosis.

  11. N-Farnesyloxy-norcantharimide inhibits progression of human leukemic Jurkat T cells through regulation of mitogen-activated protein kinase and interleukin-2 production

    PubMed Central

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

    2015-01-01

    This study investigated the anticancer effects of N-farnesyloxy-norcantharimide (NOC15), a newly synthesized norcantharidin (NCTD) analogue, on human leukemic Jurkat T cells and the signaling pathway underlying its effects. We found that the half maximal inhibitory concentration (IC50) of NOC15 on Jurkat T cells is 1.4 μmol/l, which is 11.14-fold (=15.6÷1.4) smaller than the 15.6 μmol/l of NCTD on Jurkat T cells, whereas the IC50 of NOC15 on human normal lymphoblast (HNL) is 207.9 μmol/l, which is 8.17-fold (=1698.0÷207.8) smaller than the 1698.0 μmol/l of NCTD on HNL cells. These results indicated that NOC15 exerts a higher anticancer effect on Jurkat T cells and has higher toxicity toward HNL cells than NCTD. Thus, NOC15 is 1.36-fold (=11.14÷8.17) beneficial as an anticancer agent toward Jurkat T cells compared with NCTD. Moreover, NOC15 can increase the percentage of cells in the sub-G1 phase and reduce the cell viability of Jurkat T cells, stimulate p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) of mitogen-activated protein kinases (MAPKs) signaling pathway, and inhibit calcineurin expression and interleukin-2 (IL-2) production. However, NOC15 exerted no effects on the Jun-N-terminal kinase 1/2 (JNK1/2) signaling pathway, the production of IL-8, and tumor necrosis factor-α. We conclude that the anticancer activity of the newly synthesized NOC15 is 1.36-fold beneficial than NCTD as an anticancer agent and that NOC15 can increase the percentage of cells in the sub-G1 phase through the stimulation of p38 and ERK1/2 of the MAPK signaling pathway and the inhibition of calcineurin expression and IL-2 production. The NOC15 may have the potential of being developed into an anticancer agent in the future. PMID:26288134

  12. Endoplasmic reticulium protein profiling of heat-stressed Jurkat cells by one dimensional electrophoresis and liquid chromatography tandem mass spectrometry.

    PubMed

    Zhang, Xiulian; Kuramitsu, Yasuhiro; Ma, Aiguo; Zhang, Hui; Nakamura, Kazuyuki

    2016-08-01

    Proteomic study on membrane-integrated proteins in endoplasmic reticulum (ER) fractions was performed. In this study, we examined the effects of heat stress on Jurkat cells. The ER fractions were highly purified by differential centrifugation with sodium carbonate washing and acetone methanol precipitations. The ER membrane proteins were separated by one dimensional electrophoresis (1-DE), and some of the protein bands changed their abundance by heat stress, 12 of the 14 bands containing 40 and 60 ribosomal proteins whose expression level were decreased, on the contrary, 2 of the 14 bands containing ubiquitin and eukaryotic translation initiation factor 3 were increased. Heat treatment of human Jurkat cells led to an increase in the phosphorylation of PERK and eIF2α within 30 min of exposure. This was followed by an increase in the expression of the GRP78. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, the result showed that heat stress enhanced ubiquitination modification of the microsomal proteins. The data of this study strongly suggest that heat treatment led to a significant reduction in protein expression and activated UPR, concomitant with protein hyperubiqutination in ER.

  13. Mcl-1 is up regulated by prenylated coumarin, umbelliprenin in jurkat cells.

    PubMed

    Gholami, Omid; Jeddi-Tehrani, Mahmood; Iranshahi, Mehrdad; Zarnani, Amir Hassan; Ziai, Seyed Ali

    2014-01-01

    Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the western world and exists as subtypes with very different clinical courses. Myeloid cell leukemia 1 (Mcl-1) is one member of Bcl-2 family proteins that has been shown to be expressed in various tissues and malignant cells, including CLL, where its expression is significantly associated with a failure to achieve complete remission following cytotoxic therapy. Induction of apoptosis by prenylated coumarin, umbelliprenin, in Jurkat cells was previously shown. We examined whether umbelliprenin can down-regulate Mcl-1 gene and protein in Jurkat cells. In this regard cells were incubated by umbelliprenin, and then down- regulation of Mcl-1 gene was studied by Real Time PCR method. Moreover, down-regulation of Mcl-1 protein was studied by western blot analysis. We showed that, expression of Mcl-1 mRNA was increased from 1 hour to 3 hours incubation, but this increase has a scale down pattern. Moreover umbelliprenin could inhibit Mcl-1 protein. In conclusion umbelliprenin treatment modulates Mcl-1 expression at both the transcriptional and posttranslational levels.

  14. Mcl-1 Is Up Regulated by Prenylated Coumarin, Umbelliprenin in Jurkat Cells

    PubMed Central

    Gholami, Omid; Jeddi-Tehrani, Mahmood; Iranshahi, Mehrdad; Zarnani, Amir Hassan; Ziai, Seyed Ali

    2014-01-01

    Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the western world and exists as subtypes with very different clinical courses. Myeloid cell leukemia 1 (Mcl-1) is one member of Bcl-2 family proteins that has been shown to be expressed in various tissues and malignant cells, including CLL, where its expression is significantly associated with a failure to achieve complete remission following cytotoxic therapy. Induction of apoptosis by prenylated coumarin, umbelliprenin, in Jurkat cells was previously shown. We examined whether umbelliprenin can down-regulate Mcl-1 gene and protein in Jurkat cells. In this regard cells were incubated by umbelliprenin, and then down- regulation of Mcl-1 gene was studied by Real Time PCR method. Moreover, down-regulation of Mcl-1 protein was studied by western blot analysis. We showed that, expression of Mcl-1 mRNA was increased from 1 hour to 3 hours incubation٫ but this increase has a scale down pattern. Moreover umbelliprenin could inhibit Mcl-1 protein. In conclusion umbelliprenin treatment modulates Mcl-1 expression at both the transcriptional and posttranslational levels. PMID:25587328

  15. Vitamin E synthetic derivate-TPGS-selectively induces apoptosis in jurkat t cells via oxidative stress signaling pathways: implications for acute lymphoblastic leukemia.

    PubMed

    Ruiz-Moreno, Cristian; Jimenez-Del-Rio, Marlene; Sierra-Garcia, Ligia; Lopez-Osorio, Betty; Velez-Pardo, Carlos

    2016-09-01

    D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E commonly used as a drug delivery agent. Recently, TPGS alone has been reported to induce cell death in lung, breast and prostate cancer. However, the effect of TPGS on cancer cell viability remains unclear. Thus, this study was aimed to evaluate the cytotoxic effect of TPGS on human periphral blood lymphocytes (PBL) and on T cell acute lymphocytic leukemia (ALL) Jurkat clone E6-1 cells and its possible mechanism of action. PBL and Jurkat cells were treated with TPGS (10, 20, 40, 60, and 80 μM), and morphological changes in the cell nucleus, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species levels were determined by immune-fluorescence microscopy and flow cytometry. Cellular apoptosis markers were also evaluated by immunocytochemistry. In this study, TPGS induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner with increasing nuclear DNA fragmentation, increasing cell cycle arrest, and decreasing ΔΨm. Additionally, TPGS increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, in a dose-independent fashion. TPGS increased DJ-1 Cys(106)-sulfonate, as a marker of intracellular stress and induced the activation of NF-κB, p53 and c-Jun transcription factors. Additionally, it increased the expression of apoptotic markers Bcl-2 related pro-apoptotic proteins Bax and PUMAand activated caspase-3. The antioxidant N-acetyl-L-cysteine and known pharmacological inhibitors protected the cells from the TPGS induced effects. In conclusion, TPGS selectively induces apoptosis in Jurkat cells through two independent but complementary H2O2-mediated signaling pathways. Our findings support the use of TPGS as a potential treatment for ALL. PMID:27364951

  16. Deciphering glycan linkages involved in Jurkat cell interactions with gold-coated nanofibers via sugar-displayed thiols.

    PubMed

    Du, Jian; Che, Pao-Lin; Aich, Udayanath; Tan, Elaine; Kim, Hyo Jun; Sampathkumar, Srinivasa-Gopalan; Yarema, Kevin J

    2011-09-01

    Metabolic oligosaccharide engineering (MOE) provides a method to install novel chemical functional groups into the glycocalyx of living cells. In this Letter we use this technology to compare the impact of replacing natural sialic acid, GalNAc, and GlcNAc with their thiol-bearing counterparts in Jurkat and HL-60 cells. When incubated in the presence of gold-coated nanofibers, only Jurkat cells incubated with Ac(5)ManNTGc-an analogue that installs thiols into sialosides-experienced a distinctive 'spreading' morphology. The comparison of Ac(5)ManNTGc with Ac(5)GalNTGc and Ac(5)GlcNTGc in the two cell lines implicated sialosides of N-linked glycans as critical molecular mediators of the unusual responses evoked in the Jurkat line. PMID:21684742

  17. 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.

  18. Gene expressions in Jurkat cells poisoned by a sulphur mustard vesicant and the induction of apoptosis.

    PubMed

    Zhang, Peng; Ng, Patrick; Caridha, Diana; Leach, Richard A; Asher, Ludmila V; Novak, Mark J; Smith, William J; Zeichner, Steven L; Chiang, Peter K

    2002-09-01

    1. The sulphur mustard vesicant 2-chloroethylethyl sulphide (CEES) induced apoptosis in Jurkat cells. 2. Akt (PKB), a pivotal protein kinase which can block apoptosis and promotes cell survival, was identified to be chiefly down-regulated in a dose-dependent manner following CEES treatment. Functional analysis showed that the attendant Akt activity was simultaneously reduced. 3. PDK1, an upstream effector of Akt, was also down-regulated following CEES exposure, but two other upstream effectors of Akt, PI3-K and PDK2, remained unchanged. 4. The phosphorylation of Akt at Ser(473) and Thr(308) was significantly decreased following CEES treatment, reflecting the suppressed kinase activity of both PDK1 and PDK2. 5. Concurrently, the anti-apoptotic genes, Bcl family, were down-regulated, in sharp contrast to the striking up-regulation of some death executioner genes, caspase 3, 6, and 8. 6. Based on these findings, a model of CEES-induced apoptosis was established. These results suggest that CEES attacked the Akt pathway, directly or indirectly, by inhibiting Akt transcription, translation, and post-translation modification. 7. Taken together, upon exposure to CEES, apoptosis was induced in Jurkat cells via the down-regulation of the survival factors that normally prevent the activation of the death executioner genes, the caspases. PMID:12208782

  19. Opium induces apoptosis in Jurkat cells via promotion of pro-apoptotic and inhibition of anti-apoptotic molecules

    PubMed Central

    Arababadi, Mohammad Kazemi; Asadikaram, Gholamreza

    2016-01-01

    Objective(s): The aim of this study was to determine the important molecules involved in apoptosis induction by opium in Jurkat cell line. Materials and Methods: Jurkat cells were incubated 48 hrs with 2.86×10-5 g/ml concentration of opium and apoptosis as well as expression levels of related molecules were measured. Results: Our results demonstrated that 50.3±0.2 percent of opium treated Jurkat cells were revealed apoptotic features. The levels of mRNA of several pro-apoptotic and anti-apoptotic molecules were increased and decreased, respectively, in the opium treated cells. The results also demonstrated that expression levels of BCL2, DFFA and NOL3 as anti-apoptotic molecules were increased in the opium treated cells. Conclusion: It seems that opium induces apoptosis in Jurkat cells via both intrinsic and extrinsic pathways. Although opium induces apoptosis in the cells but increased expression of some anti-apoptotic molecules may be a normal resistance of the cell for death. PMID:27081468

  20. 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.

  1. 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).

  2. 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

  3. 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.

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

    SciTech Connect

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

    2011-07-08

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

  5. Stimulation of human Jurkat cells by monoclonal antibody crosslinking of transfected-Ly-6A.2 (TAP) molecules.

    PubMed

    McGrew, J T; Rock, K L

    1991-10-01

    Monoclonal antibody crosslinking of phosphatidylinositol-anchored Ly-6A.2 molecules on the surface of murine T lymphocytes leads to cell activation and secretion of IL-2. To examine the potential activity of these molecules in human T cells we transfected the Ly-6A.2 gene into Jurkat cells. Transfection of Jurkat cells with genomic Ly-6A.2 sequences results in low levels of Ly-6A.2 on the cell surface. However, linking the Ly-6A.2 sequences to the enhancer from the human CD2 gene results in greatly increased expression of Ly-6A.2. These molecules are anchored to the membrane via a phosphatidylinositol linkage. Crosslinking of Ly-6A.2 molecules with soluble mAb stimulates the transfected Jurkat cells to produce IL-2. This stimulation is abrogated by treatment with phosphatidylinositol-specific phospholipase C. The transfected human T cells displayed the same unusual crosslinking requirements for stimulation with anti-Ly-6A.2 mAbs as previously observed for murine T cells. Crosslinking of Ly-6A.2 with soluble antibodies is stimulatory, whereas immobilized antibodies are inactive. The crosslinking requirements for antiCD3 mAb stimulation display a reciprocal pattern. These data demonstrate that the Ly-6A.2 pathway for T cell activation is conserved between human and murine T cells.

  6. 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.

  7. 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.

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

    SciTech Connect

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

    2008-09-15

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

  9. 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.

  10. 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.

  11. Involvement of Epigenetic Mechanisms in the Regulation of Secreted Phospholipase A2 Expressions in Jurkat Leukemia Cells

    PubMed Central

    Menschikowski, Mario; Hagelgans, Albert; Kostka, Heike; Eisenhofer, Graeme; Siegert, Gabriele

    2008-01-01

    Epigenetic changes provide a frequent mechanism for transcriptional silencing of genes in cancer cells. We previously established that epigenetic mechanisms are important for control of group IIA phospholipase A2 (PLA2G2A) gene transcription in human DU-145 prostate cells. In this study, we analyzed the involvement of such mechanisms in the regulation of five sPLA2 isozymes and the M-type receptor of sPLA2 (sPLA2-R) in human leukemic Jurkat cells. These cells constitutively expressed sPLA2-IB, sPLA2-III, sPLA2-X, and sPLA2-R but not sPLA2-IIA and sPLA2-V. Transcription of sPLA2-IIA and sPLA2-V was, however, detected after exposure of cells to the DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC). Expression of sPLA2-IIA was further enhanced by additional exposure to interferon-γ and blocked by inhibitors of specificity protein 1, nuclear factor κB, and Janus kinase/signal transducer and activator of transcription-dependent pathways. Sequence analysis and methylation-specific polymerase chain reaction of bisulfite-modified genomic DNA revealed two 5′-CpG sites (-111 and -82) in the sPLA2-IIA proximal promoter that were demethylated after 5-aza-dC treatment. These sites may be involved in the DNA binding of specificity protein 1 and other transcription factors. Similar findings after treatment of human U937 leukemia cells with 5-aza-dC indicate that this mechanism of PLA2G2A gene silencing is not restricted to Jurkat and DU-145 cells. These data establish that regulation of sPLA2-IIA and sPLA2-V in Jurkat and other cells involves epigenetic silencing by DNA hypermethylation. PMID:18953428

  12. 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. PMID:26736822

  13. Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line.

    PubMed

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

    2015-01-01

    Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3 h to 0.5 µM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF-κB (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit → ribotoxic stress → ER stress → calcium release from the ER into cytoplasm → T-cell activation and oxidative stress → apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell

  14. The cytotoxic macrolide FD-891 induces caspase-8-dependent mitochondrial release of cytochrome c and subsequent apoptosis in human leukemia Jurkat cells.

    PubMed

    Inaba, Susumu; Eguchi, Tadashi; Motegi, Atsushi; Mizoue, Kazutoshi; Usui, Takeo; Nagai, Kazuo; Kataoka, Takao

    2009-09-01

    The 16-membered macrolide FD-891 exerts cytotoxicity toward several cancer cell lines. In this study, we showed that FD-891 induces apoptosis in various human cancer cell lines. Human leukemia Jurkat cells were highly sensitive to FD-891, exhibiting caspase activation and mitochondrial release of cytochrome c into the cytosol at early time points after exposure to FD-891. By contrast, Jurkat cells deficient in caspase-8 were resistant to FD-891-induced apoptosis and manifested little induction of cytochrome c release as well as caspase-9 processing. Consistent with these results, the overexpression of the Bcl-2 family member Bcl-x(L) or the caspase-8 modulator c-FLIP(L) markedly prevented FD-891-induced apoptosis. These results clearly demonstrate that FD-891 triggers caspase-8-dependent mitochondrial release of cytochrome c and subsequent apoptosis in Jurkat cells.

  15. Modulation of Cytokine Production and Transcription Factors Activities in Human Jurkat T Cells by Thymol and Carvacrol

    PubMed Central

    Gholijani, Nasser; Gharagozloo, Marjan; Kalantar, Fathollah; Ramezani, Amin; Amirghofran, Zahra

    2015-01-01

    Purpose: Thymol and carvacrol, two main components of thyme, have shown anti-inflammatory effects. The aim of this study was to assess the effects of these components on Jurkat leukemia cells as an in vitro T cell model and their molecular mechanisms of activity. Methods: Cells were cultured in the presence of components and subsequently stimulated with phorbol-12-myristate-13-acetate (PMA)/calcium ionophore for evaluating interleukin (IL)-2 and interferon (IFN)-γ production. The activation of T cell transcription factors that included nuclear factors of activated T cells (NFATs), activator protein-1 (AP-1; c-Jun/c-Fos), and nuclear factor (NF)-κB were examined by Western blot analysis. Results: Thymol and carvacrol at 25 µg/ml significantly reduced IL-2 levels from 119.4 ± 8pg/ml in control cells treated only with PMA/Calcium ionophore and the solvent to 66.9 ± 6.4pg/ml (thymol) and 32.3 ± 3.6pg/ml (carvacrol) and IFN-γ from 423.7 ± 19.7pg/ml in control cells to 311.9 ± 11.6pg/ml (thymol) and 293.5 ± 16.7pg/ml (carvacrol). Western blot analyses of nuclear extracts showed that the same concentrations of components significantly reduced NFAT-2 to 44.2 ± 2.7% (thymol) and 91.4 ± 2.3% (carvacrol) of the control (p<0.05), and c-Fos to 31.2 ± 6.2% (thymol) and 27.6 ± 3.1% (carvacrol) of the control (p<0.01). No effects on NFAT-1, c-Jun and phospho-NF-κBp65 levels were observed. Conclusion: Thymol and carvacrol could contribute to modulation of T cell activity by reducing IL-2 and IFN-γ production possibly through down regulation of AP-1 and NFAT-2 transcription factors suggesting their potential usefulness for reduction of T cell overactivity in immune-mediated diseases. PMID:26793612

  16. 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

  17. Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1.

    PubMed

    Alhosin, Mahmoud; Abusnina, Abdurazzag; Achour, Mayada; Sharif, Tanveer; Muller, Christian; Peluso, Jean; Chataigneau, Thierry; Lugnier, Claire; Schini-Kerth, Valérie B; Bronner, Christian; Fuhrmann, Guy

    2010-05-01

    The salvage anti-tumoral pathway which implicates the p53-related p73 gene is not yet fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the p73-dependent pro-apoptotic pathway, by focusing on the anti-apoptotic and epigenetic integrator UHRF1 which is essential for cell cycle progression. For this purpose, we analyzed the effects of a known anti-neoplastic drug, thymoquinone (TQ), on the p53-deficient acute lymphoblastic leukemia (ALL) Jurkat cell line. Our results showed that TQ inhibits the proliferation of Jurkat cells and induces G1 cell cycle arrest in a dose-dependent manner. Moreover, TQ treatment triggers programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (DeltaPsim). TQ-induced apoptosis, confirmed by the presence of hypodiploid G0/G1 cells, is associated with a rapid and sharp re-expression of p73 and dose-dependent changes of the levels of caspase-3 cleaved subunits. These modifications are accompanied by a dramatic down-regulation of UHRF1 and two of its main partners, namely DNMT1 and HDAC1, which are all involved in the epigenetic code regulation. Knockdown of p73 expression restores UHRF1 expression, reactivates cell cycle progression and inhibits TQ-induced apoptosis. Altogether our results showed that TQ mediates its growth inhibitory effects on ALL p53-mutated cells via the activation of a p73-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets UHRF1.

  18. 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

  19. Lck Mediates Signal Transmission from CD59 to the TCR/CD3 Pathway in Jurkat T Cells

    PubMed Central

    Lipp, Anna M.; Juhasz, Kata; Paar, Christian; Ogris, Christoph; Eckerstorfer, Paul; Thuenauer, Roland; Hesse, Jan; Nimmervoll, Benedikt; Stockinger, Hannes; Schütz, Gerhard J.; Bodenhofer, Ulrich

    2014-01-01

    The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca2+ imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca2+ response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca2+ signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses. PMID:24454946

  20. 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

  1. 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.

  2. 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.

  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. Endogenous presenilin 1 redistributes to the surface of lamellipodia upon adhesion of Jurkat cells to a collagen matrix.

    PubMed

    Schwarzman, A L; Singh, N; Tsiper, M; Gregori, L; Dranovsky, A; Vitek, M P; Glabe, C G; St George-Hyslop, P H; Goldgaber, D

    1999-07-01

    Most familial early-onset Alzheimer's disease cases are caused by mutations in the presenilin 1 (PS1) gene. Subcellular localization of the endogenous PS1 is essential for understanding its function, interactions with proteins, and role in Alzheimer's disease. Although numerous studies revealed predominant localization of PS1 to endoplasmic reticulum and Golgi, there are conflicting reports on the localization of PS1 to the cell surface. We found that endogenous PS1 is highly expressed in T lymphocytes (Jurkat cells). Using a variety of methods, we present evidence that endogenous PS1 is localized to the cell surface in addition to intracellular membrane compartments. Moreover, PS1 appeared in high levels on the surface of lamellipodia upon adhesion of the cells to a collagen matrix. The redistribution of PS1 in adhered cells was strikingly similar to that of the well characterized adhesion protein CD44. Cell surface PS1 formed complexes in vivo with actin-binding protein filamin (ABP-280), which is known to form bridges between cell surface receptors and cytoskeleton and mediate cell adhesion and cell motility. Taken together, our results suggest a role of PS1 in cell adhesion and/or cell-matrix interaction.

  5. 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

  6. Nanosecond pulsed electric fields modulate the expression of Fas/CD95 death receptor pathway regulators in U937 and Jurkat Cells.

    PubMed

    Estlack, Larry E; Roth, Caleb C; Thompson, Gary L; Lambert, William A; Ibey, Bennett L

    2014-12-01

    In this publication, we demonstrate that exposure of Jurkat and U937 cells to nanosecond pulsed electrical fields (nsPEF) can modulate the extrinsic-mediated apoptotic pathway via the Fas/CD95 death receptor. An inherent difference in survival between these two cell lines in response to 10 ns exposures has been previously reported (Jurkat being more sensitive to nsPEF than U937), but the reason for this sensitivity difference remains unknown. We found that exposure of each cell line to 100, 10 ns pulses at 50 kV/cm caused a marked increase in expression of cFLIP (extrinsic apoptosis inhibitor) in U937 and FasL (extrinsic apoptosis activator) in Jurkat, respectively. Measurement of basal expression levels revealed an inherent difference between U937 cells, having a higher expression of cFLIP, and Jurkat cells, having a higher expression of FasL. From these data, we hypothesize that the sensitivity difference between the cells to nsPEF exposure may be directly related to expression of extrinsic apoptotic regulators. To validate this hypothesis, we used siRNA to knockdown cFLAR (coding for cFLIP protein) expression in U937, and FasL expression in Jurkat and challenged them to 100, 10 ns pulses at 150 kV/cm, a typical lethal dose. We observed that U937 survival was reduced nearly 60% in the knockdown population while Jurkat survival improved ~40%. These findings support the hypothesis that cell survival following 10 ns pulse exposures depends on extrinsic apoptotic regulators. Interestingly, pretreatment of U937 with a 100-pulse, 50 kV/cm exposure (to amplify cFLAR expression) significantly reduced the lethality of a 150 kV/cm, 100-pulse exposure applied 24 h later. From these data, we conclude that the observed survival differences between cells, exposed to 10 ns pulsed electric fields, is due to inherent cell biochemistry rather than the biophysics of the exposure itself. Understanding cell sensitivity to nsPEF may provide researchers/clinicians with a predicable way

  7. 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

  8. 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.

  9. 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.

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

    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.

  11. Calpain-dependent cleavage of SHP-1 and SHP-2 is involved in the dephosphorylation of Jurkat T cells induced by Entamoeba histolytica.

    PubMed

    Kim, K A; Lee, Y A; Shin, M H

    2010-03-01

    Host cell death induced by Entamoeba histolytica is an important mechanism for both host defence and microbial immune evasion during human amoebiasis. However, the signalling pathways underlying cell death induced by E. histolytica are not fully understood. This study investigated the involvement of the protein tyrosine phosphatases (PTPs) SHP-1 and SHP-2 in the dephosphorylation associated with E. histolytica-induced host cell death. Incubation with E. histolytica resulted in a marked decrease in protein tyrosine phosphorylation levels and degradation of SHP-1 or SHP-2 in Jurkat cells. Pre-treatment of cells with a calpain inhibitor, calpeptin, impeded the amoeba-induced dephosporylation and cleavage of SHP-1 or SHP-2. Additionally, inhibition of PTPs with phenylarsine oxide (PAO) attenuated Entamoeba-induced dephosphorylation and DNA fragmentation in Jurkat T cells. These results suggest that calpain-dependent cleavage of SHP-1 and SHP-2 may contribute to protein tyrosine dephosphorylation in Jurkat T cell death induced by E. histolytica. PMID:20398180

  12. 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

  13. Docosahexaenoic acid and other fatty acids induce a decrease in pHi in Jurkat T-cells

    PubMed Central

    Aires, Virginie; Hichami, Aziz; Moutairou, Kabirou; Khan, Naim Akhtar

    2003-01-01

    Docosahexaenoic acid (DHA) induced rapid (t1/2=33 s) and dose-dependent decreases in pHi in BCECF-loaded human (Jurkat) T-cells. Addition of 5-(N,N-dimethyl)-amiloride, an inhibitor of Na+/H+ exchanger, prolonged DHA-induced acidification as a function of time, indicating that the exchanger is implicated in pHi recovery. Other fatty acids like oleic acid, arachidonic acid, eicosapentaenoic acid, but not palmitic acid, also induced a fall in pHi in these cells. To assess the role of calcium in the DHA-induced acidification, we conducted experiments in Ca2+-free (0% Ca2+) and Ca2+-containing (100% Ca2+) buffer. We observed that there was no difference in the degree of DHA-induced transient acidification in both the experimental conditions, though pHi recovery was faster in 0% Ca2+ medium than that in 100% Ca2+ medium. In the presence of BAPTA, a calcium chelator, a rapid recovery of DHA-induced acidosis was observed. Furthermore, addition of CaCl2 into 0% Ca2+ medium curtailed DHA-evoked rapid pHi recovery. In 0% Ca2+ medium, containing BAPTA, DHA did not evoke increases in [Ca2+]i, though this fatty acid still induced a rapid acidification in these cells. These observations suggest that calcium is implicated in the long-lasting DHA-induced acidosis. DHA-induced rapid acidification may be due to its deprotonation in the plasma membrane (flip-flop model), as suggested by the following observations: (1) DHA with a –COOH group induced intracellular acidification, but this fatty acid with a –COOCH3 group failed to do so, and (2) DHA, but not propionic acid, -induced acidification was completely reversed by addition of fatty acid-free bovine serum albumin in these cells. These results suggest that DHA induces acidosis via deprotonation and Ca2+ mobilization in human T-cells. PMID:14645139

  14. Goniothalamin-induced oxidative stress, DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells

    PubMed Central

    Inayat-Hussain, S.H.; Chan, K.M.; Rajab, N.F.; Din, L.B.; Chow, S.C.; Kizilors, A.; Farzaneh, F.; Williams, G.T.

    2010-01-01

    Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4 h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1 h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2. PMID:20026395

  15. The Phosphotyrosine Phosphatase SHP-2 Participates in a Multimeric Signaling Complex and Regulates T Cell Receptor (TCR) coupling to the Ras/Mitogen-activated Protein Kinase (MAPK) Pathway in Jurkat T Cells

    PubMed Central

    Frearson, Julie A.; Alexander, Denis R.

    1998-01-01

    Src homology 2 (SH2) domain–containing phosphotyrosine phosphatases (SHPs) are increasingly being shown to play critical roles in protein tyrosine kinase–mediated signaling pathways. The role of SHP-1 as a negative regulator of T cell receptor (TCR) signaling has been established. To further explore the function of the other member of this family, SHP-2, in TCR-mediated events, a catalytically inactive mutant SHP-2 was expressed under an inducible promoter in Jurkat T cells. Expression of the mutant phosphatase significantly inhibited TCR-induced activation of the extracellular-regulated kinase (ERK)-2 member of the mitogen-activated protein kinase (MAPK) family, but had no effect on TCR-ζ chain tyrosine phosphorylation or TCR-elicited Ca2+ transients. Inactive SHP-2 was targeted to membranes resulting in the selective increase in tyrosine phosphorylation of three membrane-associated candidate SHP-2 substrates of 110 kD, 55-60 kD, and 36 kD, respectively. Analysis of immunoprecipitates containing inactive SHP-2 also indicated that the 110-kD and 36-kD Grb-2–associated proteins were putative substrates for SHP-2. TCR-stimulation of Jurkat T cells expressing wild-type SHP-2 resulted in the formation of a multimeric cytosolic complex composed of SHP-2, Grb-2, phosphatidylinositol (PI) 3′-kinase, and p110. A significant proportion of this complex was shown to be membrane associated, presumably as a result of translocation from the cytosol. Catalytically inactive SHP-2, rather than the wild-type PTPase, was preferentially localized in complex with Grb-2 and the p85 subunit of PI 3′-kinase, suggesting that the dephosphorylating actions of SHP-2 may regulate the association of these signaling molecules to the p110 complex. Our results show that SHP-2 plays a critical role in linking the TCR to the Ras/MAPK pathway in Jurkat T cells, and also provide some insight into the molecular interactions of SHP-2 that form the basis of this signal transduction process

  16. Comparative assessment of therapeutic safety of norcantharidin, N-farnesyloxy-norcantharimide, and N-farnesyl-norcantharimide against Jurkat T cells relative to human normal lymphoblast: A quantitative pilot study.

    PubMed

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

    2016-08-01

    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. Regulation of lymphotoxin production by the p21ras-raf-MEK-ERK cascade in PHA/PMA-stimulated Jurkat cells.

    PubMed

    Li, Y Q; Hii, C S; Costabile, M; Goh, D; Der, C J; Ferrante, A

    1999-03-15

    Although the production of lymphotoxin (LT) from activated Th1 lymphocytes has been reported extensively, the intracellular signaling mechanisms that regulate this T cell function remain totally undefined. We have examined whether the p21ras-raf-1-mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase (MEK)-ERK cascade plays a role in regulating the production of LT, because the activity of these signaling molecules is up-regulated in activated T lymphocytes. Transfection of Jurkat leukemic T cells with a dominant negative mutant of p21ras (ras17N or ras15A), raf-1 (raf 1-130), or ERK1 (Erk1-K71R) resulted in the suppression of the mitogen/phorbol ester-stimulated production/secretion of LT. This suppression was accompanied by a parallel inhibition of mitogen-stimulated ERK activation. The selective antagonist of MEK1 activation, PD98059, also attenuated the mitogen-stimulated or anti-CD3 Ab and phorbol ester-stimulated production of LT from Jurkat cells or peripheral blood T lymphocytes. This study provides, for the first time, direct evidence that the p21ras-raf-MEK-ERK cascade plays a vital role in regulating the production of LT.

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

    PubMed

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

    1997-05-16

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

  19. Involvement of Raft Aggregates Enriched in Fas/CD95 Death-Inducing Signaling Complex in the Antileukemic Action of Edelfosine in Jurkat Cells

    PubMed Central

    Gajate, Consuelo; Gonzalez-Camacho, Fernando; Mollinedo, Faustino

    2009-01-01

    Background Recent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis. Methodology/Principal Findings By a combination of genetic, biochemical, and ultrastructural approaches, we provide here compelling evidence for the involvement of lipid raft aggregates containing recruited Fas/CD95 death receptor, Fas-associated death domain-containing protein (FADD), and procaspase-8 in the induction of apoptosis in human T-cell leukemia Jurkat cells by the antitumor drug edelfosine, the prototype compound of a promising family of synthetic antitumor lipids named as synthetic alkyl-lysophospholipid analogues. Co-immunoprecipitation assays revealed that edelfosine induced the generation of the so-called death-inducing signaling complex (DISC), made up of Fas/CD95, FADD, and procaspase-8, in lipid rafts. Electron microscopy analyses allowed to visualize the formation of raft clusters and their co-localization with DISC components Fas/CD95, FADD, and procaspase-8 following edelfosine treatment of Jurkat cells. Silencing of Fas/CD95 by RNA interference, transfection with a FADD dominant-negative mutant that blocks Fas/CD95 signaling, and specific inhibition of caspase-8 prevented the apoptotic response triggered by edelfosine, hence demonstrating the functional role of DISC in drug-induced apoptosis. By using radioactive labeled edelfosine and a fluorescent analogue, we found that edelfosine accumulated in lipid rafts, forming edelfosine-rich membrane raft clusters in Jurkat leukemic T-cells. Disruption of these membrane raft domains abrogated drug uptake and drug-induced DISC assembly and apoptosis. Thus, edelfosine uptake into lipid rafts was critical for the onset of both co-aggregation of DISC in membrane rafts and subsequent apoptotic cell death. Conclusions/Significance This work shows the involvement of DISC clusters in lipid raft aggregates as a supramolecular and physical entity

  20. Epigonal conditioned media from bonnethead shark, Sphyrna tiburo, induces apoptosis in a T-cell leukemia cell line, Jurkat E6-1.

    PubMed

    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

    2013-08-26

    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.

  1. 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.

  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. PMID:20797387

  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.

  4. 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. PMID:22806078

  5. Attenuation of O(6)-methylguanine-DNA methyltransferase activity and mRNA levels by cisplatin and temozolomide in jurkat cells.

    PubMed

    D'Atri, S; Graziani, G; Lacal, P M; Nisticò, V; Gilberti, S; Faraoni, I; Watson, A J; Bonmassar, E; Margison, G P

    2000-08-01

    The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is important in cellular resistance to certain alkylating antitumor agents such as the methylating drug temozolomide (TMZ). To provide a more rational basis for clinical combinations with another commonly used drug, cisplatin, we assessed the modulation of MGMT protein and mRNA levels in the human leukemic cell line Jurkat after treatment with these agents. Cisplatin decreased MGMT activity in a time- and dose-dependent manner, with maximal suppression (50%) observed 24 h after treatment with 25 microM cisplatin. This was probably the result of decreased transcription of the MGMT gene, because there was an earlier nadir of MGMT mRNA levels after cisplatin treatment and neither cisplatin nor DNA reacted with cisplatin in vitro was able to inhibit MGMT activity in an in vitro assay. TMZ alone depleted MGMT activity in a time- and dose-dependent manner with almost complete loss of activity occurring immediately after treatment with 500 microM TMZ. Combinations of cisplatin (12.5 microM) and TMZ (250 microM) caused substantial and prolonged MGMT depletion with recovery to only 30% of pretreatment levels by 48 h. These results suggest that the clinical efficacy of TMZ and cisplatin may be improved by appropriate schedules of combinations of these agents.

  6. 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.

  7. 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.

  8. Antisense-mediated loss of calcium homoeostasis endoplasmic reticulum protein (CHERP; ERPROT213-21) impairs Ca2+ mobilization, nuclear factor of activated T-cells (NFAT) activation and cell proliferation in Jurkat T-lymphocytes.

    PubMed Central

    O'Rourke, Flavia A; LaPlante, Janice M; Feinstein, Maurice B

    2003-01-01

    We recently discovered a novel gene on chromosome 19p13.1 and its product, an integral endoplasmic reticulum (ER) membrane protein, termed CHERP (calcium homoeostasis endoplasmic reticulum protein). A monoclonal antibody against its C-terminal domain inhibits Ins(1,4,5) P (3)-induced Ca(2+) release from ER membrane vesicles of many cell types, and an antisense-mediated knockdown of CHERP in human erythroleukemia (HEL) cells greatly impaired Ca(2+) mobilization by thrombin. In the present paper, we explore further CHERP's function in Jurkat T-lymphocytes. Confocal laser immunofluorescence microscopy showed that CHERP was co-localized with the Ins(1,4,5) P (3) receptor throughout the cytoplasmic and perinuclear region, as previously found in HEL cells. Transfection of Jurkat cells with a lac I-regulated mammalian expression vector containing CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of cytoplasmic Ca(2+) (measured by fura-2 acetoxymethyl ester fluorescence) caused by phytohaemagglutinin (PHA) and thrombin. A 50% fall of CHERP decreased the PHA-induced rise of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)), but Ca(2+) influx was unaffected. Greater depletion of CHERP (>70%) did not affect the concentration of Ins(1,4,5) P (3) receptors, but diminished the rise of [Ca(2+)](i) in response to PHA to cells, decreased Ca(2+) influx and slowed the initial rate of [Ca(2+)](i) rise caused by thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase, suggesting there was also some deficit in ER Ca(2+) stores. In CHERP-depleted cells the Ca(2+)-dependent activation and translocation of the key transcription factor NFAT (nuclear factor of activated T-cells) from cytoplasm to nucleus was suppressed. Furthermore, cell proliferation was greatly slowed (as in HEL cells) along with a 60% decrease in cyclin D1, a key regulator of progression through the G(1) phase of the cell cycle. These findings

  9. 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.

  10. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    PubMed

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation.

  11. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    PubMed

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation. PMID:26581585

  12. Immunomodulatory effects of therapeutic gold compounds. Gold sodium thiomalate inhibits the activity of T cell protein kinase C.

    PubMed Central

    Hashimoto, K; Whitehurst, C E; Matsubara, T; Hirohata, K; Lipsky, P E

    1992-01-01

    Previous studies have shown that the gold compounds, gold sodium thiomalate (GST) and auranofin (AUR), which are effective in the treatment of rheumatoid arthritis, inhibit functional activities of a variety of cells, but the biochemical basis of their effect is unknown. In the current studies, human T cell proliferation and interleukin 2 production by Jurkat cells were inhibited by GST or AUR at pharmacologically relevant concentrations. Because it has been documented that protein kinase C (PKC) is involved in T cell activation, the capacity of gold compounds to inhibit PKC partially purified from Jurkat cells was assayed in vitro. GST was found to inhibit PKC in a dose-dependent manner, but AUR caused no significant inhibition of PKC at pharmacologically relevant concentrations. The inhibitory effect of GST on PKC was abolished by 2-mercaptoethanol. To investigate the effect of GST on the regulation of PKC in vivo, the levels of PKC activity in Jurkat cells were examined. Cytosolic PKC activity decreased slowly in a concentration- and time-dependent manner as a result of incubation of Jurkat cells with GST. To ascertain whether GST inhibited PKC translocation and down-regulation, PKC activities associated with the membrane and cystosolic fractions were evaluated after phorbol myristate acetate (PMA) stimulation of GST incubated Jurkat cells. Translocation of PKC was markedly inhibited by pretreatment of Jurkat cells with GST for 3 d, but the capacity of PMA to down-regulate PKC activity in Jurkat cells was not altered by GST preincubation. The functional impact of GST-mediated downregulation of PKC in Jurkat cells was examined by analyzing PMA-stimulated phosphorylation of CD3. Although GST preincubated Jurkat cells exhibited an increased density of CD3, PMA-stimulated phosphorylation of the gamma chain of CD3 was markedly inhibited. Specificity for the inhibitory effect of GST on PKC was suggested by the finding that GST did not alter the mitogen

  13. 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

  14. Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

    PubMed

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

    2013-10-01

    In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. PMID:23707954

  15. 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.

  16. Regulation of protein kinase enzymatic activity in Jurkat T cells during oxidative stress uncoupled from protein tyrosine kinases: role of oxidative changes in protein kinase activation requirements and generation of second messengers.

    PubMed

    Whisler, R L; Newhouse, Y G; Beiqing, L; Karanfilov, B K; Goyette, M A; Hackshaw, K V

    1994-12-01

    Prior studies have suggested that intracellular phosphorylation events and cellular redox mechanisms may interact in regulating a variety of cellular functions, including the transcriptional activation of gene expression. Increased activity of transcriptional factors NF kappa B and AP1 has been described in cells exposed to oxidative stress and following the direct stimulation of protein kinase C (PKC) by phorbol diesters. However, the mechanisms that may contribute to redox regulation of PKC are unknown. We studied the expression of PKC activity and several second messengers in human Jurkat T cells exposed to oxidative stress in the form of H2O2. Micromolar concentrations of H2O2 rapidly induced increased cytosolic PKC enzymatic activity in Jurkat T cells that was associated with a marked arrest of cellular proliferation. The increase in cytosolic PKC activity in cells treated with H2O2 was accompanied by elevations in intracellular free calcium ([Ca2+]i), generation of inositol phosphates, and release of arachidonic acid. Functional studies showed that H2O2 enhancement of cytosolic PKC activity required phospholipase C activity but was not primarily mediated by arachidonic acid. The response of PKC to oxidative stress displayed a lack of Ca2+ dependence and was uncoupled from the activity of protein tyrosine kinases (PTK). Furthermore, the reduced activation requirements of PKC from cells treated with H2O2 were associated with shifts in elution profiles of PKC enzymatic activity after Mono-Q chromatography. These shifts appeared to represent intrinsic changes in the conformation of PKC induced by oxidative stress because western blotting failed to reveal any PKC cleavage products or reductions in native PKC alpha or beta. These findings indicate that oxidative regulation of intracellular events can intersect phosphorylation events mediated by PKC through the release of second messengers as well as direct changes in PKC activation requirements. Moreover, redox

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Ethanol promotes T cell apoptosis through the mitochondrial pathway

    PubMed Central

    Kapasi, Aditi A; Patel, Geeta; Goenka, Anuj; Nahar, Nilay; Modi, Neeraj; Bhaskaran, Madhu; Reddy, Krishna; Franki, Nicholas; Patel, Jaimita; Singhal, Pravin C

    2003-01-01

    Clinical reports suggest that acute ethanol intoxication is often associated with lymphopenia. Previously, ethanol was reported to invoke thymocyte apoptosis. We studied the effect of ethanol on T cell apoptosis. In addition, we evaluated the molecular mechanism of ethanol-induced T cell apoptosis. Human T cells harvested from healthy subjects after an alcohol drinking binge showed enhanced T cell apoptosis (before, 0·4 ± 0·2% versus after, 19·6 ± 2·5% apoptotic lymphocytes/field; P < 0·001). In in vitro studies, ethanol in a concentration of 50 mm and higher enhanced the apoptosis of Jurkat cells. DNA isolated from ethanol-treated Jurkat cells displayed integer multiples of 180 base pairs. Ethanol decreased Jurkat cell expression of Bcl-2, whereas ethanol increased Jurkat cell expression of Bax. Jurkat cells treated with ethanol also showed translocation of cytochrome C into cytosol. Moreover, a caspase-9 inhibitor partially inhibited ethanol-induced Jurkat cell apoptosis. In in vivo studies, after binge drinking, T cell expression of Bcl-2 also decreased. In addition, binge drinking induced the cleavage of caspase-3, suggesting activation of caspase-3 in T cells. These results suggest that ethanol promotes T cell apoptosis through the activation of intrinsic or mitochondrial pathway. PMID:12603597

  19. 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

  20. 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.

  1. Entamoeba histolytica-induced dephosphorylation in host cells.

    PubMed

    Teixeira, José E; Mann, Barbara J

    2002-04-01

    Activation of host cell protein tyrosine phosphatases (PTPases) and protein dephosphorylation is an important mechanism used by various microorganisms to deactivate or kill host defense cells. To determine whether protein tyrosine dephosphorylation played a role in signaling pathways affecting Entamoeba histolytica-mediated host cell killing, we investigated the involvement of PTPases during the attachment of E. histolytica to target cells. We observed a rapid decrease in cellular protein tyrosine levels in Jurkat cells, as measured with an antiphosphotyrosine monoclonal antibody, following adherence to E. histolytica. Ameba-induced protein dephosphorylation was contact dependent and required intact parasite, since blocking amebic adherence with galactose inhibited tyrosine dephosphorylation and amebic lysates had no effect on phosphotyrosine levels. Moreover, disruption of amebic adherence with galactose promoted recovery of phosphorylation in Jurkat cells, indicating that dephosphorylation precedes target cell death. The evidence suggests that ameba-induced dephosphorylation is mediated by host cell phosphatases. Prior treatment of Jurkat cells with phenylarsine oxide, a PTPase inhibitor, inhibited ameba-induced dephosphorylation. We also found proteolytic cleavage of the PTPase 1B (PTP1B) in Jurkat cells after contact with amebae. The calcium-dependent protease calpain is responsible for PTP1B cleavage and enzymatic activation. Pretreatment of Jurkat cells with calpeptin, a calpain inhibitor, blocked PTP1B cleavage and inhibited ameba-induced dephosphorylation. In addition, inhibition of Jurkat cell PTPases with phenylarsine oxide blocked Jurkat cell apoptosis induced by E. histolytica. These results suggest that E. histolytica-mediated host cell death occurs by a mechanism that involves PTPase activation. PMID:11895943

  2. The Effects of Intense Submicrosecond Electrical Pulses on Cells

    PubMed Central

    Deng, Jingdong; Schoenbach, Karl H.; Buescher, E. Stephen; Hair, Pamela S.; Fox, Paula M.; Beebe, Stephen J.

    2003-01-01

    A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 μs–60 ns, electric field intensities 3–150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude. PMID:12668479

  3. 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.

  4. Inhibition of Cancer Cell Proliferation and Antiradical Effects of Decoction, Hydroalcoholic Extract, and Principal Constituents of Hemidesmus indicus R. Br.

    PubMed

    Statti, Giancarlo; Marrelli, Mariangela; Conforti, Filomena; Spagnoletti, Antonella; Tacchini, Massimo; Fimognari, Carmela; Brognara, Eleonora; Gambari, Roberto; Sacchetti, Gianni; Guerrini, Alessandra

    2015-06-01

    Indian Sarsaparilla (Hemidesmus indicus R. Br.) is widely used in Indian traditional medicine. In the present work, we explored the effects of decoction, traditional Ayurvedic preparation, and hydroalcoholic extract, a phytocomplex more traditionally studied and commercialized as food supplement in western medicine, from the roots as possible source of chemicals with new functional potential linked to their nutritional uses. The antiproliferative and antioxidant properties were assayed. To test antiproliferative affects, different cancer cell lines, growing both as monolayers (CaCo2, MCF-7, A549, K562, MDA-MB-231, Jurkat, HepG2, and LoVo) and in suspension (K562 and Jurkat) were used. The decoction showed strong activity on HepG2 cells, while the hydroalcoholic extracts were active on HepG2, LoVo, MCF-7, K562, and Jurkat cell lines. Weak inhibition of cancer cell proliferation was observed for the principal constituents of the preparations: 2-hydroxy-4-methoxybenzaldehyde, 2-hydroxy-4-methoxybenzoic acid, and 3-hydroxy-4-methoxybenzaldehyde that were tested alone. The antiradical activity was tested with 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt tests and inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages. Interesting result has also been obtained for hydroalcoholic extract regarding genoprotective potential (58.79% of inhibition at 37.5 µg/mL). PMID:25753739

  5. 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

  6. 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

  7. In Vitro Effects of Propranolol on T Helper Type 1 Cytokine Profile in Human Leukemic T Cells

    PubMed Central

    Hajighasemi, Fatemeh; Mirshafiey, Abbas

    2016-01-01

    Introduction: Cytokines are a large group of proteins play a key role in inflammation. Down-regulation of pro-inflammatory cytokines has beneficial effect on heart function. Propranolol, as a non selective beta-adrenergic blocker, has been extensively used for treatment of many cardiovascular problems such as arrhythmias and heart malfunction. In addition anti-inflammatory effects of propranolol have been revealed. In this study the propranolol effect on T helper type 1 cytokine profile in human leukemic T cells has been assessed in vitro. Materials and methods: Human leukemic T cells (Molt-4 and Jurkat) were cultured in complete RPMI medium. The cells were then incubated with different concentrations of propranolol (0.03- 30 µM) in the presence or absence of PHA (10 µg/ml) for 48 hours. The supernatants of cell culture media were collected and used for cytokines assay. Results: Propranolol significantly decreased the T helper type 1 cytokine profile [Interleukin-2 (IL-2) and Interferon- γ (IFN-γ)] production in PHA stimulated Molt-4 and Jurkat cells, after 48 hour of incubation time, dose-dependently compared to untreated control cells. Conclusion: Our data showed a dose dependent inhibitory effect of propranolol on the IL-2 and IFN-γ production in human leukemic Molt-4 and Jurkat cells. The anti- inflammatory effect of propranolol reported by other investigators may be in part due to its suppressive effect on production of inflammatory cytokines such as IL-2 and IFN-γ. So, propranolol along with its chronic long-term usage in cardiovascular problems may have potential implication in treatment of inflammatory-based disorders. PMID:27252810

  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.

  9. 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.

  10. 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.

  11. 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

  12. Chitosan-modified cobalt oxide nanoparticles stimulate TNF-α-mediated apoptosis in human leukemic cells.

    PubMed

    Chattopadhyay, Sourav; Dash, Sandeep Kumar; Kar Mahapatra, Santanu; Tripathy, Satyajit; Ghosh, Totan; Das, Balaram; Das, Debasis; Pramanik, Panchanan; Roy, Somenath

    2014-03-01

    The objective of this study was to develop chitosan-based delivery of cobalt oxide nanoparticles to human leukemic cells and investigate their specific induction of apoptosis. The physicochemical properties of the chitosan-coated cobalt oxide nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, X-ray diffraction, and Fourier transform infrared spectroscopy. The solubility of chitosan-coated cobalt oxide nanoparticles was higher at acidic pH, which helps to release more cobalt ions into the medium. Chitosan-coated cobalt oxide nanoparticles showed good compatibility with normal cells. However, our results showed that exposure of leukemic cells (Jurkat cells) to chitosan-coated cobalt oxide nanoparticles caused an increase in reactive oxygen species generation that was abolished by pretreatment of cells with the reactive oxygen species scavenger N-acetyl-L-cysteine. The apoptosis of Jurkat cells was confirmed by flow-cytometric analysis. Induction of TNF-α secretion was observed from stimulation of Jurkat cells with chitosan-coated cobalt oxide nanoparticles. We also tested the role of TNF-α in the induction of Jurkat cell death in the presence of TNF-α and caspase inhibitors. Treatment of leukemic cells with a blocker had a greater effect on cancer cell viability. From our findings, oxidative stress and caspase activation are involved in cancer cell death induced by chitosan-coated cobalt oxide nanoparticles.

  13. 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.

  14. The effects of omega-3 Fatty acids on matrix metalloproteinase-9 production and cell migration in human immune cells: implications for multiple sclerosis.

    PubMed

    Shinto, Lynne; Marracci, Gail; Bumgarner, Lauren; Yadav, Vijayshree

    2011-01-01

    In multiple sclerosis (MS), compromised blood-brain barrier (BBB) integrity contributes to inflammatory T cell migration into the central nervous system. Matrix metalloproteinase-9 (MMP-9) is associated with BBB disruption and subsequent T cell migration into the CNS. The aim of this paper was to evaluate the effects of omega-3 fatty acids on MMP-9 levels and T cell migration. Peripheral blood mononuclear cells (PBMC) from healthy controls were pretreated with two types of omega-3 fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Cell supernatants were used to determine MMP-9 protein and activity levels. Jurkat cells were pretreated with EPA and DHA and were added to fibronectin-coated transwells to measure T cell migration. EPA and DHA significantly decreased MMP-9 protein levels, MMP-9 activity, and significantly inhibited human T cell migration. The data suggest that omega-3 fatty acids may benefit patients with multiple sclerosis by modulating immune cell production of MMP-9.

  15. Tetramethylpyrazine inhibits the proliferation of acute lymphocytic leukemia cell lines via decrease in GSK-3β.

    PubMed

    Wang, Xiao-Jing; Xu, You-Hua; Yang, Gui-Cun; Chen, Hong-Xia; Zhang, Ping

    2015-05-01

    Tetramethylpyrazine (TMP) has been proven to be an anticancer agent in many studies. However, its effectiveness in acute lymphoblastic leukemia (ALL) and its molecular mechanisms are still unclear. The present study aimed to evaluate the effect of TMP against Jurkat and SUP-B15 ALL cell lines and to investigate the possible detailed mechanism of action of TMP. A Cell Counting Kit-8 (CCK-8) assay was employed to examine the proliferation of Jurkat and SUP-B15 cells. Flow cytometric analysis was conducted to detect the cell cycle distribution and apoptotic rate. The expression of total glycogen synthase kinase-3β (GSK-3β), cox-2, survivin, bcl-2 and p27 RNA and protein levels was detected by quantitative real-time PCR and western blot assay, respectively. Additionally, western blot analysis was used to determine the whole-cell and nuclear protein levels of GSK-3β downstream transcription factors, NF-κB (p65) and c-myc. TMP inhibited the proliferation of Jurkat and SUP-B15 cells in a dose- and time-dependent manner, with IC₅₀ values of 120 and 200 µg/ml, respectively at 48 h. TMP induced the apoptosis of Jurkat and SUP-B15 cells and synergistically blocked cell cycle progression at the G0/G1 phase. Cells treated with TMP exhibited significantly attenuated GSK-3β, NF-κB (p65) and c-myc expression, followed by downregulation of bcl-2, cox-2 and survivin and an upregulation of p27. The results showed that TMP induced apoptosis and caused cell cycle arrest in Jurkat and SUP-B15 cells through the downregulation of GSK-3β, which may have further prevented the induced translocation of NF-κB and c-myc from the cytoplasm to the nucleus.

  16. 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.

  17. 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.

  18. Effects of silver nanoparticles and gold nanoparticles on IL-2, IL-6, and TNF-α production via MAPK pathway in leukemic cell lines.

    PubMed

    Parnsamut, C; Brimson, S

    2015-01-01

    Silver nanopaticles (AgNPs) and gold nanoparticles (AuNPs) have various applications in medical healthcare and various biological properties such as anti-inflammation, anti-cancer, and anti-angiogenesis. In this study, we investigated the effect of AgNPs and AuNPs on cytokine production via the mitogen-activated protein kinase pathway in leukemic cell lines (T-lymphocytic Jurkat and monocytic U937 cells). We found that both AgNPs and AuNPs inhibited cell proliferation of leukemic cell lines. AgNPs inhibited TNF-α, while AuNPs inhibited interleukin-2 production in Jurkat cells, in which inhibition of cytokines is involved in the extracellular-signal regulated protein kinase but not the c-Jun N-terminal kinase pathway. In U937 cells, AuNPs inhibited interleukin-6 but stimulated TNF-α production in a concentration-dependent manner through the c-Jun N-terminal kinase but not the extracellular-signal regulated protein kinase pathway. Our study showed that each leukemic cell line treated with nanoparticles exhibited a distinct signaling pathway response to inhibit or stimulate cytokine production, leading to anti-cell proliferation. The effects of AgNPs and AuNPs on leukemic cell lines may have a significant impact on leukemia treatment in the future. PMID:25966134

  19. 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.

  20. Effects of arsenic sulfide (As2S2) on B and T lymphoma cell lines and possible underlying mechanisms.

    PubMed

    Li, Xianglu; Liu, Xinyu; Wang, Ling; Lv, Xiao; Li, Peipei; Lu, Kang; Wang, Xin

    2013-07-01

    Lymphoma is a hematological malignancy that originates from lymph nodes and lymphoid tissues and is divided into Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL), based on its histopathological characteristics. The aim of this study was to investigate the effects of arsenic sulfide (As2S2), the main ingredient of realgar, on the proliferation and apoptosis of the Raji B-cell lymphoma and Jurkat T-cell lymphoma lines, comparing the sensitivity between the two cell lines and investigating the possible underlying mechanisms. The two lymphoma cell lines were cultured in vitro, using different concentrations of As2S2 for different time periods. The cell proliferation was detected using the Cell Counting kit-8 (CCK-8). Apoptosis was assessed via flow cytometry. Expression levels of the apoptosis-associated genes [Homo sapiens Bcl-2-associated X protein (BAX), Homo sapiens B-cell CLL/lymphoma 2 (Bcl-2), Homo sapiens Bcl-2-like protein 1 (BCL2L1, Bcl-xL), Homo sapiens v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, c-Myc) and Homo sapiens pim-1 oncogene (PIM)] were measured via the reverse transcription polymerase chain reaction (RT-PCR) method. The results demonstrated that As2S2 inhibited proliferation and induced apoptosis in the two lymphoma cell lines in a time- and concentration-dependent manner, with the Raji cells being more sensitive to As2S2 compared to Jurkat cells. As2S2 may also alter the expression levels of different apoptosis-associated genes, with the alterations of the mRNA expression levels being different between Raji and Jurkat cells. These findings indicated that As2S2 may inhibit the proliferation and promote the apoptosis of non-Hodgkin lymphoma (NHL) cell lines and that B-cell lymphoma cell lines are more sensitive compared to T-cell lymphoma cell lines. The possible underlying mechanism is that As2S2 alters the expression levels of the apoptosis-associated genes and activates apoptosis-associated signaling pathways.

  1. [Ribonuclease binase induces death in T-cell acute lymphoblastic leukemia cells by apoptosis].

    PubMed

    Burnysheva, K M; Petrushanko, I Yu; Spirin, P V; Prassolov, V S; Makarov, A A; Mitkevich, V A

    2016-01-01

    Bacterial ribonuclease binase is a potential anticancer agent. In the present study, we have determined the toxic effect of binase towards cell lines of T-cell acute lymphoblastic leukemia Jurkat and CEMss. We have shown that binase induces apoptosis in these cells. At the same time, binase does not cause toxic effects in leukocytes of healthy donors, which suggests that binase activity towards leukemic cells is selective. We have found that the treatment of cancer cells with binase leads to a reduction in reactive oxygen species and transcription factor NFκB levels, and demonstrated that these effects are a common feature of the action of RNases on cancer cells.

  2. Inhibition of gamma-secretase affects proliferation of leukemia and hepatoma cell lines through Notch signaling.

    PubMed

    Suwanjunee, Saipin; Wongchana, Wipawee; Palaga, Tanapat

    2008-06-01

    Notch signaling is a well-conserved pathway playing crucial roles in regulating cell fate decision, proliferation, and apoptosis during the development of multiple cell lineages. Aberration in Notch signaling is associated with tumorigenesis of tissues from various origins. To investigate the role Notch signaling plays in the proliferation of cancer cell lines, the expression profiles of Notch1 in six human cancer cell lines (Jurkat, HepG2, SW620, KATOIII, A375, BT474) were examined. All cell lines differentially expressed Notch1, and only Jurkat and SW620 expressed cleaved Notch1 (Val1744). Among the six cell lines tested, only Jurkat and HepG2 showed a decrease in cell proliferation during 4 days of treatment with a gamma-secretase inhibitor (GSI). This is the first report on the anti-proliferative effects of GSI on a human hepatoma cell line. These two cell lines expressed Notch1-3, Jagged1, Jagged2, Dlk1 and Hes1. GSI treatment led to a decrease in Hes1 expression in both cell lines. Surprisingly, GSI treatment resulted in the accumulation of Notch1 protein upon treatment. During this period, GSI treatment did not induce apoptosis, but caused cell cycle arrest in both cell lines. This was also correlated with decreased c-myc expression. Forced expression of activated intracellular Notch1 completely abrogated GSI sensitivity in both cell lines. These results clearly demonstrate that Notch signaling positively regulates cell proliferation in Jurkat and HepG2 cell lines and that GSI treatment inhibits tumor cell proliferation through the suppression of Notch signaling. PMID:18418214

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:27104236

  4. 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

    2016-05-01

    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. PMID:27104236

  5. 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

  6. 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

  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

    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. Involvement of glyco-receptors in Galactoxylomannan-induced T cell death

    PubMed Central

    Pericolini, Eva; Gabrielli, Elena; Cenci, Elio; De Jesus, Magdia; Bistoni, Francesco; Casadevall, Arturo; Vecchiarelli, Anna

    2010-01-01

    The major virulence factor of Cryptococcus neoformans is its capsular polysaccharide which is also released into tissues. The shed polysaccharide is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins. In a previous study, we demonstrated a direct interaction of purified soluble GalXM with T cells that induced their apoptosis. In this study, we focus on the mechanisms involved in the apoptotic effect of GalXM. In our experimental system, we analyzed the effect of GalXM on purified human T cells and Jurkat cells, a T cell line routinely used for apoptotic studies. Our results reveal that GalXM activates the extrinsic and intrinsic apoptotic pathways through the cleavage and recruitment of caspase-8. Caspase-8 elicits the downstream executioner caspases 3, 6, and 7 both directly and indirectly, via Bid cleavage and caspase-9 activation. These effects appeared to be primarily mediated by the interaction of GalXM with the glyco-receptors, which differed in human T and Jurkat cells. CD45 was primarily involved in Jurkat cells apoptosis while CD7 and CD43 mediated human T cell apoptosis. Our results highlight a new mechanism by which a microbial product can contribute to virulence through direct interaction with T cell glyco-receptors, thereby triggering lymphocyte apoptosis. PMID:19414751

  9. 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.

  10. 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.

  11. 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

  12. 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. PMID:25813896

  13. Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: A contribution to the optimization of gene and drug delivery

    NASA Astrophysics Data System (ADS)

    Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Congiu Castellano, Agostina

    2011-12-01

    Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

  14. Grassypeptolides As Natural Inhibitors of Dipeptidyl Peptidase 8 and T-Cell Activation

    PubMed Central

    Kwan, Jason C.; Liu, Yanxia; Ratnayake, Ranjala; Hatano, Ryo; Kuribara, Akiko; Morimoto, Chiko; Ohnuma, Kei; Paul, Valerie J.; Ye, Tao

    2014-01-01

    Natural products made by marine cyanobacteria are often highly modified peptides and depsipeptides that have the potential to act as inhibitors for proteases. In the interest of finding novel protease inhibition activity and selectivity grassypeptolide A (1) was screened against a panel of proteases and found to selectively inhibit DPP8 over DPP4. Grassypeptolides were also found to inhibit IL-2 production and proliferation in activated T-cells, consistent with a putative role of DPP8 in the immune system. These effects were also observed in Jurkat cells, and DPP activity in Jurkat cell cytosol was shown to be inhibited by grassypeptolides. In silico docking suggests two possible binding modes of grassypeptolides – both at the active site of DPP8 and at one of the entrances to the internal cavity. Collectively these results suggest that grassypeptolides may be useful tool compounds in the study of DPP8 function. PMID:24591193

  15. Nitric oxide and bcl-2 mediated the apoptosis induced by nickel(II) in human T hybridoma cells

    SciTech Connect

    Guan Fuqin; Zhang Dongmei; Wang Xinchang; Chen Junhui . E-mail: jhchen@nju.edu.cn

    2007-05-15

    Although effects of nickel(II) on the immune system have long been recognized, little is known about the effects of nickel(II) on the induction of apoptosis and related signaling events in T cells. In the present study, we investigated the roles and signaling pathways of nickel(II) in the induction of apoptosis in a human T cell line jurkat. The results showed that the cytotoxic effects of Ni involved significant morphological changes and chromosomal condensation (Hoechst 33258 staining). Analyses of hypodiploid cells and FITC-Annexin V and PI double staining showed significant increase of apoptosis in jurkat cells 6, 12 and 24 h after nickel(II) treatment. Flow cytometry analysis also revealed that the loss of mitochondrial membrane potential (MMP) occurred concomitantly with the onset of NiCl{sub 2}-induced apoptosis. Induction of apoptotic cell death by nickel was mediated by reduction of bcl-2 expression. Furthermore, nickel stimulated the generation of nitric oxide (NO). These results suggest that nickel(II) chloride induces jurkat cells apoptosis via nitric oxide generation, mitochondrial depolarization and bcl-2 suppression.

  16. 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.

  17. Umbelliprenin Induces Apoptosis in CLL Cell Lines.

    PubMed

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V-FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate.

  18. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  19. 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.

  20. 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

  1. 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.

  2. Study of antitumor effect of selected vanadium and molybdenum organometallic complexes in human leukemic T-cells.

    PubMed

    Šebestová, Lucie; Havelek, Radim; Řezáčová, Martina; Honzíček, Jan; Kročová, Zuzana; Vinklárek, Jaromír

    2015-12-01

    This work describes cytotoxic effect of non-platinum metal-based compounds on the human T-leukemic cells with different p53 status (p53 wild-type MOLT-4 and p53-deficient Jurkat cells). The cytotoxic and apoptosis-inducing effect of the vanadium complex [(η(5)-C5H5)2V(5-NH2-phen)]OTf (V1) and molybdenum complex [(η(3)-C3H5)Mo(CO)2(phen)Cl] (Mo1) were studied using flow cytometry, spectrophotometry and Western blotting. We found that the cytotoxic effect of both tested complexes after 24 h is higher against the both examined cell lines than that of cis-platin (cis-DDP). At later investigated time intervals of 48 and 72 h, the cytotoxic effect of the cis-DDP increased but the values of the cytotoxicity of the tested V1 and Mo1 complexes remained unchanged, with the cytotoxicity of V1 comparable to that of cis-DDP. Furthermore we observed that the apoptotic process was induced by the activation of the caspases 9 (intrinsic pathway) and 8 (extrinsic pathway) in cells exposed to evaluated complexes. In case of the p53 wild-type MOLT-4 cells, the expression of the tumor-suppressor protein p53 and its form phosphorylated at the serine 15 increased after both V1 and Mo1 treatment, similar to the effect of cis-DDP.

  3. Study of antitumor effect of selected vanadium and molybdenum organometallic complexes in human leukemic T-cells.

    PubMed

    Šebestová, Lucie; Havelek, Radim; Řezáčová, Martina; Honzíček, Jan; Kročová, Zuzana; Vinklárek, Jaromír

    2015-12-01

    This work describes cytotoxic effect of non-platinum metal-based compounds on the human T-leukemic cells with different p53 status (p53 wild-type MOLT-4 and p53-deficient Jurkat cells). The cytotoxic and apoptosis-inducing effect of the vanadium complex [(η(5)-C5H5)2V(5-NH2-phen)]OTf (V1) and molybdenum complex [(η(3)-C3H5)Mo(CO)2(phen)Cl] (Mo1) were studied using flow cytometry, spectrophotometry and Western blotting. We found that the cytotoxic effect of both tested complexes after 24 h is higher against the both examined cell lines than that of cis-platin (cis-DDP). At later investigated time intervals of 48 and 72 h, the cytotoxic effect of the cis-DDP increased but the values of the cytotoxicity of the tested V1 and Mo1 complexes remained unchanged, with the cytotoxicity of V1 comparable to that of cis-DDP. Furthermore we observed that the apoptotic process was induced by the activation of the caspases 9 (intrinsic pathway) and 8 (extrinsic pathway) in cells exposed to evaluated complexes. In case of the p53 wild-type MOLT-4 cells, the expression of the tumor-suppressor protein p53 and its form phosphorylated at the serine 15 increased after both V1 and Mo1 treatment, similar to the effect of cis-DDP. PMID:26391003

  4. 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.

  5. Inhibition of proliferation by agricultural plant extracts in seven human adult T-cell leukaemia (ATL)-related cell lines.

    PubMed

    Kai, Hisahiro; Akamatsu, Ena; Torii, Eri; Kodama, Hiroko; Yukizaki, Chizuko; Sakakibara, Yoichi; Suiko, Masahito; Morishita, Kazuhiro; Kataoka, Hiroaki; Matsuno, Koji

    2011-07-01

    Adult T-cell leukaemia (ATL) is caused by human T-cell leukaemia virus type I (HTLV-I) infection and is resistant to conventional chemotherapy. We evaluated the inhibitory effects of agricultural plants on the proliferation of seven ATL-related human leukaemia cells, using three ATL cell lines (ED, Su9T01 and S1T), two human T-cell lines transformed by HTLV-I infection (HUT-102 and MT-2) and two HTLV-I-negative human T-cell acute lymphoblastic leukaemia cell lines (Jurkat and MOLT-4). A total of 52 samples of 80% ethanol extracts obtained from 30 types of agricultural plants were examined. On the basis of IC(50) values, we selected samples with greater activity than genistein, which was used as a positive control. The highest inhibitory effect was observed with extracts from leaves of Vaccinium virgatum Aiton (blueberry) on four cell lines (ED, Su9T01, HUT-102 and Jurkat); seeds of Momordica charantia L. (bitter gourd) exhibited the second highest activity. The bitter gourd seeds suppressed the proliferation of three cell lines (Su9T01, HUT-102 and Jurkat). The extracts from edible parts of Ipomea batatas LAM. (sweet potato), edible parts of Colocasia esculenta (L.) Schott (taro), skin of taro and seeds of Prunus mume Sieb. et Zucc. (mume) showed markedly greater inhibitory effects on Su9T01 than genistein. These findings suggest that ATL-preventative bioactive compounds may exist in these agricultural plants, which are considered to be functional foods. PMID:21293936

  6. 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.

  7. 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. PMID:20648630

  8. 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.

  9. Inhibitory effects of naringenin on tumor growth in human cancer cell lines and sarcoma S-180-implanted mice.

    PubMed

    Kanno, Syu-Ichi; Tomizawa, Ayako; Hiura, Takako; Osanai, Yuu; Shouji, Ai; Ujibe, Mayuko; Ohtake, Takaharu; Kimura, Katsuhiko; Ishikawa, Masaaki

    2005-03-01

    We have investigated the effect of naringenin (NGEN) on tumor growth in various human cancer cell lines and sarcoma S-180-implanted mice. NGEN showed cytotoxicity in cell lines derived from cancer of the breast (MCF-7, MDA-MB-231), stomach (KATOIII, MKN-7), liver (HepG2, Hep3B, Huh7), cervix (Hela, Hela-TG), pancreas (PK-1), and colon (Caco-2) as well as leukemia (HL-60, NALM-6, Jurkat, U937). NGEN-induced cytotoxicity was low in Caco-2 and high in leukemia cells compared to other cell lines. NGEN dose-dependently induced apoptosis, with hypodiploid cells detected in both Caco-2 and HL-60 by flow cytometric analysis. In vivo, NGEN inhibited tumor growth in sarcoma S-180-implanted mice, following intraperitoneal or peroral injection once a day for 5 d. Naringin (NG) also inhibited tumor growth by peroral injection but not intraperitoneal injection. NGEN, one of the most abundant flavonoids in citrus fruits, may have a potentially useful inhibitory effect on tumor growth. PMID:15744083

  10. A clinically attainable dose of L-asparaginase targets glutamine addiction in lymphoid cell lines.

    PubMed

    Sugimoto, Koichi; Suzuki, Hiroshi I; Fujimura, Tsutomu; Ono, Asami; Kaga, Naoko; Isobe, Yasushi; Sasaki, Makoto; Taka, Hikari; Miyazono, Kohei; Komatsu, Norio

    2015-11-01

    L-asparaginase (L-ASNase) is an important branch of chemotherapy for acute lymphoblastic leukemia (ALL) and some types of non-Hodgkin's lymphoma, including natural killer (NK)-cell lymphoma. Although it mediates hydrolysis of asparagine (Asn) and glutamine (Gln), which are variably required for cancer cell survival, the relative contribution of Asn and Gln depletion to the anti-tumor activity in therapeutic doses is unclear in ALL and malignant lymphoma. Here we demonstrate that L-ASNase exerts cytotoxicity through targeting the Gln addiction phenotype in lymphoid cell lines. A clinically attainable intermediate dose of L-ASNase induced massive apoptosis in ALL Jurkat and mantle cell lymphoma Jeko cell lines, while a low dose of L-ASNase effectively killed NK-cell lymphoma cells. In the lymphoid cell lines Jurkat and Jeco, deprivation of Gln but not Asn specifically suppressed cell growth and survival, and phenocopied the action of L-ASNase. L-ASNase treatment and Gln deprivation dramatically disrupted the refilling of the tricarboxylic acid (TCA) cycle by intracellular glutamate (Glu) and disturbed the mitochondrial integrity, which were alleviated by various anaplerotic TCA cycle intermediates, suggesting a direct contribution of glutaminase activity of L-ASNase. The action of L-ASNase differs between Jurkat cells and NK-cell lymphoma cells, according to their dependence on Gln and Asn. Furthermore, we observed that high expression of glutaminase GLS1 is associated with increased sensivity to L-ASNase in pediatric B lineage ALL. Our results redefine L-ASNase as a therapeutic agent targeting Gln addiction in certain lymphoid cells and offer an additional basis for predicting L-ASNase sensitivity and engineering selective L-ASNase derivatives for leukemia and lymphoma.

  11. Cadmium-coordinated supramolecule suppresses tumor growth of T-cell leukemia in mice.

    PubMed

    Zhou, Xiaoping; Koizumi, Yukio; Zhang, Muxin; Natsui, Miyuki; Koyota, Souichi; Yamada, Manabu; Kondo, Yoshihiko; Hamada, Fumio; Sugiyama, Toshihiro

    2015-05-01

    Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal-coordinating ability has been postulated to contribute to the cytotoxic effects of anti-tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p-alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium-coordinated thiacalix[4]arene tetrasulfate (TC4ATS-Cd) exhibits an anti-proliferative effect against T-cell leukemia cells. Cadmium exhibited cytotoxicity with IC50 values ranging from 36 to 129 μM against epithelia-derived cancer cell lines, while TC4ATS-Cd elicited no significant cytotoxicity (IC50 > 947 μM). However, a number of T-cell leukemia cell lines exhibited marked sensitivity to TC4ATS-Cd. In Jurkat cells, toxicity of TC4ATS-Cd occurred with an IC50 of 6.9 μM, which is comparable to that of 6.5 μM observed for cadmium alone. TC4ATS-Cd induced apoptotic cell death through activation of caspase-3 in Jurkat cells. In a xenograft model, TC4ATS-Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS-Cd-treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium-treated mice. These results suggest that cadmium-coordinated supramolecules may have therapeutic potential for treatment of T-cell leukemia.

  12. Cadmium-coordinated supramolecule suppresses tumor growth of T-cell leukemia in mice

    PubMed Central

    Zhou, Xiaoping; Koizumi, Yukio; Zhang, Muxin; Natsui, Miyuki; Koyota, Souichi; Yamada, Manabu; Kondo, Yoshihiko; Hamada, Fumio; Sugiyama, Toshihiro

    2015-01-01

    Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal-coordinating ability has been postulated to contribute to the cytotoxic effects of anti-tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p-alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium-coordinated thiacalix[4]arene tetrasulfate (TC4ATS-Cd) exhibits an anti-proliferative effect against T-cell leukemia cells. Cadmium exhibited cytotoxicity with IC50 values ranging from 36 to 129 μM against epithelia-derived cancer cell lines, while TC4ATS-Cd elicited no significant cytotoxicity (IC50 > 947 μM). However, a number of T-cell leukemia cell lines exhibited marked sensitivity to TC4ATS-Cd. In Jurkat cells, toxicity of TC4ATS-Cd occurred with an IC50 of 6.9 μM, which is comparable to that of 6.5 μM observed for cadmium alone. TC4ATS-Cd induced apoptotic cell death through activation of caspase-3 in Jurkat cells. In a xenograft model, TC4ATS-Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS-Cd-treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium-treated mice. These results suggest that cadmium-coordinated supramolecules may have therapeutic potential for treatment of T-cell leukemia. PMID:25735932

  13. Novel Derivative of Benzofuran Induces Cell Death Mostly by G2/M Cell Cycle Arrest through p53-dependent Pathway but Partially by Inhibition of NF-κB*

    PubMed Central

    Manna, Sunil K.; Bose, Julie S.; Gangan, Vijay; Raviprakash, Nune; Navaneetha, Thota; Raghavendra, Pongali B.; Babajan, Banaganapalli; Kumar, Chitta S.; Jain, Swatantra K.

    2010-01-01

    The Dracaena resin is widely used in traditional medicine as an anticancer agent, and benzofuran lignan is the active component. In this report, we provide evidence that the synthetic derivative of benzofuran lignan (Benfur) showed antitumor activities. It induced apoptosis in p53-positive cells. Though it inhibited endotoxin-induced nuclear factor κB (NF-κB) activation in both p53-positive and -negative cells, the activation of caspase 3 was observed in p53-positive cells. It showed partial cell death effect in both p53-positive and -negative cells through inhibition of NF-κB. Cell cycle analysis using flow cytometry showed that treatment with this novel benozofuran lignan derivative to Jurkat T-cells, but not U-937 cells, resulted in a G2/M arrest in a dose- and time-dependent manner. It increased amounts of p21, p27, and cyclin B, but not phospho-Rb through p53 nuclear translocation in Jurkat T-cells, but not in U-937 cells. It inhibited amounts of MDM2 (murine double minute 2) by repressing the transcription factor Sp1, which was also proved in silico. It induced cell death in tumor cells, but not in primary T-cells. Overall, our data suggest that Benfur-mediated cell death is partially dependent upon NF-κB, but predominantly dependent on p53. Thus, this novel benzofuran lignan derivative can be effective chemopreventive or chemotherapeutic agent against malignant T-cells. PMID:20472557

  14. Dichloromethane fraction of Melissa officinalis induces apoptosis by activation of intrinsic and extrinsic pathways in human leukemia cell lines.

    PubMed

    Ebrahimnezhad Darzi, Salimeh; Amirghofran, Zahra

    2013-06-01

    Various components from medicinal plants are currently used in cancer therapy because of their apoptosis-inducing effects. The present study has aimed to investigate the growth inhibitory and apoptotic effects of Melissa officinalis on tumor cells. We prepared different fractions of this plant to investigate their inhibitory effects on two leukemia cell lines, Jurkat and K562. Fractions with the highest inhibitory effects were examined for induction of apoptosis by the annexin V/propidium iodide assay and cell cycle changes by flow cytometry. Real-time polymerase chain reaction evaluated the changes in expression of apoptosis-related genes. Among different fractions, dichloromethane and n-hexane dose-dependent showed the strongest inhibitory effects on both K562 and Jurkat cells. The dichloromethane fraction significantly induced apoptosis at concentration of 50 µg/ml on Jurkat (85.66 ± 4.9%) and K562 cells (65.04 ± 0.93%) at 24 h after treatment (p < 0.002). According to cell cycle analysis, more than 70% of the cells accumulated in the sub-G1 phase when cultured in the presence of the dichloromethane fraction. This fraction up-regulated Fas and Bax mRNA expression as well as the Bax/Bcl-2 ratio according to cell type, showing its effect on the activation of both extrinsic and intrinsic pathways of apoptosis. The expression of apoptosis-related genes did not significantly change following treatment with the n-hexane fraction. These data indicated that the dichloromethane fraction of M. officinalis had the ability to induce apoptosis and change apoptosis-related gene expression in leukemia cells.

  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. Real-Time Analysis of Imatinib- and Dasatinib-Induced Effects on Chronic Myelogenous Leukemia Cell Interaction with Fibronectin

    PubMed Central

    Obr, Adam; Röselová, Pavla; Grebeňová, Dana; Kuželová, Kateřina

    2014-01-01

    Attachment of stem leukemic cells to the bone marrow extracellular matrix increases their resistance to chemotherapy and contributes to the disease persistence. In chronic myelogenous leukemia (CML), the activity of the fusion BCR-ABL kinase affects adhesion signaling. Using real-time monitoring of microimpedance, we studied in detail the kinetics of interaction of human CML cells (JURL-MK1, MOLM-7) and of control BCR-ABL-negative leukemia cells (HEL, JURKAT) with fibronectin-coated surface. The effect of two clinically used kinase inhibitors, imatinib (a relatively specific c-ABL inhibitor) and dasatinib (dual ABL/SRC family kinase inhibitor), on cell binding to fibronectin is described. Both imatinib and low-dose (several nM) dasatinib reinforced CML cell interaction with fibronectin while no significant change was induced in BCR-ABL-negative cells. On the other hand, clinically relevant doses of dasatinib (100 nM) had almost no effect in CML cells. The efficiency of the inhibitors in blocking the activity of BCR-ABL and SRC-family kinases was assessed from the extent of phosphorylation at autophosphorylation sites. In both CML cell lines, SRC kinases were found to be transactivated by BCR-ABL. In the intracellular context, EC50 for BCR-ABL inhibition was in subnanomolar range for dasatinib and in submicromolar one for imatinib. EC50 for direct inhibition of LYN kinase was found to be about 20 nM for dasatinib and more than 10 µM for imatinib. Cells pretreated with 100 nM dasatinib were still able to bind to fibronectin and SRC kinases are thus not necessary for the formation of cell-matrix contacts. However, a minimal activity of SRC kinases might be required to mediate the increase in cell adhesivity induced by BCR-ABL inhibition. Indeed, active (autophosphorylated) LYN was found to localize in cell adhesive structures which were visualized using interference reflection microscopy. PMID:25198091

  17. Calcitriol stimulates prolactin expression in non-activated human peripheral blood mononuclear cells: breaking paradigms.

    PubMed

    Díaz, Lorenza; Martínez-Reza, Isela; García-Becerra, Rocío; González, Leticia; Larrea, Fernando; Méndez, Isabel

    2011-08-01

    Calcitriol, the hormonal form of vitamin D(3), exerts immunomodulatory effects through the vitamin D(3) receptor (VDR) and increases prolactin (PRL) expression in the pituitary and decidua. Nevertheless, the effects of calcitriol upon lymphocyte PRL have not been evaluated. Therefore, we investigated calcitriol effects upon PRL in resting and phytohemagglutinin-activated human peripheral blood mononuclear cells (PBMNC) and Jurkat T lymphoma cells. Immunoblots showed constitutive expression of the 50-kDa VDR species in activated PBMNC and Jurkat cells, while a 75-kDa species was recognized in both resting and activated-PBMNC. Only in resting PBMNC calcitriol significantly stimulated PRL expression in a dose-dependent manner. The positive control CYP24A1, a highly VDR-responsive gene, was stimulated by calcitriol, effect that was stronger in resting than in activated-PBMNC (P<0.05), and without effect in Jurkat cells. Calcitriol upregulation of PRL and CYP24A1 was significantly inhibited by the VDR antagonist TEI-9647. EMSA showed that resting PBMNC contain a protein that binds to DR3-type VDRE. Cell activation reduced basal CYP24A1 while induced CYP27B1, VDR and pregnane X receptor (PXR) expression. In summary, calcitriol stimulated PRL and CYP24A1 gene expression in quiescent lymphocytes through a VDR-mediated mechanism. Our results suggest that the 75-kDa VDR species could be participating in calcitriol-mediated effects, and that activation induces factors such as PXR that restrain VDR transcriptional processes. This study supports the presence of a functional VDR in quiescent lymphocytes, providing evidence to reevaluate the VDR paradigm that assumes that lymphocytes respond to calcitriol only after activation. Altogether, our results offer new insights into the mechanisms whereby PRL is regulated in immune cells.

  18. 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.

  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. 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.

  1. 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.

  2. The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies.

    PubMed

    Manger, B; Weiss, A; Imboden, J; Laing, T; Stobo, J D

    1987-10-15

    Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.

  3. 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

  4. Antitumor activities of valproic acid on Epstein-Barr virus-associated T and natural killer lymphoma cells.

    PubMed

    Iwata, Seiko; Saito, Takashi; Ito, Yoshinori; Kamakura, Maki; Gotoh, Kensei; Kawada, Jun-Ichi; Nishiyama, Yukihiro; Kimura, Hiroshi

    2012-02-01

    Epstein-Barr virus (EBV), which infects B cells, T cells, and natural killer (NK) cells, is associated with multiple lymphoid malignancies. Recently, histone deacetylase (HDAC) inhibitors have been reported to have anticancer effects against various tumor cells. In the present study, we evaluated the killing effect of valproic acid (VPA), which acts as an HDAC inhibitor, on EBV-positive and -negative T and NK lymphoma cells. Treatment of multiple T and NK cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3 and KHYG1) with 0.1-5 mM of VPA inhibited HDAC, increased acetylated histone levels and reduced cell viability. No significant differences were seen between EBV-positive and -negative cell lines. Although VPA induced apoptosis in some T and NK cell lines (SNT16, Jurkat and KHYG1) and cell cycle arrest, it did not induce lytic infection in EBV-positive T or NK cell lines. Because the killing effect of VPA was modest (1 mM VPA reduced cell viability by between 22% and 56%), we tested the effects of the combination of 1 mM of VPA and 0.01 μM of the proteasome inhibitor bortezomib. The combined treated of cells with VPA and bortezomib had an additive killing effect. Finally, we administered VPA to peripheral blood mononuclear cells from three patients with EBV-associated T or NK lymphoproliferative diseases. In these studies, VPA had a greater killing effect against EBV-infected cells than uninfected cells, and the effect was increased when VPA was combined with bortezomib. These results indicate that VPA has antitumor effects on T and NK lymphoma cells and that VPA and bortezomib may have synergistic effects, irrespective of the presence of EBV. PMID:22017376

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

    PubMed Central

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

    2016-01-01

    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. PMID:27666896

  6. 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. PMID:24986306

  7. 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.

  8. Mechanistic Study of the Phytocompound, 2- β -D-Glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne in Human T-Cell Acute Lymphocytic Leukemia Cells by Using Combined Differential Proteomics and Bioinformatics Approaches.

    PubMed

    Shiau, Jeng-Yuan; Yin, Shu-Yi; Chang, Shu-Lin; Hsu, Yi-Jou; Chen, Kai-Wei; Kuo, Tien-Fen; Feng, Ching-Shan; Yang, Ning-Sun; Shyur, Lie-Fen; Yang, Wen-Chin; Wen, Tuan-Nan

    2015-01-01

    Bidens pilosa, a medicinal herb worldwide, is rich in bioactive polyynes. In this study, by using high resolution 2-dimensional gel electrophoresis coupled with mass spectrometry analysis, as many as 2000 protein spots could be detected and those whose expression was specifically up- or downregulated in Jurkat T cells responsive to the treatment with 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHTT) can be identified. GHTT treatment can upregulate thirteen proteins involved in signal transduction, detoxification, metabolism, energy pathways, and channel transport in Jurkat cells. Nine proteins, that is, thioredoxin-like proteins, BH3 interacting domain death agonist (BID protein involving apoptosis), methylcrotonoyl-CoA carboxylase beta chain, and NADH-ubiquinone oxidoreductase, were downregulated in GHTT-treated Jurkat cells. Further, bioinformatics tool, Ingenuity software, was used to predict signaling pathways based on the data obtained from the differential proteomics approach. Two matched pathways, relevant to mitochondrial dysfunction and apoptosis, in Jurkat cells were inferred from the proteomics data. Biochemical analysis further verified both pathways involving GHTT in Jurkat cells. These findings do not merely prove the feasibility of combining proteomics and bioinformatics methods to identify cellular proteins as key players in response to the phytocompound in Jurkat cells but also establish the pathways of the proteins as the potential therapeutic targets of leukemia.

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

    SciTech Connect

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

    2013-08-30

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

  10. Rhizoctonia Bataticola Lectin (RBL) Induces Caspase-8-Mediated Apoptosis in Human T-Cell Leukemia Cell Lines but Not in Normal CD3 and CD34 Positive Cells

    PubMed Central

    Pujari, Radha; Eligar, Sachin M.; Kumar, Natesh; Barkeer, Srikanth; Reddy, Vishwanath; Swamy, Bale M.; Inamdar, Shashikala R.; Shastry, Padma

    2013-01-01

    We have previously demonstrated immunostimulatory activity of a fungal lectin, Rhizoctonia bataticola lectin (RBL), towards normal human peripheral blood mononuclear cells. The present study aimed to explore the anticancer activities of RBL using human leukemic T-cell lines, Molt-4, Jurkat and HuT-78. RBL exhibited significant binding (>90%) to the cell membrane that was effectively inhibited by complex glycoproteins such as mucin (97% inhibition) and asialofetuin (94% inhibition) but not simple sugars such as N-acetyl-D-galactosamine, glucose and sucrose. RBL induced a dose and time dependent inhibition of proliferation and induced cytotoxicity in the cell lines. The percentage of apoptotic cells, as determined by hypodiploidy, was 33% and 42% in Molt-4 and Jurkat cells, respectively, compared to 3.11% and 2.92% in controls. This effect was associated with a concomitant decrease in the G0/G1 population. Though initiator caspase-8 and -9 were activated upon exposure to RBL, inhibition of caspase-8 but not caspase-9 rescued cells from RBL-induced apoptosis. Mechanistic studies revealed that RBL induced cleavage of Bid, loss of mitochondrial membrane potential and activation of caspase-3. The expression of the anti-apoptotic proteins Bcl-2 and Bcl-X was down regulated without altering the expression of pro-apoptotic proteins- Bad and Bax. In contrast to leukemic cells, RBL did not induce apoptosis in normal PBMC, isolated CD3+ve cells and undifferentiated CD34+ve hematopoietic stem and progenitor cells (HSPCs). The findings highlight the differential effects of RBL on transformed and normal hematopoietic cells and suggest that RBL may be explored for therapeutic applications in leukemia. PMID:24244478

  11. Inhibition of type I insulin-like growth factor receptor tyrosine kinase by picropodophyllin induces apoptosis and cell cycle arrest in T lymphoblastic leukemia/lymphoma

    PubMed Central

    Huang, Zhiwei; Fang, Zhijia; Zhen, Hong; Zhou, Li; Amin, Hesham M.; Shi, Ping

    2015-01-01

    It has been recently shown that IGF-IR contributes significantly to the survival of T lymphoblastic leukemia/lymphoma (T-LBL) cells, and it was therefore suggested that IGF-IR could represent a legitimate therapeutic target in this aggressive disease. Picropodphyllin (PPP) is a potent, selective inhibitor of IGF-IR that is currently used with notable success in clinical trials that include patients with aggressive types of epithelial tumors. In the present study, we tested the effects of PPP on Jurkat and Molt-3 cells; two prototype T-LBL cell lines. Our results demonstrate that PPP efficiently induced apoptotic cell death and cell cycle arrest of these two cells. These effects were attributable to alterations of downstream target proteins. By using proteomic analysis, 7 different proteins were found to be affected by PPP treatment of Jurkat cells. These proteins are involved in various aspects of cellular metabolism, cytoskeleton organization, and signal transduction pathways. The results suggest that PPP affects multiple signaling molecules and inhibits fundamental pathways that control cell growth and survival. Our study also provides novel evidence that PPP could be potentially utilized for the treatment of the aggressive T-LBL. PMID:24206093

  12. Constitutive NO synthase regulates the Na+/Ca2+ exchanger in human T cells: role of [Ca2+]i and tyrosine phosphorylation.

    PubMed

    Kiang, Juliann G; McClain, David E; Warke, Vishal G; Krishnan, Sandeep; Tsokos, George C

    2003-08-01

    For many types of cells, heat stress leads to an increase in intracellular free calcium concentration ([Ca2+](i)) that has been shown to trigger a wide variety of cellular responses. In T lymphocytes, for example, heat stress stimulates pathways that make them more susceptible to Fas/CD95-mediated apoptosis. Because of our interest in understanding more about the response of lymphocytes to various stressors, we used human peripheral and Jurkat T lymphocytes to investigate the effect of heat stress on calcium homeostasis. We found that peripheral and Jurkat T cells both exhibit cNOs activity but not iNOs activity. Heat stress increased NO production, which was inhibited by LNNA (a cNOs inhibitor) but not L-NIL (an iNOs inhibitor). Heat stress increased [Ca2+](i) in Jurkat T cells by decreasing the K(m) of the cell surface membrane Na+/Ca2+ exchanger for extracellular Ca2+. Heating also increased cNOs phosphorylation at tyrosine residues. In cells incubated with LNNA, heat stress promoted an increase in [Ca2+](i) and a decrease in [Na+](i) greater than in cells heated without LNNA, a larger decrease in K(m) of the Na+/Ca2+ exchanger for extracellular Ca2+, and decreased phosphorylation of cNOs. Our results suggest that cNOs plays an important regulatory role after heat stress. Heating appears to increase the phosphorylation of cNOs that is complexed with the Na+/Ca2+ exchanger to decrease its activity. This process is related to increased expression of Fas/CD95 on the cell surface, which might explain the apoptotic diathesis of lymphocytes after heat stress.

  13. Cytotoxicity Mediated by the Fas Ligand (FasL)-activated Apoptotic Pathway in Stem Cells*

    PubMed Central

    Mazar, Julia; Thomas, Molly; Bezrukov, Ludmila; Chanturia, Alexander; Pekkurnaz, Gulcin; Yin, Shurong; Kuznetsov, Sergei A.; Robey, Pamela G.; Zimmerberg, Joshua

    2009-01-01

    Whereas it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. Here, we test the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and find both Fas and FasL expression by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(−/−) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues. PMID:19531476

  14. Two structurally identical mannose-specific jacalin-related lectins display different effects on human T lymphocyte activation and cell death.

    PubMed

    Benoist, Hervé; Culerrier, Raphaël; Poiroux, Guillaume; Ségui, Bruno; Jauneau, Alain; Van Damme, Els J M; Peumans, Willy J; Barre, Annick; Rougé, Pierre

    2009-07-01

    Plant lectins displaying similar single sugar-binding specificity and identical molecular structure might present various biological effects. To explore this possibility, the effects on human lymphocytes of two mannose-specific and structurally closely related lectins, Morniga M from Morus nigra and artocarpin from Artocarpus integrifolia were investigated. In silico analysis revealed that Morniga M presents a more largely open carbohydrate-binding cavity than artocarpin, probably allowing interactions with a broader spectrum of carbohydrate moieties. In vitro, Morniga M interacted strongly with the lymphocyte surface and was uptaken quickly by cells. Morniga M and artocarpin triggered the proliferation and activation of human T and NK lymphocytes. A minority of B lymphocytes was activated in artocarpin-treated culture, whereas Morniga M favored the emergence of CD4+ CD8+ T lymphocytes. Moreover, cell death occurred in activated PBMC, activated T lymphocytes, and Jurkat T leukemia cells incubated with Morniga M only. The biological effects of both lectins were dependent on carbohydrate recognition. The Morniga M-induced cell death resulted, at least in part, from caspase-dependent apoptosis and FADD-dependent receptor-mediated cell death. Finally, Morniga M, but not artocarpin, triggered AICD of T lymphocytes. In conclusion, both lectins trigger lymphocyte activation, but only Morniga M induces cell death. In spite of similar in vitro mannose-binding specificities and virtually identical structure, only Morniga M probably interacts with carbohydrate moieties bound to molecules able to induce cell death. The present data suggest that subtle alterations in N-glycans can distinguish activation and cell death molecules at the lymphocyte surface.

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

    PubMed

    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 T(538) 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

  16. 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

  17. Quantitative Effect of Suboptimal Codon Usage on Translational Efficiency of mRNA Encoding HIV-1 gag in Intact T Cells

    PubMed Central

    Ngumbela, Kholiswa C.; Ryan, Kieran P.; Sivamurthy, Rohini; Brockman, Mark A.; Gandhi, Rajesh T.; Bhardwaj, Nina; Kavanagh, Daniel G.

    2008-01-01

    Background The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1) are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. Methodology and Principal Findings To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per µg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. Conclusions We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA. PMID:18523584

  18. 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.

  19. [Comparative analysis of natural and synthetic antimutagens as regulators of gene expression in human cells under exposure to ionizing radiation].

    PubMed

    Mikhailov, V F; Shishkina, A A; Vasilyeva, I M; Shulenina, L V; Raeva, N F; Rogozhin, E A; Startsev, M I; Zasukhina, G D; Gromov, S P; Alfimov, M V

    2015-02-01

    This paper studies the effect of plant peptides of thionine Ns-W2 extracted from seeds of fennel flower (Nigella sativa) and β-purothionine from wheat germs (Triticum kiharae), as well as a synthetic antimutagen (crown-compound), on the expression of several genes involved in the.control of cellular homeostasis, processes of carcinogenesis, and radiation response in human rhabdomyosarcoma cells (RD cells), T-lymphoblastoid cell line Jurkat, and blood cells. All of these agents acted as antimutagens-anticarcinogens, reducing the expression of genes involved in carcinogenesis (genes of families MMP, TIMP, and IAP and G-protein genes) in a tumor cell. A pronounced reduction in the mRNA level of these genes was caused by thionine Ns-W2, and the least effect was demonstrated by β-purothionine. Antimutagens had very little effect on the mRNA levels of the several studied genes in normal blood cells. PMID:25966580

  20. 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

  1. 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

  2. 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; dos Santos Silva, Maria Claudia; 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.

  3. Effect of a nutrient mixture on matrix metalloproteinase-9 dimers in various human cancer cell lines.

    PubMed

    Roomi, M W; Kalinovsky, T; Rath, M; Niedzwiecki, A

    2014-03-01

    Strong clinical and experimental evidence demonstrates association of elevated levels of matrix metalloproteinase MMP-9 with cancer progression, metastasis and shortened patient survival, as it plays a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. MMP-9 is secreted in both the monomeric and dimeric form. Although there is little research on MMP-9 dimers, some studies have shown the dimer to be associated with more aggressive tumor progression. Our objective was to study the relative secretion patterns of MMP-9 monomer and dimer in a variety of cancer cell lines and the effect of a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract on MMP-9 secretion. The cancer cell lines were grown in their respective media, supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with NM at 0,10, 50, 100, 500 and 1000 µg/ml. Parallel sets of cultures were treated with PMA (100 ng/ml) for induction of MMP-9. Cell MMP-9 secretion was assayed by gelatinase zymography. MMP-9 dimer secretion patterns of cancer cells fell into different categories. We observed no MMP-9 dimer in prostate DU-145 and PC-3, pancreatic MIA-Pa-Ca2, colon HCT-116, bladder T-24, head and neck FaDu, glioblastoma A-172, T-98 and LN-18 and leukemia HL-60, Jurkat, and Raji cell lines. MMP-dimer secretion only with PMA induction was seen in breast MCF-7 and MDA-MB-231, uterine SK-UT-1, lung A-549, tongue SC-25, melanoma A2058, osteosarcoma U-2OS, rhabdomyosarcoma, fibrosarcoma HT-1080, chondrosarcoma SW-1350 and liposarcoma SW-872. Cervical HeLa and DoTc 2 4510, renal 786-0 and HCC SK-Hep-1 cells exhibited MMP-9 dimer without PMA treatment and increased secretion with PMA treatment. Sarcomas had the highest levels of MMP-9 monomer and dimer with and without PMA among these cancer cell lines. Cervical, uterine and male

  4. Molecular Characteristics of CTA056, a Novel Interleukin-2-Inducible T-Cell Kinase Inhibitor that Selectively Targets Malignant T Cells and Modulates Oncomirs

    PubMed Central

    Guo, Wenchang; Liu, Ruiwu; Ono, Yoko; Ma, Ai-Hong; Martinez, Anthony; Sanchez, Eduardo; Wang, Yan; Huang, Wenzhe; Mazloom, Anisha; Li, Jixian; Ning, Jinying; Maverakis, Emanual; Lam, Kit S.

    2012-01-01

    Interleukin-2-inducible T-cell kinase (Itk) is a member of the Btk (Bruton's tyrosine kinase) family of tyrosine kinases. Itk plays an important role in normal T-cell functions and in the pathophysiology of both autoimmune diseases and T-cell malignancies. Here, we describe the initial characterization of a selective inhibitor, 7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one (CTA056), that was developed through screening a 9600-compound combinatorial solution phase library, followed by molecular modeling, and extensive structure-activity relationship studies. CTA056 exhibits the highest inhibitory effects toward Itk, followed by Btk and endothelial and epithelial tyrosine kinase. Among the 41 cancer cell lines analyzed, CTA056 selectively targets acute lymphoblastic T-cell leukemia and cutaneous T-cell lymphoma. Normal T cells are minimally affected. Incubation of Jurkat and MOLT-4 cells with CTA056 resulted in the inhibition of the phosphorylation of Itk and its effectors including PLC-γ, Akt, and extracellular signal-regulated kinase, as well as the decreased secretion of targeted genes such as interleukin-2 and interferon-γ. Jurkat cells also underwent apoptosis in a dose-dependent manner when incubated with CTA056. The potent apoptosis-inducing potential of CTA056 is reflected by the significant modulation of microRNAs involved in survival pathways and oncogenesis. The in vitro cytotoxic effect on malignant T cells is further validated in a xenograft model. The selective expression and activation of Itk in malignant T cells, as well as the specificity of CTA056 for Itk, make this molecule a potential therapeutic agent for the treatment of T-cell leukemia and lymphoma. PMID:22899868

  5. A microfluidic systems biology approach for live single-cell mitochondrial ROS imaging.

    PubMed

    Kniss, Ariel; Lu, Hang; Jones, Dean P; Kemp, Melissa L

    2013-01-01

    Most current studies of reactive oxygen species (ROS) production report globally averaged measurements within the cell; however, ROS can be produced in distinct subcellular locations and have local effects in their immediate vicinity. A microfluidic platform for high-throughput single-cell imaging allows mitochondrial ROS production to be monitored as varying in both space and time. Using this systems biology approach, single-cell variability can be viewed within a population. We discuss single-cell monitoring of contributors to mitochondrial redox state-mitochondrial hydrogen peroxide or superoxide-through the use of a small molecule probe or targeted fluorescent reporter protein. Jurkat T lymphoma cells were stimulated with antimycin A and imaged in an arrayed microfluidic device over time. Differences in single-cell responses were observed as a function of both inhibitor concentration and type of ROS measurement used.

  6. 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

  7. Triazene compounds induce apoptosis in O6-alkylguanine-DNA alkyltransferase deficient leukemia cell lines.

    PubMed

    Tentori, L; Graziani, G; Gilberti, S; Lacal, P M; Bonmassar, E; D'Atri, S

    1995-11-01

    Previous studies demonstrated that triazene compounds (TZC) possess antitumor, antimetastatic and immunosuppressive activity, and induce novel antigenic properties in neoplastic cells. Moreover, TZC showed marked antitumor activity in patients with acute myelogenous leukemias (AML). In most cases leukemic blasts with low levels of the repair enzyme O6-alkyl-guanine-DNA alkyltransferase (OGAT) were highly susceptible to TZC. Therefore the cytotoxic effects of TZC against human leukemic cells and the influence of OGAT modulation were investigated. Five leukemia cell lines were treated with the in vitro active derivative of dacarbazine: 5-(3-methyl-1-triazeno) imidazole-4-carboxamide (MTIC), or with temozolomide (TZM), which is readily cleaved to form the linear triazene MTIC in aqueous solution. The results showed that treatment with TZC at concentrations ranging between 62.5 and 250 microM significantly inhibited cell growth of U-937 and K-562 leukemia cell lines, both with undetectable OGAT activity. Growth inhibition was accompanied by DNA fragmentation and reduction of cell volume characteristic of cell undergoing apoptosis. In contrast, Daudi, HL-60 and Jurkat leukemia cell lines, characterized by high levels of the repair enzyme, were resistant to concentrations of TZC up to 500 microM. Treatment of resistant lines with O6-benzylguanine (BG, a specific inhibitor of OGAT) rendered HL-60 and Daudi but not Jurkat cells sensitive to cytotoxic effects and apoptosis mediated by MTIC. The results presented suggest that: (1) apoptosis is involved in cytotoxic activity of TZC; (2) OGAT could have a role in preventing programmed cell death induced by TZC; and (3) treatment with BG could potentiate cytotoxic and apoptotic effects of TZC on leukemic cell lines when high level of OGAT activity is the main factor involved in resistance to TZC.

  8. Molecular Cytotoxic Mechanisms of 1-(3,4,5-Trihydroxyphenyl)-dodecylbenzoate in Human Leukemia Cell Lines

    PubMed Central

    Maioral, M. F.; Bubniak, L.D.S.; Marzarotto, M.A.L.; De Moraes, A.C.R.; Leal, P.; Nunes, R.; Yunes, R. A.; Santos-Silva, M. C.

    2016-01-01

    Recent studies have shown that gallic acid and its alkylesters induce apoptosis in different cell lines. Since new compounds with biological activity and less cytotoxicity to normal cells are necessary for cancer therapy, the aim of this study was to evaluate the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. The cell viability was determined by MTT method. The apoptosis induction was assessed by bromide and acridine orange staining and by Annexin V-FITC Apoptosis Detection kit. The cell cycle analysis was carried out by flow cytometry using propidium iodide. Cytometric analysis was also performed to evaluate the expression of the following proteins: AIF, p53, Bcl-2 and Bax. The mitochondrial potential was also assessed by flow cytometry using MitoView633 kit. The results showed that the compound significantly reduced the cell viability of K562 and Jurkat cells in a concentration and time dependent manner (IC50 of 30 μM). The compound induced cell cycle arrest in G0/G1phase and significantly increased the proportion of cells in the sub-G0/G1phase. Apoptosis was confirmed by the sight of morphological characteristics of apoptosis and by phosphatidylserine externalization (73.47±5.71% of cells expressing annexin). The results also showed that the compound promotes a modification in Bax:Bcl-2 ratio and increases p53 expression. Thus, it is possible to conclude that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate induces apoptosis by inhibiting the antiapoptotic protein Bcl-2 and by increasing the release of AIF, Bax and p53. In addition, it blocks the cell cycle at G0/G1, stopping cell proliferation. So far, the results suggest that this compound may have a potential therapeutic effect against leukemia cells. PMID:27168690

  9. 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.

  10. 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

  11. 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.

  12. The Interaction of CD154 with the α5β1 Integrin Inhibits Fas-Induced T Cell Death

    PubMed Central

    Yacoub, Daniel; Salti, Suzanne; Alaaeddine, Nada; Aoudjit, Fawzi; Hassan, Ghada S.; Mourad, Walid

    2016-01-01

    CD154, a critical regulator of the immune response, is usually associated with chronic inflammatory, autoimmune diseases as well as malignant disorders. In addition to its classical receptor CD40, CD154 is capable of binding other receptors, members of the integrin family, the αIIbβ3, αMβ2 and α5β1. Given the role attributed to integrins and particularly the β1 integrins in inhibiting apoptotic events in normal as well as malignant T cells, we were highly interested in investigating the role of the CD154/α5β1 interaction in promoting survival of malignant T cells contributing as such to tumor development and/or propagation. To support our hypothesis, we first show that soluble CD154 binds to the T-cell acute lymphoblastic leukemia cell line, Jurkat E6.1 in a α5β1-dependent manner. Binding of soluble CD154 to α5β1 integrin of Jurkat cells leads to the activation of key survival proteins, including the p38 and ERK1/2 mitogen-activated protein kinases (MAPKs), phosphoinositide 3 kinase (PI-3K), and Akt. Interestingly, soluble CD154 significantly inhibits Fas-mediated apoptosis in T cell leukemia-lymphoma cell lines, Jurkat E6.1 and HUT78 cells, an important hallmark of T cell survival during malignancy progression. These anti-apoptotic effects were mainly mediated by the activation of the PI-3K/Akt pathway but also involved the p38 and the ERK1/2 MAPKs cascades. Our data also demonstrated that the CD154-triggered inhibition of the Fas-mediated cell death response was dependent on a suppression of caspase-8 cleavage, but independent of de novo protein synthesis or alterations in Fas expression on cell surface. Together, our results highlight the impact of the CD154/α5β1 interaction in T cell function/survival and identify novel targets for the treatment of malignant disorders, particularly of T cell origin. PMID:27391025

  13. 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.

  14. 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

  15. 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.

  16. Apoptosis induction by Epican Forte in HTLV-1 positive and negative malignant T-cells.

    PubMed

    Harakeh, S; Diab-Assaf, M; Niedzwiecki, A; Khalife, J; Abu-El-Ardat, K; Rath, M

    2006-07-01

    The effects of a novel nutrient formulation Epican Forte (EF) were evaluated on proliferation and induction of apoptosis using non-cytotoxic concentrations against HTLV-1 positive (HuT-102 & C91-PL) and negative (CEM & Jurkat) cells. EF showed anti-proliferative effect as determined by MTT assay and TGF mRNA protein expression using RT-PCR. EF resulted in the down-regulation of TGF-alpha and an up-regulation in TGF-beta2. EF caused a significant increase in apoptotic cells in the preG1 phase. These results were confirmed using Cell Death ELISA and Annexin V-FITC. Induction of apoptosis was caused by an up-regulation of p53, p21 and Bax protein levels and a down-regulation of Bcl-2alpha protein expression level. PMID:16427125

  17. Paracrine effects of haematopoietic cells on human mesenchymal stem cells

    PubMed Central

    Zhou, Shuanhu

    2015-01-01

    Stem cell function decline during ageing can involve both cell intrinsic and extrinsic mechanisms. Bone and blood formation are intertwined in bone marrow, therefore haematopoietic cells and bone cells could be extrinsic factors for each other. In this study, we assessed the paracrine effects of extrinsic factors from haematopoietic cells on human mesenchymal stem cells (MSCs). Our data showed that haematopoietic cells stimulate proliferation, osteoblast differentiation and inhibit senescence of MSCs; TNF-α, PDGF-β, Wnt1, 4, 6, 7a and 10a, sFRP-3 and sFRP-5 are dominantly expressed in haematopoietic cells; the age-related increase of TNF-α in haematopoietic cells may perform as a negative factor in the interactions of haematopoietic cells on MSCs via TNF-α receptors and then activating NF-κB signaling or Wnt/β-catenin signaling to induce senescence and reduce osteoblast differentiation in MSCs. In conclusion, our data demonstrated that there are paracrine interactions of haematopoietic cells on human MSCs; immunosenescence may be one of the extrinsic mechanisms by which skeletal stem cell function decline during human skeletal ageing. PMID:26030407

  18. Protein tyrosine kinase inhibition and cell proliferation: is the [3H]-thymidine uptake assay representative of the T-lymphocyte proliferation rate?

    PubMed

    Spinozzi, F; Pagliacci, M C; Agea, E; Migliorati, G; Riccardi, C; Bertotto, A; Nicoletti, I

    1995-01-01

    T-cell growth is controlled to a large degree by extracellular signals that bind to specific receptors on the surface of cells. A number of these receptors have intrinsic protein tyrosine kinase (PTK) activity. Their action on second messenger generation, and thus on cell proliferation, has been indirectly demonstrated by the decrease in [3H]-thymidine (TdR) uptake that follows co-stimulation of T-cells with mitogens and PTK inhibitors such as genistein (GEN). In this paper we report that the [3H]-TdR uptake assay is not a valid and reliable tool for investigating the proliferative activity of certain T-cell lines. In fact, a concomitant assessment of both [3H]-TdR uptake and cell cycle progression demonstrated that GEN is able to block G2/M progression of Jurkat T-lymphocytes even at doses (5 micrograms/ml) that do not influence [3H]-TdR uptake. Pretreatment with sodium o-vanadate (100 nM) could not reverse the GEN-related cell cycle perturbation, but was able to restore optimal [3H]-TdR uptake. Finally, GEN treatment was able to induce concentration-dependent apoptotic cell death of Jurkat T-cells. The control of cell activation, proliferation and programmed cell death is undoubtedly influenced by receptor-associated PTKs. The final effect on cell survival is almost entirely dependent on the activation state of the cell. The [3H]-TdR uptake assay seems to be inadequate for a correct interpretation of the expected results. PMID:7655707

  19. Group I PAK Inhibitor IPA-3 Induces Cell Death and Affects Cell Adhesivity to Fibronectin in Human Hematopoietic Cells

    PubMed Central

    Kuželová, Kateřina; Grebeňová, Dana; Holoubek, Aleš; Röselová, Pavla; Obr, Adam

    2014-01-01

    P21-activated kinases (PAKs) are involved in the regulation of multiple processes including cell proliferation, adhesion and migration. However, the current knowledge about their function is mainly based on results obtained in adherent cell types. We investigated the effect of group I PAK inhibition using the compound IPA-3 in a variety of human leukemic cell lines (JURL-MK1, MOLM-7, K562, CML-T1, HL-60, Karpas-299, Jurkat, HEL) as well as in primary blood cells. IPA-3 induced cell death with EC50 ranging from 5 to more than 20 μM. Similar range was found for IPA-3-mediated dephosphorylation of a known PAK downstream effector, cofilin. The cell death was associated with caspase-3 activation, PARP cleavage and apoptotic DNA fragmentation. In parallel, 20 μM IPA-3 treatment induced rapid and marked decrease of the cell adhesivity to fibronectin. Per contra, partial reduction of PAK activity using lower dose IPA-3 or siRNA resulted in a slight increase in the cell adhesivity. The changes in the cell adhesivity were also studied using real-time microimpedance measurement and by interference reflection microscopy. Significant differences in the intracellular IPA-3 level among various cell lines were observed indicating that an active mechanism is involved in IPA-3 transport. PMID:24664099

  20. LDEF solar cell radiation effects analysis

    NASA Technical Reports Server (NTRS)

    Rives, Carol J.; Azarewicz, Joseph L.; Massengill, Lloyd

    1993-01-01

    Because of the extended time that the Long Duration Exposure Facility (LDEF) mission stayed in space, the solar cells on the satellite experienced greater environments than originally planned. The cells showed an overall degradation in performance that is due to the combined effects of the various space environments. The purpose of this analysis is to calculate the effect of the accumulated radiation on the solar cells, thereby helping Marshall Space Flight Center (MSFC) to unravel the relative power degradation from the different environments.

  1. [Effects of beryllium chloride on cultured cells].

    PubMed

    Sakaguchi, T; Sakaguchi, S; Nakamura, I; Kagami, M

    1984-05-01

    The effects of beryllium on cultured cells were investigated. Three cell-lines (HeLa-S3, Vero, HEL-R66) were used in these experiments and they were cultured in Eagle's MEM plus 5 or 10% FBS (Fetal Bovine Serum) containing beryllium in various concentrations. HeLa cells or Vero cells were able to grow in the medium with 10 micrograms Be/ml (1.1 mM). On the other hand, the growth of HEL cells were strongly inhibited, even when cultured in the medium with 1 microgram Be/ml (1.1 X 10(-1) mM) and the number of living cells showed markedly low level as compared to that of the control samples cultured in the medium without beryllium. The cytotoxic effects of beryllium on these cells, which were cultured for three days in the medium with beryllium, were observed. None of cytotoxic effects were found on HeLa cells cultured with 0.5 micrograms/ml (5.5 X 10(-2) mM) and on Vero cells cultured with 0.05 micrograms Be/ml (5.5 X 10(-3) mM), while HEL cells received cytotoxic effects even when cultured in the medium containing 0.05 micrograms Be/ml (5.5 X 10(-3) mM), and these effects on the cells appeared strong when cultured in the medium without FBS. It was revealed from these experiments that HEL cells are very sensitive in terms of toxic effects of beryllium. Therefore, there cells can be used for the toxicological study on low level concentrations of the metal.

  2. Downregulation of NPM-ALK by siRNA causes anaplastic large cell lymphoma cell growth inhibition and augments the anti cancer effects of chemotherapy in vitro.

    PubMed

    Hsu, Faye Yuan-yi; Zhao, Yi; Anderson, W French; Johnston, Patrick B

    2007-06-01

    The fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), results from the chromosome translocation t(2;5)(p23;q25) and is present in 50-70 percent of anaplastic large-cell lymphomas (ALCLs). NPM-ALK is a constitutively activated kinase that transforms cells through stimulating several mitogenic signaling pathways. To examine if the NPM-ALK is a potential therapeutic target in ALCL, we used siRNA to specifically downregulate the expression of the NPM-ALK in ALCL cell lines. In this report, we demonstrated viability loss in t(2;5)-positive ALCL cell lines, SUDHL-1 and Karpas 299 cells, but not in lymphoma cell lines without the chromosome translocation, Jurkat and Granta 519 cells. Further study demonstrated that the downregulation of NPM-ALK resulted in decreased cell proliferation and increased cell apoptosis. When used in combination with chemotherapeutic agents, such as doxorubicin, the inhibition of the NPM-ALK augments the chemosensitivity of the tumor cells. These results revealed the importance of continuous expression of NPM-ALK in maintaining the growth of ALCL cells. Our data also suggested that the repression of the fusion gene might be a potential novel therapeutic strategy for NPM-ALK positive ALCLs.

  3. Effect of protein deficiency on suppressor cells.

    PubMed Central

    Khorshidi, M; Mohagheghpour, N

    1979-01-01

    The effects of moderate protein deficiency on the in vitro response of spleen cells to phytohemagglutinin in A/Jax mice were studied. The response of spleen cells from protein-deficient mice to phytohemagglutinin was found to be enhanced as compared with that of cells from control animals. Since inadequate development or function of suppressor cells in the protein-deficient mice offered a possible explanation for the enhanced lymphoproliferative activity, cocultures of spleen cells from protein-deficient and control animals were tested for their responses to phytohemagglutinin. Suppression of [3H]thymidine incorporation was detected in coculture of 25% mitomycin-treated spleen cells from control animals and 75% spleen cells from protein-deficient mice. The suppressor (regulator) elements in control spleens were found to reside in the adherent cell population. PMID:313906

  4. Differential inhibition of DNA synthesis in human T cells by the cigarette tar components hydroquinone and catechol.

    PubMed

    Li, Q; Aubrey, M T; Christian, T; Freed, B M

    1997-08-01

    Hydroquinone (HQ), catechol, and phenol exist in microgram quantities in cigarette tar and represent the predominant form of human exposure to benzene. Exposure of human T lymphoblasts (HTL) in vitro to 50 microM HQ or 50 microM catechol decreased IL-2-dependent DNA synthesis and cell proliferation by >90% with no effect on cell viability. Phenol had no effect on HTL proliferation at concentrations up to 1 mm. The addition of HQ or catechol to proliferating HTL blocked 3H-TdR uptake by >90% within 2 hr without significantly affecting 3H-UR uptake, suggesting that both compounds inhibit a rate-limiting step in DNA synthesis. However, the effects of HQ and catechol appear to involve different mechanisms. Ferric chloride (FeCl3) reversed the inhibitory effect of catechol, but not HQ, corresponding with the known ability of catechol to chelate iron. HQ, but not catechol, caused a decrease in transferrin receptor (TfR, CD71) expression, comparable to the level observed in IL-2-starved cells. HQ also inhibited DNA synthesis in cultures of transformed Jurkat T lymphocytes, primary and transformed fibroblasts, and mink lung epithelial cells, indicating that its antiproliferative effect was not restricted to IL-2 mediated proliferation. However, DNA synthesis by primary lymphocytes was more sensitive to HQ (IC50 = 6 microM) than that of the transformed Jurkat T cell line (IC50 = 37 microM) or primary human fibroblasts (IC50 = 45 microM), suggesting that normal lymphocytes may be particularly sensitive to HQ. The effects of HQ and catechol on DNA synthesis could be partially reversed by a combination of adenosine deoxyribose and guanosine deoxyribose, suggesting that both compounds may inhibit ribonucleotide reductase.

  5. Discovery of novel small molecule cell type-specific enhancers of NF-kappaB nuclear translocation.

    PubMed

    Gong, Gangli; Xie, Yuli; Liu, Yidong; Rinderspacher, Alison; Deng, Shi-Xian; Feng, Yan; Zhu, Zhengxiang; Tang, Yufei; Wyler, Michael; Aulner, Nathalie; Toebben, Udo; Smith, Deborah H; Branden, Lars; Chung, Caty; Schürer, Stephan; Vidović, Dusica; Landry, Donald W

    2009-02-15

    An IKKbeta inhibitor reported to block NF-kappaB transcriptional activities in Jurkat T cells, was found to enhance NF-kappaB translocation in HUVEC cells. These studies suggested a noncanonical NF-kappaB signaling pathway independent of IKKbeta in HUVEC cells.

  6. Cardiac cell therapy: boosting mesenchymal stem cells effects.

    PubMed

    Samper, E; Diez-Juan, A; Montero, J A; Sepúlveda, P

    2013-06-01

    Acute myocardial infarction is a major problem of world public health and available treatments have limited efficacy. Cardiac cell therapy is a new therapeutic strategy focused on regeneration and repair of the injured cardiac muscle. Among different cell types used, mesenchymal stem cells (MSC) have been widely tested in preclinical studies and several clinical trials have evaluated their clinical efficacy in myocardial infarction. However, the beneficial effects of MSC in humans are limited due to poor engraftment and survival of these cells, therefore ways to overcome these obstacles should improve efficacy. Different strategies have been used, such as genetically modifying MSC, or preconditioning the cells with factors that potentiate their survival and therapeutic mechanisms. In this review we compile the most relevant approaches used to improve MSC therapeutic capacity and to understand the molecular mechanisms involved in MSC mediated cardiac repair.

  7. Cell cycle-specific effects of lovastatin.

    PubMed Central

    Jakóbisiak, M; Bruno, S; Skierski, J S; Darzynkiewicz, Z

    1991-01-01

    Lovastatin (LOV), the drug recently introduced to treat hypercholesteremia, inhibits the synthesis of mevalonic acid. The effects of LOV on the cell cycle progression of the human bladder carcinoma T24 cell line expressing activated p21ras were investigated. At a concentration of 2-10 microM, LOV arrested cells in G1 and also prolonged--or arrested a minor fraction of cells in--the G2 phase of the cell cycle; at a concentration of 50 microM, LOV was cytotoxic. The cytostatic effects were reversed by addition of exogenous mevalonate. Cells arrested in the cycle by LOV were viable for up to 72 hr and did not show any changes in RNA or protein content or chromatin condensation, which would be typical of either unbalanced growth or deep quiescence. The expression of the proliferation-associated nuclear proteins Ki-67 and p105 in these cells was reduced by up to 72% and 74%, respectively, compared with exponentially growing control cells. After removal of LOV, the cells resumed progression through the cycle; they entered S phase asynchronously after a lag of approximately 6 hr. Because mevalonate is essential for the posttranslational modification (isoprenylation) of p21ras, which in turn allows this protein to become attached to the cell membrane, the data suggest that the LOV-induced G1 arrest may be a consequence of the loss of the signal transduction capacity of p21ras. Indeed, while exposure of cells to LOV had no effect on the cellular content of p21ras (detected immunocytochemically), it altered the intracellular location of this protein, causing its dissociation from the cell membrane and translocation toward the cytoplasm and nucleus. However, it is also possible that inhibition of isoprenylation of proteins other than p21ras (e.g., nuclear lamins) by LOV may be responsible for the observed suppression of growth of T24 cells. Images PMID:1673788

  8. Cytotoxic effect of magnetic iron oxide nanoparticles synthesized via seaweed aqueous extract

    PubMed Central

    Namvar, Farideh; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Baharara, Javad; Mahdavi, Mahnaz; Amini, Elaheh; Chartrand, Max Stanley; Yeap, Swee Keong

    2014-01-01

    Magnetic iron oxide nanoparticles (Fe3O4 MNPs) are among the most useful metal nanoparticles for multiple applications across a broad spectrum in the biomedical field, including the diagnosis and treatment of cancer. In previous work, we synthesized and characterized Fe3O4 MNPs using a simple, rapid, safe, efficient, one-step green method involving reduction of ferric chloride solution using brown seaweed (Sargassum muticum) aqueous extract containing hydroxyl, carboxyl, and amino functional groups mainly relevant to polysaccharides, which acts as a potential stabilizer and metal reductant agent. The aim of this study was to evaluate the in vitro cytotoxic activity and cellular effects of these Fe3O4 MNPs. Their in vitro anticancer activity was demonstrated in human cell lines for leukemia (Jurkat cells), breast cancer (MCF-7 cells), cervical cancer (HeLa cells), and liver cancer (HepG2 cells). The cancer cells were treated with different concentrations of Fe3O4 MNPs, and an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to test for cytotoxicity, resulting in an inhibitory concentration 50 (IC50) value of 23.83±1.1 μg/mL (HepG2), 18.75±2.1 μg/mL (MCF-7), 12.5±1.7 μg/mL (HeLa), and 6.4±2.3 μg/mL (Jurkat) 72 hours after treatment. Therefore, Jurkat cells were selected for further investigation. The representative dot plots from flow cytometric analysis of apoptosis showed that the percentages of cells in early apoptosis and late apoptosis were increased. Cell cycle analysis showed a significant increase in accumulation of Fe3O4 MNP-treated cells at sub-G1 phase, confirming induction of apoptosis by Fe3O4 MNPs. The Fe3O4 MNPs also activated caspase-3 and caspase-9 in a time-response fashion. The nature of the biosynthesis and therapeutic potential of Fe3O4 MNPs could pave the way for further research on the green synthesis of therapeutic agents, particularly in nanomedicine, to assist in the treatment of cancer. PMID

  9. Cytotoxic effect of magnetic iron oxide nanoparticles synthesized via seaweed aqueous extract.

    PubMed

    Namvar, Farideh; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Baharara, Javad; Mahdavi, Mahnaz; Amini, Elaheh; Chartrand, Max Stanley; Yeap, Swee Keong

    2014-01-01

    Magnetic iron oxide nanoparticles (Fe3O4 MNPs) are among the most useful metal nanoparticles for multiple applications across a broad spectrum in the biomedical field, including the diagnosis and treatment of cancer. In previous work, we synthesized and characterized Fe3O4 MNPs using a simple, rapid, safe, efficient, one-step green method involving reduction of ferric chloride solution using brown seaweed (Sargassum muticum) aqueous extract containing hydroxyl, carboxyl, and amino functional groups mainly relevant to polysaccharides, which acts as a potential stabilizer and metal reductant agent. The aim of this study was to evaluate the in vitro cytotoxic activity and cellular effects of these Fe3O4 MNPs. Their in vitro anticancer activity was demonstrated in human cell lines for leukemia (Jurkat cells), breast cancer (MCF-7 cells), cervical cancer (HeLa cells), and liver cancer (HepG2 cells). The cancer cells were treated with different concentrations of Fe3O4 MNPs, and an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to test for cytotoxicity, resulting in an inhibitory concentration 50 (IC50) value of 23.83±1.1 μg/mL (HepG2), 18.75±2.1 μg/mL (MCF-7), 12.5±1.7 μg/mL (HeLa), and 6.4±2.3 μg/mL (Jurkat) 72 hours after treatment. Therefore, Jurkat cells were selected for further investigation. The representative dot plots from flow cytometric analysis of apoptosis showed that the percentages of cells in early apoptosis and late apoptosis were increased. Cell cycle analysis showed a significant increase in accumulation of Fe3O4 MNP-treated cells at sub-G1 phase, confirming induction of apoptosis by Fe3O4 MNPs. The Fe3O4 MNPs also activated caspase-3 and caspase-9 in a time-response fashion. The nature of the biosynthesis and therapeutic potential of Fe3O4 MNPs could pave the way for further research on the green synthesis of therapeutic agents, particularly in nanomedicine, to assist in the treatment of cancer. PMID

  10. Effects of Helicobacter suis γ- Glutamyl Transpeptidase on Lymphocytes: Modulation by Glutamine and Glutathione Supplementation and Outer Membrane Vesicles as a Putative Delivery Route of the Enzyme

    PubMed Central

    Zhang, Guangzhi; Ducatelle, Richard; Pasmans, Frank; D’Herde, Katharina; Huang, Liping; Smet, Annemieke; Haesebrouck, Freddy; Flahou, Bram

    2013-01-01

    Helicobacter (H.) suis colonizes the stomach of the majority of pigs as well as a minority of humans worldwide. Infection causes chronic inflammation in the stomach of the host, however without an effective clearance of the bacteria. Currently, no information is available about possible mechanisms H. suis utilizes to interfere with the host immune response. This study describes the effect on various lymphocytes of the γ-glutamyl transpeptidase (GGT) from H. suis. Compared to whole cell lysate from wild-type H. suis, lysate from a H. suis ggt mutant strain showed a decrease of the capacity to inhibit Jurkat T cell proliferation. Incubation of Jurkat T cells with recombinantly expressed H. suis GGT resulted in an impaired proliferation, and cell death was shown to be involved. A similar but more pronounced inhibitory effect was also seen on primary murine CD4+ T cells, CD8+ T cells, and CD19+ B cells. Supplementation with known GGT substrates was able to modulate the observed effects. Glutamine restored normal proliferation of the cells, whereas supplementation with reduced glutathione strengthened the H. suis GGT-mediated inhibition of proliferation. H. suis GGT treatment abolished secretion of IL-4 and IL-17 by CD4+ T cells, without affecting secretion of IFN-γ. Finally, H. suis outer membrane vesicles (OMV) were identified as a possible delivery route of H. suis GGT to lymphocytes residing in the deeper mucosal layers. Thus far, this study is the first to report that the effects on lymphocytes of this enzyme, not only important for H. suis metabolism but also for that of other Helicobacter species, depend on the degradation of two specific substrates: glutamine and reduced glutatione. This will provide new insights into the pathogenic mechanisms of H. suis infection in particular and infection with gastric helicobacters in general. PMID:24147103

  11. Charged MVB protein 5 is involved in T-cell receptor signaling.

    PubMed

    Wi, Sae Mi; Min, Yoon; Lee, Ki-Young

    2016-01-29

    Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)-mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5(KD)) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5(KD) Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5(KD) Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling.

  12. Protease inhibitors effectively block cell-to-cell spread of HIV-1 between T cells

    PubMed Central

    2013-01-01

    Background The Human Immunodeficiency Virus type-1 (HIV-1) spreads by cell-free diffusion and by direct cell-to-cell transfer, the latter being a significantly more efficient mode of transmission. Recently it has been suggested that cell-to-cell spread may permit ongoing virus replication in the presence of antiretroviral therapy (ART) based on studies performed using Reverse Transcriptase Inhibitors (RTIs). Protease Inhibitors (PIs) constitute an important component of ART; however whether this class of inhibitors can suppress cell-to-cell transfer of HIV-1 is unexplored. Here we have evaluated the inhibitory effect of PIs during cell-to-cell spread of HIV-1 between T lymphocytes. Results Using quantitative assays in cell line and primary cell systems that directly measure the early steps of HIV-1 infection we find that the PIs Lopinavir and Darunavir are equally potent against both cell-free and cell-to-cell spread of HIV-1. We further show that a protease resistant mutant maintains its resistant phenotype during cell-to-cell spread and is transmitted more efficiently than wild-type virus in the presence of drug. By contrast we find that T cell-T cell spread of HIV-1 is 4–20 fold more resistant to inhibition by the RTIs Nevirapine, Zidovudine and Tenofovir. Notably, varying the ratio of infected and uninfected cells in co-culture impacted on the degree of inhibition, indicating that the relative efficacy of ART is dependent on the multiplicity of infection. Conclusions We conclude that if the variable effects of antiviral drugs on cell-to-cell virus dissemination of HIV-1 do indeed impact on viral replication and maintenance of viral reservoirs this is likely to be influenced by the antiviral drug class, since PIs appear particularly effective against both modes of HIV-1 spread. PMID:24364896

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

    PubMed

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

    2016-03-01

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

  14. Cytotoxicity effects of amiodarone on cultured cells.

    PubMed

    Golli-Bennour, Emna El; Bouslimi, Amel; Zouaoui, Olfa; Nouira, Safa; Achour, Abdellatif; Bacha, Hassen

    2012-07-01

    Amiodarone is a potent anti-arrhythmic drug used for the treatment of cardiac arrhythmias. Although, the effects of amiodarone are well characterized on post-ischemic heart and cardiomyocytes, its toxicity on extra-cardiac tissues is still poorly understood. To this aim, we have monitored the cytotoxicity effects of this drug on three cultured cell lines including hepatocytes (HepG2), epithelial cells (EAhy 926) and renal cells (Vero). We have investigated the effects of amiodarone on (i) cell viabilities, (ii) heat shock protein expressions (Hsp 70) as a parameter of protective and adaptive response and (iii) oxidative damage.Our results clearly showed that amiodarone inhibits cell proliferation, induces an over-expression of Hsp 70 and generates significant amount of reactive oxygen species as measured by lipid peroxidation occurrence. However, toxicity of amiodarone was significantly higher in renal and epithelial cells than in hepatocytes. Vitamin E supplement restores the major part of cell mortalities induced by amiodarone showing that oxidative damage is the predominant toxic effect of the drug.Except its toxicity for the cardiac system, our findings demonstrated that amiodarone can target other tissues. Therefore, kidneys present a high sensibility to this drug which may limit its use with subjects suffering from renal disorders.

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

    PubMed Central

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

    2016-01-01

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

  16. Effects of ultrasound upon endothelial cell ultrastructure

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

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

  17. Formic acid and acetic acid induce a programmed cell death in pathogenic Candida species.

    PubMed

    Lastauskienė, Eglė; Zinkevičienė, Auksė; Girkontaitė, Irutė; Kaunietis, Arnoldas; Kvedarienė, Violeta

    2014-09-01

    Cutaneous fungal infections are common and widespread. Antifungal agents used for the treatment of these infections often have undesirable side effects. Furthermore, increased resistance of the microorganisms to the antifungal drugs becomes the growing problem. Accordingly, the search for natural antifungal compounds continues to receive attention. Apoptosis is highly regulated programmed cell death. During yeast cell apoptosis, amino acids and peptides are released and can stimulate regeneration of human epithelium cells. Thus, detection of chemical compounds inducing apoptosis in yeast and nontoxic for humans is of great medical relevance. The aim of this study was to detect chemical compound inducing apoptosis in pathogenic Candida species with the lowest toxicity to the mammalian cells. Five chemical compounds--acetic acid, sodium bicarbonate, potassium carbonate, lithium acetate, and formic acid--were tested for evaluation of antifungal activity on C. albicans, C. guilliermondii, and C. lusitaniae. The results showed that acetic acid and formic acid at the lowest concentrations induced yeast cells death. Apoptosis analysis revealed that cells death was accompanied by activation of caspase. Minimal inhibitory concentrations of potassium carbonate and sodium bicarbonate induced Candida cells necrosis. Toxicity test with mammalian cell cultures showed that formic acid has the lowest effect on the growth of Jurkat and NIH 3T3 cells. In conclusion, our results show that a low concentration of formic acid induces apoptosis-like programmed cell death in the Candida yeast and has a minimal effect on the survivability of mammalian cells, suggesting potential applications in the treatment of these infections. PMID:24752490

  18. Molecular difference between WASP and N-WASP critical for chemotaxis of T-cells towards SDF-1α.

    PubMed

    Jain, Neeraj; Thanabalu, Thirumaran

    2015-01-01

    Wiskott-Aldrich Syndrome protein (WASP) integrates cell signaling pathways to the actin cytoskeleton, which play a critical role in T-cell activation and migration. Hematopoietic cells express both WASP and neural-WASP (N-WASP) which share similar domain structure, yet WASP deficiency causes Wiskott-Aldrich syndrome, suggesting that N-WASP present in the cells is not able to carry out all the functions of WASP. We have identified a unique internal thirty amino acid region (I30) in WASP, which regulates its function in chemotaxis of Jurkat T-cells. Deletion of the I30 region altered the WASP's closed conformation and impaired its ability to rescue the chemotactic defect of WASP-deficient (Jurkat(WKD)) T-cells. Expression of N-WASP in Jurkat(WKD) T-cells using WASP promoter restored the migration velocity without correcting the chemotactic defect. However, insertion of I30 region in N-WASP (N-WASP-I30) enabled N-WASP to rescue the chemotactic defect of Jurkat(WKD) T-cells. N-WASP-I30-EGFP displayed a punctate localization in contrast to the predominant nuclear localization of N-WASP-EGFP. Thus, our study has demonstrated that the I30 region of WASP is critical for localization and chemotaxis. This suggests that N-WASP's failure to compensate for WASP in rescuing chemotaxis could be due to the absence of this I30 region.

  19. Ethanol cytotoxic effect on trophoblast cells.

    PubMed

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

    2014-03-01

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

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

    PubMed

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

    2013-02-01

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

  1. Canthin-6-one displays antiproliferative activity and causes accumulation of cancer cells in the G2/M phase.

    PubMed

    Dejos, Camille; Voisin, Pierre; Bernard, Marianne; Régnacq, Matthieu; Bergès, Thierry

    2014-11-26

    Canthinones are natural substances with a wide range of biological activities, including antipyretic, antiparasitic, and antimicrobial. Antiproliferative and/or cytotoxic effects of canthinones on cancer cells have also been described, although their mechanism of action remains ill defined. To gain better insight into this mechanism, the antiproliferative effect of a commercially available canthin-6-one (1) was examined dose-dependently on six cancer cell lines (human prostate, PC-3; human colon, HT-29; human lymphocyte, Jurkat; human cervix, HeLa; rat glioma, C6; and mouse embryonic fibroblasts, NIH-3T3). Cytotoxic effects of 1 were investigated on the same cancer cell lines by procaspase-3 cleavage and on normal human skin fibroblasts. Strong antiproliferative effects of the compound were observed in all cell lines, whereas cytotoxic effects were very dependent on cell type. A better definition of the mechanism of action of 1 was obtained on PC-3 cells, by showing that it decreases BrdU incorporation into DNA by 60% to 80% and mitotic spindle formation by 70% and that it causes a 2-fold accumulation of cells in the G2/M phase of the cell cycle. Together, the data suggest that the primary effect of canthin-6-one (1) is antiproliferative, possibly by interfering with the G2/M transition. Proapoptotic effects might result from this disturbance of the cell cycle. PMID:25379743

  2. PTEN expression in PTEN-null leukaemic T cell lines leads to reduced proliferation via slowed cell cycle progression.

    PubMed

    Seminario, Maria-Cristina; Precht, Patricia; Wersto, Robert P; Gorospe, Myriam; Wange, Ronald L

    2003-11-01

    The balance of activities between the proto-oncogene phosphoinositide 3-kinase (PI3K) and the tumour suppressor gene PTEN has been shown to affect cellular growth and proliferation, as well as tumorigenesis. Previously, PTEN expression in the PTEN-null Jurkat T cell leukaemia line was shown to cause reduced proliferation without cell cycle arrest. Here, we further these investigations by determining the basis for this phenomenon. By BrdU pulse-chase and cell cycle arrest and release assays, we find that PTEN expression reduced proliferation by slowing progression through all phases of the cell cycle. This was associated with reduced levels of cyclins A, B1 and B2, cdk4, and cdc25A and increased p27KIP1 expression. Apoptosis played no role in the antiproliferative effect of PTEN, since only marginal increases in the rate of apoptosis were detected upon PTEN expression, and inhibitors of effector caspases did not restore proliferative capacity. Active Akt blocked the antiproliferative effects of PTEN, indicating that PTEN mediates its effects through conventional PI3K-linked signalling pathways. Similar results were obtained from a different PTEN-null leukaemia T cell line, CEM. Together, these results show that PTEN expression in leukaemic T cells leads to reduced proliferation via an apoptosis-independent mechanism involving slower passage through the cell cycle.

  3. RACK-1 overexpression protects against goniothalamin-induced cell death

    PubMed Central

    Inayat-Hussain, S.H.; Wong, L.T.; Chan, K.M.; Rajab, N.F.; Din, L.B.; Harun, R.; Kizilors, A.; Saxena, N.; Mourtada-Maarabouni, M.; Farzaneh, F.; Williams, G.T.

    2009-01-01

    Goniothalamin, a styryllactone, has been shown to induce cytotoxicity via apoptosis in several tumor cell lines. In this study, we have examined the potential role of several genes, which were stably transfected into T-cell lines and which regulate apoptosis in different ways, on goniothalamin-induced cell death. Overexpression of full-length receptor for activated protein C-kinase 1 (RACK-1) and pc3n3, which up-regulates endogenous RACK-1, in both Jurkat and W7.2 T cells resulted in inhibition of goniothalamin-induced cell death as assessed by MTT and clonogenic assays. However, overexpression of rFau (antisense sequence to Finkel–Biskis–Reilly murine sarcoma virus-associated ubiquitously expressed gene) in W7.2 cells did not confer resistance to goniothalamin-induced cell death. Etoposide, a clinically used cytotoxic agent, was equipotent in causing cytotoxicity in all the stable transfectants. Assessment of DNA damage by Comet assay revealed goniothalamin-induced DNA strand breaks as early as 1 h in vector control but this effect was inhibited in RACK-1 and pc3n3 stably transfected W7.2 cells. This data demonstrate that RACK-1 plays a crucial role in regulating cell death signalling pathways induced by goniothalamin. PMID:19698770

  4. ERYTHROPOIETIN EFFECTS ON FETAL MOUSE ERYTHROID CELLS

    PubMed Central

    Chui, David H. K.; Djaldetti, Meir; Marks, Paul A.; Rifkind, Richard A.

    1971-01-01

    The effect of the hormone, erythropoietin, on cultures of erythroblasts derived from the livers of fetal C57BL/6J mice was examined. An increase both in the content and in the rate of synthesis of normal adult mouse globin chains was detected in hormone-treated cultures. The rate of protein synthesis by individual erythroblasts does not increase in response to the hormone, whereas the absolute number of hemoglobin-synthesizing cells does increase and accounts for the observed stimulation of hemoglobin synthesis. The principal effect of erythropoietin appears to be upon the population of immature erythroid precursor cells which persists in the presence of the hormone, the cells maintaining their ability to replicate, and their capacity to differentiate into hemoglobinizing erythroblasts. In the absence of hormone, already committed erythroblasts continue their development, but erythropoiesis is not sustained. PMID:5128349

  5. Radiation effects on the cell-cell communication of mammalian cells

    NASA Astrophysics Data System (ADS)

    Depriest, Kendall Russell

    Recent observations of bystander effects in unirradiated cell populations have focused attention on cell-cell communication, particularly gap junction intercellular communication (GJIC), as a means through which the bystander effect may be transmitted. The bystander expression of CDKN1A in unirradiated AG1522 human fibroblast cells observed in another laboratory was verified. The dose response of the bystander effect in the AG1522 cells showed that the effect had reached its maximum at the lowest alpha-particle fluence tested, 0.013 alpha/nuclei. To test potential mechanisms for communication to bystander cells, the fluorescence recovery after photobleaching technique was used. Only the rat liver epithelial cell line (Clone 9) exhibited GJIC based upon a fluorescence recovery after photobleaching assay, and there was no change in the rate constant for GJIC following exposure to low LET or high LET radiation. The fibroblast cell lines (AG1521, AG1522, and GM5758) showed no evidence of GJIC in three separate assays including immunohistochemistry. Lindane, an inhibitor of GJIC, eliminated the bystander expression of CDKN1A in AG1522 cells while octanol, another inhibitor of GJIC, did not change the bystander expression of the protein. The two chemicals act in different ways to disrupt GJIC and each one may alter other functions as well, so the elimination of the bystander effect by lindane apparently indicates that lindane is interfering with a bystander signaling mechanism that is not mediated by gap junctions. The lack of connexin localization in the cell membrane of the fibroblast cell lines and the elimination of the bystander expression by lindane, but not octanol, indicates that the bystander effect must be mediated by a non-GJIC mechanism. The experimental evidence suggests that the mediator of the bystander expression of CDKN1A in human diploid fibroblasts is most likely an extracellular signal, such as a cytokine, that acts in a calcium-dependent signal

  6. Productive persistent infection of hematopoietic cells by human foamy virus.

    PubMed Central

    Yu, S F; Stone, J; Linial, M L

    1996-01-01

    Human foamy virus can establish persistent infections in human hematopoietic cell lines, such as H92.1.7 (erythroblastoid cells), Jurkat (CD4+ T cells), and U937 (myeloid-monocytic cells). The infection is characterized by constant production of infectious viruses (for > 2 1/2 years) with no cytopathic effects on the host cells. Electron microscopy of the infected cells showed a viral morphology similar to that observed for particles produced after acute infection. We have detected, in addition to the full-length form of bel1, a previously described deletion in the bel1 gene of the proviral DNA in these cells. RNA containing this 301-bp deletion, which mapped to the splice donor and acceptor sites of the intron of the bet gene, was also found in encapsidated virion RNA. However, the presence of this defective provirus harboring the deletion in bel1 does not prevent productive persistence in these chronically infected cells, since the virus titer does not decrease during cultivation. PMID:8551590

  7. Method for the production of human T-T cell hybrids and production suppressor factor by human T-T cell hybrids

    SciTech Connect

    Platsoucas, C.

    1989-06-27

    This patent describes a method for production of human T-T cell hybrids which produce Suppressor Factor wherein cells of lymphoid origin are fused with comprises: (a) mixing cells from a first parent cell line comprising a non-mutagenized Jurkat lymoblastoid T cell line, wherein the Jurkat lymphoblastoid cells are not sensitive and cannot be killed by hypoxanthine-aminopterin-thymidine medium, with a second parent cell comprising mitogen or alloantigen activated peripheral blood leukocyte T cells or purified T-cells, (b) allowing the first and second parent cells to fuse in the presence of polyethylene glycol for about 10-20 minutes with gentle agitation to generate hybrids in the cell mixture, (c) incubating the cell mixture containing the hybrids and the first and second parent cells, after removal of the polyethylene glycol, for periods of between one to sixty days at 37{sup 0} in 5% CO/sub 2/, (d) selecting for the hybrids by separating the hybrids from the first parent Jurkat lymphoblastoid cells by coloning in agar medium wherein the hybrids form colonies, (e) recovering the hybrids that form colonies in agar medium and expanding them in culture, and (f) determining the presence of Suppressor Factor in the culture and recovering the T-T cell hybrids which produce suppressor factor.

  8. Celecoxib prevents curcumin-induced apoptosis in a hematopoietic cancer cell model.

    PubMed

    Sobolewski, Cyril; Muller, Florian; Cerella, Claudia; Dicato, Mario; Diederich, Marc

    2015-10-01

    Molecules targeting pro-inflammatory pathways have demonstrated beneficial effects in cancer treatment. More recently, combination of natural and synthetic anti-inflammatory drugs was suggested as an appealing strategy to inhibit tumor growth. Herein, we show that curcumin, a polyphenol from Curcuma longa and celecoxib induce apoptosis in hematopoietic cancer cell lines (Hel, Jurkat, K562, Raji, and U937). Further investigations on the most sensitive cell line, U937, indicated that these effects were tightly associated with an accumulation of the cells in S and G2/M for curcumin and in G0/G1 phase of cell cycle for celecoxib, respectively. The effect of celecoxib on cell cycle is associated with an induction of p27 and the down-regulation of cyclin D1. However, in the case of combination experiments, the pretreatment of U937 cells with celecoxib at non-apoptogenic concentrations counteracted curcumin-induced apoptosis. We found that this effect correlated with the prevention of the accumulation in S and G2/M phase of cell cycle induced by curcumin. Similar results have been obtained when celecoxib and curcumin were co-administrated at the same time. Overall our data suggest that this natural and synthetic drug combination is detrimental for cell death induction.

  9. Bismuth(III) complexes with 2-acetylpyridine- and 2-benzoylpyridine-derived hydrazones: Antimicrobial and cytotoxic activities and effects on the clonogenic survival of human solid tumor cells.

    PubMed

    Ferreira, Isabella P; Piló, Elisa D L; Recio-Despaigne, Angel A; Da Silva, Jeferson G; Ramos, Jonas P; Marques, Lucas B; Prazeres, Pedro H D M; Takahashi, Jacqueline A; Souza-Fagundes, Elaine M; Rocha, Willian; Beraldo, Heloisa

    2016-07-01

    Complexes [Bi(2AcPh)Cl2]·0.5H2O (1), [Bi(2AcpClPh)Cl2] (2), [Bi(2AcpNO2Ph)Cl2] (3), [Bi(2AcpOHPh)Cl2]·2H2O (4), [Bi(H2BzPh)Cl3]·2H2O (5), [Bi(H2BzpClPh)Cl3] (6), [Bi(2BzpNO2Ph)Cl2]·2H2O (7) and [Bi(H2BzpOHPh)Cl3]·2H2O (8) were obtained with 2-acetylpyridine phenylhydrazone (H2AcPh), its -para-chloro-phenyl- (H2AcpClPh), -para-nitro-phenyl (H2AcpNO2Ph) and -para-hydroxy-phenyl (H2AcpOHPh) derivatives, as well as with the 2-benzoylpyridine phenylhydrazone analogues (H2BzPh, H2BzpClPh, H2BzpNO2Ph, H2BzpOHPh). Upon coordination to bismuth(III) antibacterial activity against Gram-positive and Gram-negative bacterial strains significantly improved except for complex (4). The cytotoxic effects of the compounds under study were evaluated on HL-60, Jurkat and THP-1 leukemia, and on MCF-7 and HCT-116 solid tumor cells, as well as on non-malignant Vero cells. In general, 2-acetylpyridine-derived hydrazones proved to be more potent and more selective as cytotoxic agents than the corresponding 2-benzoylpyridine-derived counterparts. Exposure of HCT-116 cells to H2AcpClPh, H2AcpNO2Ph and complex (3) led to 99% decrease of the clonogenic survival. The IC50 values of these compounds were three-fold smaller when cells were cultured in soft-agar (3D) than when cells were cultured in monolayer (2D), suggesting that they constitute interesting scaffolds, which should be considered in further studies aiming to develop new drug candidates for the treatment of colon cancer. PMID:27209169

  10. Effect of Carbon Nanotubes on Mammalian Cells

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  11. High electrical field effects on cell membranes.

    PubMed

    Pliquett, U; Joshi, R P; Sridhara, V; Schoenbach, K H

    2007-05-01

    Electrical charging of lipid membranes causes electroporation with sharp membrane conductance increases. Several recent observations, especially at very high field strength, are not compatible with the simple electroporation picture. Here we present several relevant experiments on cell electrical responses to very high external voltages. We hypothesize that, not only are aqueous pores created within the lipid membranes, but that nanoscale membrane fragmentation occurs, possibly with micelle formation. This effect would produce conductivity increases beyond simple electroporation and display a relatively fast turn-off with external voltage. In addition, material loss can be expected at the anode side of cells, in agreement with published experimental reports at high fields. Our hypothesis is qualitatively supported by molecular dynamics simulations. Finally, such cellular responses might temporarily inactivate voltage-gated and ion-pump activity, while not necessarily causing cell death. This hypothesis also supports observations on electrofusion.

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

    PubMed Central

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

    2016-01-01

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

  13. Nanosecond pulsed electric fields modulate cell function through intracellular signal transduction mechanisms.

    PubMed

    Beebe, Stephen J; Blackmore, Peter F; White, Jody; Joshi, Ravindra P; Schoenbach, Karl H

    2004-08-01

    These studies describe the effects of nanosecond (10-300 ns) pulsed electric fields (nsPEF) on mammalian cell structure and function. As the pulse durations decrease, effects on the plasma membrane (PM) decrease and effects on intracellular signal transduction mechanisms increase. When nsPEF-induced PM electroporation effects occur, they are distinct from classical PM electroporation effects, suggesting unique, nsPEF-induced PM modulations. In HL-60 cells, nsPEF that are well below the threshold for PM electroporation and apoptosis induction induce effects that are similar to purinergic agonistmediated calcium release from intracellular stores, which secondarily initiate capacitive calcium influx through store-operated calcium channels in the PM. NsPEF with durations and electric field intensities that do or do not cause PM electroporation, induce apoptosis in mammalian cells with a well-characterized phenotype typified by externalization of phosphatidylserine on the outer PM and activation of caspase proteases. Treatment of mouse fibrosarcoma tumors with nsPEF also results in apoptosis induction. When Jurkat cells were transfected by electroporation and then treated with nsPEF, green fluorescent protein expression was enhanced compared to electroporation alone. The results indicate that nsPEF activate intracellular mechanisms that can determine cell function and fate, providing an important new tool for probing signal transduction mechanisms that modulate cell structure and function and for potential therapeutic applications for cancer and gene therapy.

  14. [Cytotoxicity of polyphenolic/flavonoid compounds in a leukaemia cell culture].

    PubMed

    Josipović, Pavle; Orsolić, Nada

    2008-12-01

    Flavonoid components of propolis are biologically active substances with antioxidative, immunostimulative, immunomodulative, and anti-inflamatory properties. The aim of the study was to investigate their cytotoxic effect on different leukaemia cell lines. For this purpose we used five different flavonoids (quercetin, caffeic acid, chrysin, naringenin, and naringin) and five types of leukemia cell lines (MOLT, JURKAT, HL-60, RAJI and U937). Cells were cultured at 37 degrees C in the RPMI-1640 medium supplemented with 10% FCS in humified atmosphere with 5% of CO2. Flavonoids were added in the following concentrations: 100 microg mL(-1), 50 microg mL(-1), 25 microg mL(-1), or 12.5 microg mL(-1). The results show different dose- and cell-type-dependent cytotoxicity. Among the flavonoids, quercetin showed the strongest cytotoxic effect in all cell lines. Caffeic acid and chrisyn also expressed a high level of cytotoxicty. Treatment of U937 and HL-60 cell lines with low concentrations of chrisyn or naringenin stimulated cell proliferation. These results suggest a biphase effect of the tested compounds on monocyte cell lines. Cytotoxicity and growth stimulation mechanisms caused directly by flavonoids should further be investigated on the molecular level.

  15. Biological effects in hit and non-hit cells

    NASA Astrophysics Data System (ADS)

    Hall, E. J.; Hei, T. K.; Geard, C. R.; Brenner, D. J.; Mitchell, S. A.

    It had long been considered axiomatic that heritable biological effects of radiation required direct damage to DNA. This is no longer the case. The bystander effect refers to the induction of biological effects in cells that are not directly traversed by a charged particle, but are close to cells that are. Experiments suggest that the effect is due to a molecule secreted by irradiated cells which is capable of transferring damage to distant cells. The magnitude of the effect is much larger if cells are in gap junction communication. In cell cultures, a bystander effect has been shown for cell lethality, chromosomal aberrations, mutation, oncogenic transformation and upregulation of gene expression. A similar effect has been observed in artificial 3-dimensional skin cultures. Bystander studies imply that the target for the biological effects of radiation is larger than the cell, and this has implications for biologically based models of carcinogenesis at low doses where not all cells receive a direct hit.

  16. Cellular Stress Induced by Resazurin Leads to Autophagy and Cell Death Via Production of Reactive Oxygen Species and Mitochondrial Impairment

    PubMed Central

    Erikstein, Bjarte Skoe; Hagland, Hanne Røland; Nikolaisen, Julie; Kulawiec, Mariola; Singh, Keshav K.; Gjertsen, Bjørn Tore; Tronstad, Karl Johan

    2010-01-01

    Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin-induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL-60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL-60 cells which also were more sensitive and contained autophagic vesicles. Studies in Rho0 cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti-proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance. PMID:20568117

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

    PubMed Central

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

    2016-01-01

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

  18. Cytotoxic Effects of Loperamide Hydrochloride on Canine Cancer Cells

    PubMed Central

    REGAN, Rebecca Cohen; GOGAL, Robert Michael; BARBER, James Perry; TUCKFIELD, Richard Cary; HOWERTH, Elizabeth Wynne; LAWRENCE, Jessica Ann

    2014-01-01

    Loperamide is a peripheral opiate agonist that can cause apoptosis and G2/M arrest in human cancer cell lines and may sensitize cells to chemotherapy. The objectives of this study were to investigate the effects of loperamide on viability, apoptosis and cell cycle kinetics in canine cancer cells and to establish whether the drug sensitizes cells to doxorubicin. Cell viability was assessed using Alamar Blue. Cell death and cell cycle were studied using flow cytometry with 7-Aminoactinomycin-D (7-AAD) and propidium iodide (PI), respectively. Loperamide decreased cell viability in a dose-dependent fashion and was most effective against canine osteosarcoma cells. In all cell lines, it induced a dose and time dependent apoptosis and resulted in accumulation in G0/G1. When co-incubated with doxorubicin, loperamide induced a synergistic cell kill in canine carcinoma cells. Investigation is warranted into the role of loperamide in the treatment of canine cancer. PMID:25649936

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-10

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

  1. Hypertonic stress regulates T cell function via pannexin-1 hemichannels and P2X receptors

    PubMed Central

    Woehrle, Tobias; Yip, Linda; Manohar, Monali; Sumi, Yuka; Yao, Yongli; Chen, Yu; Junger, Wolfgang G.

    2010-01-01

    Hypertonic saline (HS) resuscitation increases T cell function and inhibits posttraumatic T cell anergy, which can reduce immunosuppression and sepsis in trauma patients. We have previously shown that HS induces the release of cellular ATP and enhances T cell function. However, the mechanism by which HS induces ATP release and the subsequent regulation of T cell function by ATP remain poorly understood. In the present study, we show that inhibition of the gap junction hemichannel pannexin-1 (Panx1) blocks ATP release in response to HS, and HS exposure triggers significant changes in the expression of all P2X-type ATP receptors in Jurkat T cells. Blocking or silencing of Panx1 or of P2X1, P2X4, or P2X7 receptors blunts HS-induced p38 MAPK activation and the stimulatory effects of HS on TCR/CD28-induced IL-2 gene transcription. Moreover, treatment with HS or agonists of P2X receptors overcomes T cell suppression induced by the anti-inflammatory cytokine IL-10. These findings indicate that Panx1 hemichannels facilitate ATP release in response to hypertonic stress and that P2X1, P2X4, and P2X7 receptor activation enhances T cell function. We conclude that HS and P2 receptor agonists promote T cell function and thus, could be used to improve T cell function in trauma patients. PMID:20884646

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    van Gijsel-Bonnello, Manuel; Acar, Niyazi; Molino, Yves; Bretillon, Lionel; Khrestchatisky, Michel; de Reggi, Max; Gharib, Bouchra

    2015-10-01

    Pantethine, a natural low-molecular-weight thiol, shows a broad activity in a large range of essential cellular pathways. It has been long known as a hypolipidemic and hypocholesterolemic agent. We have recently shown that it exerts a neuroprotective action in mouse models of cerebral malaria and Parkinson's disease through multiple mechanisms. In the present study, we looked at its effects on membrane lipid rafts that serve as platforms for molecules engaged in cell activity, therefore providing a target against inappropriate cell response leading to a chronic inflammation. We found that pantethine-treated cells showed a significant change in raft fatty acid composition and cholesterol content, with ultimate downregulation of cell adhesion, CXCL12-driven chemotaxis, and transendothelial migration of various T cell types, including human Jurkat cell line and circulating effector T cells. The mechanisms involved include the alteration of the following: (i) CXCL12 binding to its target cells; (ii) membrane dynamics of CXCR4 and CXCR7, the two CXCL12 receptors; and (iii) cell redox status, a crucial determinant in the regulation of the chemokine system. In addition, we considered the linker for activation of T cells molecule to show that pantethine effects were associated with the displacement from the rafts of the acylated signaling molecules which had their palmitoylation level reduced.. In conclusion, the results presented here, together with previously published findings, indicate that due to its pleiotropic action, pantethine can downregulate the multifaceted process leading to pathogenic T cell activation and migration.

  4. Variable responses of different human cancer cells to the lichen compounds parietin, atranorin, usnic acid and gyrophoric acid.

    PubMed

    Bačkorová, M; Bačkor, M; Mikeš, J; Jendželovský, R; Fedoročko, P

    2011-02-01

    One of the ways for searching for potentially new anti-cancer drugs is the testing of various naturally synthesized compounds. Lichens are a source of unique chemical agents of which some have already been proved to be effective against various cancer in vitro models. Our study reports on the sensitivity of up to nine human cancer cell lines (A2780, HeLa, MCF-7, SK-BR-3, HT-29, HCT-116 p53(+/+), HCT-116 p53(-/-), HL-60 and Jurkat) to the anti-proliferative/cytotoxic effects of four typical secondary metabolites of lichens (parietin, atranorin, usnic acid and gyrophoric acid). Variations in the dynamics of tumour cell line populations were evaluated by the MTT, clonogenic and viability assays, cell proliferation and detachment, cell cycle transition and apoptotic nuclear morphology, thereby confirming their concentration- and time-dependent cytotoxicity. However, in comparison with parietin and gyrophoric acid, the suppression of viability and cell proliferation by usnic acid or atranorin was found to be more efficient at equitoxic doses and correlated more strongly with an increased number of floating cells or a higher apoptotic index. Moreover, the analysis of cell cycle distribution also revealed an accumulation of cells in S-phase. This study has confirmed a differential sensitivity of cancer cell lines to lichen secondary metabolites. PMID:20837130

  5. Label-free and noninvasive optical detection of the distribution of nanometer-size mitochondria in single cells

    NASA Astrophysics Data System (ADS)

    Su, Xuantao; Qiu, Yuanyuan; Marquez-Curtis, Leah; Gupta, Manisha; Capjack, Clarence E.; Rozmus, Wojciech; Janowska-Wieczorek, Anna; Tsui, Ying Y.

    2011-06-01

    A microfluidic flow cytometric technique capable of obtaining information on nanometer-sized organelles in single cells in a label-free, noninvasive optical manner was developed. Experimental two-dimensional (2D) light scattering patterns from malignant lymphoid cells (Jurkat cell line) and normal hematopoietic stem cells (cord blood CD34+ cells) were compared with those obtained from finite-difference time-domain simulations. In the simulations, we assumed that the mitochondria were randomly distributed throughout a Jurkat cell, and aggregated in a CD34+ cell. Comparison of the experimental and simulated light scattering patterns led us to conclude that distinction from these two types of cells may be due to different mitochondrial distributions. This observation was confirmed by conventional confocal fluorescence microscopy. A method for potential cell discrimination was developed based on analysis of the 2D light scattering patterns. Potential clinical applications using mitochondria as intrinsic biological markers in single cells were discussed in terms of normal cells (CD34+ cell and lymphocytes) versus malignant cells (THP-1 and Jurkat cell lines).

  6. Cell Biology of Thiazide Bone Effects

    NASA Astrophysics Data System (ADS)

    Gamba, Gerardo; Riccardi, Daniela

    2008-09-01

    The thiazide-sensitive Na+:Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian kidney. The activity of NCC is not only related to salt metabolism, but also to calcium and magnesium homeostasis due to the inverse relationship between NCC activity and calcium reabsorption. Hence, the thiazide-type diuretics that specifically block NCC have been used for years, not only for treatment of hypertension and edematous disease, but also for the management of renal stone disease. Epidemiological studies have shown that chronic thiazide treatment is associated with higher bone mineral density and reduced risk of bone fractures, which can only partly be explained in terms of their effects on the kidney. In this regard, we have recently shown that NCC is expressed in bone cells and that inhibition of NCC in bone, either by thiazides or by reduction of NCC protein with specific siRNA, is associated with increased mineralization in vitro. These observations open a field of study to begin to understand the cell biology of the beneficial effects of thiazides in bone.

  7. Calreticulin Binds to Fas Ligand and Inhibits Neuronal Cell Apoptosis Induced by Ischemia-Reperfusion Injury

    PubMed Central

    Chen, Beilei; Wu, Zhengzheng; Xu, Jun; Xu, Yun

    2015-01-01

    Background. Calreticulin (CRT) can bind to Fas ligand (FasL) and inhibit Fas/FasL-mediated apoptosis of Jurkat T cells. However, its effect on neuronal cell apoptosis has not been investigated. Purpose. We aimed to evaluate the neuroprotective effect of CRT following ischemia-reperfusion injury (IRI). Methods. Mice underwent middle cerebral artery occlusion (MCAO) and SH-SY5Y cells subjected to oxygen glucose deprivation (OGD) were used as models for IRI. The CRT protein level was detected by Western blotting, and mRNA expression of CRT, caspase-3, and caspase-8 was measured by real-time PCR. Immunofluorescence was used to assess the localization of CRT and FasL. The interaction of CRT with FasL was verified by coimmunoprecipitation. SH-SY5Y cell viability was determined by MTT assay, and cell apoptosis was assessed by flow cytometry. The measurement of caspase-8 and caspase-3 activity was carried out using caspase activity assay kits. Results. After IRI, CRT was upregulated on the neuron surface and bound to FasL, leading to increased viability of OGD-exposed SH-SY5Y cells and decreased activity of caspase-8 and caspase-3. Conclusions. This study for the first time revealed that increased CRT inhibited Fas/FasL-mediated neuronal cell apoptosis during the early stage of ischemic stroke, suggesting it to be a potential protector activated soon after IRI. PMID:26583143

  8. Cell separation by an aqueous two-phase system in a microfluidic device.

    PubMed

    Tsukamoto, Masatoshi; Taira, Shu; Yamamura, Shohei; Morita, Yasutaka; Nagatani, Naoki; Takamura, Yuzuru; Tamiya, Eiichi

    2009-10-01

    We generated an aqueous two-phase laminar flow in a microfluidic chip and used the system to isolate leukocyte and erythrocyte cells from whole blood cells. The microfluidic system reduced the effect of gravity in the aqueous two-phase system (ATPS). Poly(ethylene glycol) (PEG) and dextran (Dex) solutions were used as the two phases, and the independent flow rates of the solutions were both 2 microL/min. When hydrophobic and hydrophilic polystyrene beads were introduced into the microfluidic device, the hydrophilic beads moved to the Dex layer and the hydrophobic beads to the interface between the two phases. In the case of living cells, Jurkat cells and erythrocytes moved more efficiently to the PEG and Dex layers, respectively, than they move in a conventional ATPS. When whole blood cells were inserted into the microfluidic chip, leukocytes could be separated from erythrocytes because erythrocytes moved to the Dex layer while leukocytes remained outside of this layer in the microfluidic system. The reported microfluidic chip for the whole blood cell separation can effectively be integrated into a Micro Total Analysis System designed for cell-based clinical, forensic, and environmental analyses.

  9. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    SciTech Connect

    Cowan, Robert W.; Ghert, Michelle; Singh, Gurmit

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These

  10. Cytotoxic effects of four antibiotics on endothelial cells.

    PubMed

    Lanbeck, P; Paulsen, O

    1995-12-01

    Intravenous administration of antibiotics often causes local pain and thrombophlebitis at the site of injection. An in vitro model that could predict these effects would be of great value. In this study the effects of four antibiotics, benzylpenicillin, cefuroxime, dicloxacillin and erythromycin, have been evaluated on three types of endothelial cells in culture. The cell types employed were primary culture from human umbilical vein, primary culture from bovine aorta, and the cell line EA-hy 926, a hybride endothelial cell. These cells were exposed to antibiotics for 24 hr and subsequently toxic effects on cells were evaluated by three different assays. Benzylpenicillin was atoxic in all types of cells and in all assays, in contrast to the other antibiotics. The other three antibiotics exerted dose dependent toxic effects in all investigated cells when DNA-synthesis and total cell protein were used as toxicity assays but the results varied between the cell types. There were no significant differences between the effects of cefuroxime, dicloxacillin and erythromycin on bovine endothelial cells. In the other cell types, however, there were significant differences between some drugs but the outcome depended on the cell type. It is concluded that it is possible to show differences between the effect of antibiotics on endothelial cells, but the result depends on the cell type employed.

  11. A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death

    PubMed Central

    Furness, SGB; Hare, DL; Kourakis, A; Turnley, AM; Wookey, PJ

    2016-01-01

    We have discovered that the accumulation of an anti-calcitonin receptor (anti-CTR) antibody conjugated to a fluorophore (mAb2C4:AF568) provides a robust signal for cells undergoing apoptotic programmed cell death (PCD). PCD is an absolute requirement for normal development of metazoan organisms. PCD is a hallmark of common diseases such as cardiovascular disease and tissue rejection in graft versus host pathologies, and chemotherapeutics work by increasing PCD. This robust signal or high fluorescent events were verified by confocal microscopy and flow cytometry in several cell lines and a primary culture in which PCD had been induced. In Jurkat cells, GBM-L2 and MG63 cells, the percentage undergoing PCD that were positive for both mAb2C4:AF568 and annexin V ranged between 70 and >90%. In MG63 cells induced for the preapoptotic cell stress response (PACSR), the normal expression of α-tubulin, a key structural component of the cytoskeleton, and accumulation of mAb2C4:AF568 were mutually exclusive. Our data support a model in which CTR is upregulated during PACSR and recycles to the plasma membrane with apoptosis. In cells committed to apoptosis (α-tubulin negative), there is accumulation of the CTR-ligand mAb2C4:AF568 generating a high fluorescent event. The reagent mAb2C4:AF568 effectively identifies a novel event linked to apoptosis. PMID:27777788

  12. Restoration of WNT4 inhibits cell growth in leukemia-derived cell lines

    PubMed Central

    2013-01-01

    Background WNT signaling pathways are significantly altered during cancer development. Vertebrates possess two classes of WNT signaling pathways: the “canonical” WNT/β-catenin signaling pathway, and the “non-canonical” pathways including WNT/Ca2+ and WNT/Planar cell polarity [PCP] signaling. WNT4 influences hematopoietic progenitor cell expansion and survival; however, WNT4 function in cancer development and the resulting implications for oncogenesis are poorly understood. The aim of this study was twofold: first, to determine the expression of WNT4 in mature peripheral blood cells and diverse leukemia-derived cells including cell lines from hematopoietic neoplasms and cells from patients with leukemia; second, to identify the effect of this ligand on the proliferation and apoptosis of the blast-derived cell lines BJAB, Jurkat, CEM, K562, and HL60. Methods We determined WNT4 expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) and T- and B-lymphocytes from healthy individuals, as well as from five leukemia-derived cell lines and blasts derived from patients with leukemia. To analyze the effect of WNT4 on cell proliferation, PBMCs and cell lines were exposed to a commercially available WNT4 recombinant human protein. Furthermore, WNT4 expression was restored in BJAB cells using an inducible lentiviral expression system. Cell viability and proliferation were measured by the addition of WST-1 to cell cultures and counting cells; in addition, the progression of the cell cycle and the amount of apoptosis were analyzed in the absence or presence of WNT4. Finally, the expression of WNT-pathway target genes was measured by qRT-PCR. Results WNT4 expression was severely reduced in leukemia-derived cell lines and blasts derived from patients with leukemia. The exposure of cell lines to WNT4 recombinant protein significantly inhibited cell proliferation; inducing WNT4 expression in BJAB

  13. Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

    PubMed

    Ledderose, Carola; Woehrle, Tobias; Ledderose, Stephan; Strasser, Katharina; Seist, Richard; Bao, Yi; Zhang, Jingping; Junger, Wolfgang G

    2016-09-01

    T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca(2+) in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca(2+) signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia.

  14. Cutting off the power: inhibition of leukemia cell growth by pausing basal ATP release and P2X receptor signaling?

    PubMed

    Ledderose, Carola; Woehrle, Tobias; Ledderose, Stephan; Strasser, Katharina; Seist, Richard; Bao, Yi; Zhang, Jingping; Junger, Wolfgang G

    2016-09-01

    T cells respond to antigen stimulation with the rapid release of cellular ATP, which stimulates an autocrine feedback mechanism that regulates calcium influx through P2X receptors. This autocrine purinergic feedback mechanism plays an essential role in the activation of T cells resulting in cell proliferation and clonal expansion. We recently reported that increases in mitochondrial ATP production drive this stimulation-induced purinergic signaling mechanism but that low-level mitochondrial ATP production fuels basal T cell functions required to maintain vigilance of unstimulated T cells. Here we studied whether defects in these purinergic signaling mechanisms are involved in the unwanted proliferation of leukemia T cells. We found that acute leukemia T cells (Jurkat) possess a larger number and more active mitochondria than their healthy counterparts. Jurkat cells have higher intracellular ATP concentrations and generat more extracellular ATP than unstimulated T cells from healthy donors. As a result, increased purinergic signaling through P2X1 and P2X7 receptors elevates baseline levels of cytosolic Ca(2+) in Jurkat cells. We found that pharmacological inhibition of this basal purinergic signaling mechanism decreases mitochondrial activity, Ca(2+) signaling, and cell proliferation. Similar results were seen in the leukemic cell lines THP-1, U-937, and HL-60. Combined treatment with inhibitors of P2X1 or P2X7 receptors and the chemotherapeutic agent 6-mercaptopurine completely blocked Jurkat cell proliferation. Our results demonstrate that increased mitochondrial metabolism promotes autocrine purinergic signaling and uncontrolled proliferation of leukemia cells. These findings suggest that deranged purinergic signaling can result in T cell malignancy and that therapeutic targeting aimed at purinergic signaling is a potential strategy to combat T cell leukemia. PMID:27020575

  15. G2/M Cell Cycle Arrest and Tumor Selective Apoptosis of Acute Leukemia Cells by a Promising Benzophenone Thiosemicarbazone Compound

    PubMed Central

    Cabrera, Maia; Gomez, Natalia; Remes Lenicov, Federico; Echeverría, Emiliana; Shayo, Carina; Moglioni, Albertina; Fernández, Natalia; Davio, Carlos

    2015-01-01

    Anti-mitotic therapies have been considered a hallmark in strategies against abnormally proliferating cells. Focusing on the extensively studied family of thiosemicarbazone (TSC) compounds, we have previously identified 4,4’-dimethoxybenzophenone thiosemicarbazone (T44Bf) as a promising pharmacological compound in a panel of human leukemia cell lines (HL60, U937, KG1a and Jurkat). Present findings indicate that T44Bf-mediated antiproliferative effects are associated with a reversible chronic mitotic arrest caused by defects in chromosome alignment, followed by induced programmed cell death. Furthermore, T44Bf selectively induces apoptosis in leukemia cell lines when compared to normal peripheral blood mononuclear cells. The underlying mechanism of action involves the activation of the mitochondria signaling pathway, with loss of mitochondrial membrane potential and sustained phosphorylation of anti-apoptotic protein Bcl-xL as well as increased Bcl-2 (enhanced phosphorylated fraction) and pro-apoptotic protein Bad levels. In addition, ERK signaling pathway activation was found to be a requisite for T44Bf apoptotic activity. Our findings further describe a novel activity for a benzophenone thiosemicarbazone and propose T44Bf as a promising anti-mitotic prototype to develop chemotherapeutic agents to treat acute leukemia malignancies. PMID:26360247

  16. G2/M Cell Cycle Arrest and Tumor Selective Apoptosis of Acute Leukemia Cells by a Promising Benzophenone Thiosemicarbazone Compound.

    PubMed

    Cabrera, Maia; Gomez, Natalia; Remes Lenicov, Federico; Echeverría, Emiliana; Shayo, Carina; Moglioni, Albertina; Fernández, Natalia; Davio, Carlos

    2015-01-01

    Anti-mitotic therapies have been considered a hallmark in strategies against abnormally proliferating cells. Focusing on the extensively studied family of thiosemicarbazone (TSC) compounds, we have previously identified 4,4'-dimethoxybenzophenone thiosemicarbazone (T44Bf) as a promising pharmacological compound in a panel of human leukemia cell lines (HL60, U937, KG1a and Jurkat). Present findings indicate that T44Bf-mediated antiproliferative effects are associated with a reversible chronic mitotic arrest caused by defects in chromosome alignment, followed by induced programmed cell death. Furthermore, T44Bf selectively induces apoptosis in leukemia cell lines when compared to normal peripheral blood mononuclear cells. The underlying mechanism of action involves the activation of the mitochondria signaling pathway, with loss of mitochondrial membrane potential and sustained phosphorylation of anti-apoptotic protein Bcl-xL as well as increased Bcl-2 (enhanced phosphorylated fraction) and pro-apoptotic protein Bad levels. In addition, ERK signaling pathway activation was found to be a requisite for T44Bf apoptotic activity. Our findings further describe a novel activity for a benzophenone thiosemicarbazone and propose T44Bf as a promising anti-mitotic prototype to develop chemotherapeutic agents to treat acute leukemia malignancies. PMID:26360247

  17. Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines

    PubMed Central

    Montaner, Beatriz; Navarro, Sira; Piqué, Maria; Vilaseca, Marta; Martinell, Marc; Giralt, Ernest; Gil, Joan; Pérez-Tomás, Ricardo

    2000-01-01

    The effects of supernatant from the bacterial strain Serratia marcescens 2170 (CS-2170) on the viability of different haematopoietic cancer cell lines (Jurkat, NSO, HL-60 and Ramos) and nonmalignant cells (NIH-3T3 and MDCK) was studied. We examined whether this cytotoxic effect was due to apoptosis, and we purified the molecule responsible for this effect and determined its chemical structure.Using an MTT assay we showed a rapid (4 h) decrease in the number of viable cells. This cytotoxic effect was due to apoptosis, according to the fragmentation pattern of DNA, Hoechst 33342 staining and FACS analysis of the phosphatidylserine externalization. This apoptosis was blocked by using the caspase inhibitor Z-VAD.fmk, indicating the involvement of caspases.Prodigiosin is a red pigment produced by various bacteria including S. marcescens. Using mutants of S. marcescens (OF, WF and 933) that do not synthesize prodigiosin, we further showed that prodigiosin is involved in this apoptosis. This evidence was corroborated by spectroscopic analysis of prodigiosin isolated from S. marcescens.These results indicate that prodigiosin, an immunosuppressor, induces apoptosis in haematopoietic cancer cells with no marked toxicity in nonmalignant cells, raising the possibility of its therapeutic use as an antineoplastic drug. PMID:11015311

  18. Prodigiosin from the supernatant of Serratia marcescens induces apoptosis in haematopoietic cancer cell lines.

    PubMed

    Montaner, B; Navarro, S; Piqué, M; Vilaseca, M; Martinell, M; Giralt, E; Gil, J; Pérez-Tomás, R

    2000-10-01

    The effects of supernatant from the bacterial strain Serratia marcescens 2170 (CS-2170) on the viability of different haematopoietic cancer cell lines (Jurkat, NSO, HL-60 and Ramos) and nonmalignant cells (NIH-3T3 and MDCK) was studied. We examined whether this cytotoxic effect was due to apoptosis, and we purified the molecule responsible for this effect and determined its chemical structure. Using an MTT assay we showed a rapid (4 h) decrease in the number of viable cells. This cytotoxic effect was due to apoptosis, according to the fragmentation pattern of DNA, Hoechst 33342 staining and FACS analysis of the phosphatidylserine externalization. This apoptosis was blocked by using the caspase inhibitor Z-VAD.fmk, indicating the involvement of caspases. Prodigiosin is a red pigment produced by various bacteria including S. marcescens. Using mutants of S. marcescens (OF, WF and 933) that do not synthesize prodigiosin, we further showed that prodigiosin is involved in this apoptosis. This evidence was corroborated by spectroscopic analysis of prodigiosin isolated from S. marcescens. These results indicate that prodigiosin, an immunosuppressor, induces apoptosis in haematopoietic cancer cells with no marked toxicity in nonmalignant cells, raising the possibility of its therapeutic use as an antineoplastic drug.

  19. Biotinylation of histones in human cells. Effects of cell proliferation.

    PubMed

    Stanley, J S; Griffin, J B; Zempleni, J

    2001-10-01

    An enzymatic mechanism has been proposed by which biotinidase may catalyze biotinylation of histones. Here, human cells were found to covalently bind biotin to histones H1, H2A, H2B, H3, and H4. Cells respond to proliferation with increased biotinylation of histones; biotinylation increases early in the cell cycle and remains increased during the cycle. Notwithstanding the catalytic role of biotinidase in biotinylation of histones, mRNA encoding biotinidase and biotinidase activity did not parallel the increased biotinylation of histones in proliferating cells. Biotinylation of histones might be regulated by enzymes other than biotinidase or by the rate of histone debiotinylation.

  20. New betulinic acid derivatives induce potent and selective antiproliferative activity through cell cycle arrest at the S phase and caspase dependent apoptosis in human cancer cells.

    PubMed

    Santos, Rita C; Salvador, Jorge A R; Cortés, Roldán; Pachón, Gisela; Marín, Silvia; Cascante, Marta

    2011-06-01

    New semisynthetic derivatives of betulinic acid (BA) RS01, RS02 and RS03 with 18-45 times improved cytotoxic activity against HepG2 cells, were tested for their ability to induce apoptosis and cell cycle arrest in HepG2, HeLa and Jurkat cells. All the compounds induced significant increase in the population at the S phase more effectively than BA. RS01, RS02 and RS03 were also found to be potent inducers of apoptosis with RS01 being markedly more potent than BA, suggesting that the introduction of the imidazolyl moiety is crucial for enhancing the induction of apoptosis and the cell cycle arrest. The mechanism of apoptosis induction has been studied in HepG2 cells and found to be mediated by activation of the postmitochondrial caspases-9 and -3 cascade and possibly by mitochondrial amplification loop involving caspase-8. These facts were corroborated by detection of mitochondrial cytochrome c release and DNA fragmentation. Because RS01, RS02 and RS03 exhibited significant improved antitumor activity with respect to BA, they may be promising new agents for the treatment of cancer. In particular, RS01 is the most promising compound with an IC(50) value 45 times lower than BA on HepG2 cells and 61 times lower than the one found for the non-tumoral Chang liver cells.

  1. Study of radiation effects on mammalian cells in vitro

    NASA Technical Reports Server (NTRS)

    Sinclair, W. K.

    1968-01-01

    Radiation effect on single cells and cell populations of Chinese hamster lung tissue is studied in vitro. The rate and position as the cell progresses through the generation cycle shows division delay, changes in some biochemical processes in the cell, chromosomal changes, colony size changes, and loss of reproductive capacity.

  2. A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

    PubMed Central

    Chi, Shen; Weiss, Arthur; Wang, Haopeng

    2016-01-01

    CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens. PMID:27057542

  3. Culture at a Higher Temperature Mildly Inhibits Cancer Cell Growth but Enhances Chemotherapeutic Effects by Inhibiting Cell-Cell Collaboration.

    PubMed

    Zhu, Shengming; Wang, Jiangang; Xie, Bingkun; Luo, Zhiguo; Lin, Xiukun; Liao, D Joshua

    2015-01-01

    Acute febrile infections have historically been used to treat cancer. To explore the underlying mechanism, we studied chronic effects of fever on cancer cell growth and chemotherapeutic efficacy in cell culture. We found that culturing cancer cells at 39°C mildly inhibited cell growth by arresting the cells at the G1 phase of the cell cycle. When cells were seeded in culture dishes at a lower density, e.g. about 1000-2000 cells per 35-mm dish, the growth inhibition was much greater, manifested as many fewer cell colonies in the 39°C dishes, compared with the results at a higher density seeding, e.g. 20,000 cells per dish, suggesting that cell-cell collaboration as the Allee effect in cell culture is inhibited at 39°C. Withdrawal of cells from serum enhanced the G1 arrest at 39°C and, for some cell lines such as A549 lung cancer cells, serum replenishment failed to quickly drive the cells from the G1 into the S and G2-M phases. Therapeutic effects of several chemotherapeutic agents, including clove bud extracts, on several cancer cell lines were more potent at 39°C than at 37°C, especially when the cells were seeded at a low density. For some cell lines and some agents, this enhancement is long-lasting, i.e. continuing after the cessation of the treatment. Collectively these results suggest that hyperthermia may inhibit cancer cell growth by G1 arrest and by inhibition of cell-cell collaboration, and may enhance the efficacy of several chemotherapeutic agents, an effect which may persist beyond the termination of chemotherapy. PMID:26495849

  4. Culture at a Higher Temperature Mildly Inhibits Cancer Cell Growth but Enhances Chemotherapeutic Effects by Inhibiting Cell-Cell Collaboration

    PubMed Central

    Zhu, Shengming; Wang, Jiangang; Xie, Bingkun; Luo, Zhiguo; Lin, Xiukun; Liao, D. Joshua

    2015-01-01

    Acute febrile infections have historically been used to treat cancer. To explore the underlying mechanism, we studied chronic effects of fever on cancer cell growth and chemotherapeutic efficacy in cell culture. We found that culturing cancer cells at 39°C mildly inhibited cell growth by arresting the cells at the G1 phase of the cell cycle. When cells were seeded in culture dishes at a lower density, e.g. about 1000–2000 cells per 35-mm dish, the growth inhibition was much greater, manifested as many fewer cell colonies in the 39°C dishes, compared with the results at a higher density seeding, e.g. 20,000 cells per dish, suggesting that cell-cell collaboration as the Allee effect in cell culture is inhibited at 39°C. Withdrawal of cells from serum enhanced the G1 arrest at 39°C and, for some cell lines such as A549 lung cancer cells, serum replenishment failed to quickly drive the cells from the G1 into the S and G2-M phases. Therapeutic effects of several chemotherapeutic agents, including clove bud extracts, on several cancer cell lines were more potent at 39°C than at 37°C, especially when the cells were seeded at a low density. For some cell lines and some agents, this enhancement is long-lasting, i.e. continuing after the cessation of the treatment. Collectively these results suggest that hyperthermia may inhibit cancer cell growth by G1 arrest and by inhibition of cell-cell collaboration, and may enhance the efficacy of several chemotherapeutic agents, an effect which may persist beyond the termination of chemotherapy. PMID:26495849

  5. The role of TRPV1 in the CD4+ T cell-mediated inflammatory response of allergic rhinitis.

    PubMed

    Samivel, Ramachandran; Kim, Dae Woo; Son, Hye Ran; Rhee, Yun-Hee; Kim, Eun Hee; Kim, Ji Hye; Bae, Jun-Sang; Chung, Young-Jun; Chung, Phil-Sang; Raz, Eyal; Mo, Ji-Hun

    2016-01-01

    Transient receptor potential vanilloid 1 (TRPV1), which has been identified as a molecular target for the activation of sensory neurons by various painful stimuli, was reported to regulate the signaling and activation of CD4+ T cells. However, the role of TRPV1 in CD4+ T cell in allergic rhinitis remains poorly understood. In this study, TRPV1 expression was localized in CD4+ T cells. Both knockout and chemical inhibition of TRPV1 suppressed Th2/Th17 cytokine production in CD4 T cells and Jurkat T cells, respectively, and can suppress T cell receptor signaling pathways including NF-κB, MAP kinase, and NFAT. In TRPV1 knockout allergic rhinitis (AR) mice, eosinophil infiltration, Th2/Th17 cytokines in the nasal mucosa, and total and ova-specific IgE levels in serum decreased, compared with wild-type AR mice. The TRPV1 antagonists, BCTC or theobromine, showed similar inhibitory immunologic effects on AR mice models. In addition, the number of TRPV1+/CD4+ inflammatory cells increased in the nasal mucosa of patients with AR, compared with that of control subjects. Thus, TRPV1 activation on CD4+ T cells is involved in T cell receptor signaling, and it could be a novel therapeutic target in AR.

  6. The role of TRPV1 in the CD4+ T cell-mediated inflammatory response of allergic rhinitis

    PubMed Central

    Son, Hye Ran; Rhee, Yun-Hee; Kim, Eun Hee; Kim, Ji Hye; Bae, Jun-Sang; Chung, Young-Jun; Chung, Phil-Sang; Raz, Eyal; Mo, Ji-Hun

    2016-01-01

    Transient receptor potential vanilloid 1 (TRPV1), which has been identified as a molecular target for the activation of sensory neurons by various painful stimuli, was reported to regulate the signaling and activation of CD4+ T cells. However, the role of TRPV1 in CD4+ T cell in allergic rhinitis remains poorly understood. In this study, TRPV1 expression was localized in CD4+ T cells. Both knockout and chemical inhibition of TRPV1 suppressed Th2/Th17 cytokine production in CD4 T cells and Jurkat T cells, respectively, and can suppress T cell receptor signaling pathways including NF-κB, MAP kinase, and NFAT. In TRPV1 knockout allergic rhinitis (AR) mice, eosinophil infiltration, Th2/Th17 cytokines in the nasal mucosa, and total and ova-specific IgE levels in serum decreased, compared with wild-type AR mice. The TRPV1 antagonists, BCTC or theobromine, showed similar inhibitory immunologic effects on AR mice models. In addition, the number of TRPV1+/CD4+ inflammatory cells increased in the nasal mucosa of patients with AR, compared with that of control subjects. Thus, TRPV1 activation on CD4+ T cells is involved in T cell receptor signaling, and it could be a novel therapeutic target in AR. PMID:26700618

  7. Differentiation state determines neural effects on microvascular endothelial cells

    SciTech Connect

    Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  8. The physiological concentration of ferrous iron (II) alters the inhibitory effect of hydrogen peroxide on CD45, LAR and PTP1B phosphatases.

    PubMed

    Kuban-Jankowska, Alicja; Gorska, Magdalena; Jaremko, Lukasz; Jaremko, Mariusz; Tuszynski, Jack A; Wozniak, Michal

    2015-12-01

    Hydrogen peroxide is an important regulator of protein tyrosine phosphatase activity via reversible oxidation. However, the role of iron in this reaction has not been yet elucidated. Here we compare the influence of hydrogen peroxide and the ferrous iron (reagent for Fenton reaction) on the enzymatic activity of recombinant CD45, LAR, PTP1B phosphatases and cellular CD45 in Jurkat cells. The obtained results show that ferrous iron (II) is potent inhibitor of CD45, LAR and PTP1B, but the inhibitory effect is concentration dependent. We found that the higher concentrations of ferrous iron (II) increase the inactivation of CD45, LAR and PTP1B phosphatase caused by hydrogen peroxide, but the addition of the physiological concentration (500 nM) of ferrous iron (II) has even a slightly preventive effect on the phosphatase activity against hydrogen peroxide.

  9. Cell electroporation with a three-dimensional microelectrode array on a printed circuit board.

    PubMed

    Xu, Youchun; Su, Shisheng; Zhou, Changcheng; Lu, Ying; Xing, Wanli

    2015-04-01

    Electroporation is a commonly used approach to rapidly introduce exogenous molecules into cells without permanent damage. Compared to classical electroporation protocols, microchip-based electroporation approaches have the advantages of high transfection efficiency and low consumption, but they also commonly rely on costly and tedious microfabrication technology. Hence, it is desirable to develop a novel, more affordable, and effective approach to facilitate cell electroporation. In this study, we utilized a standard printed circuit board (PCB) technology to fabricate a chip with an interdigitated array of electrodes for electroporation of suspended cells. The electrodes (thickness ~35 μm) fabricated by PCB technology are much thicker than the two-dimensional (2D) planar electrodes (thickness < 1 μm) fabricated by conventional microfabrication techniques and possess a smooth corner edge. Numerical simulations showed that the three-dimensional (3D) electrodes fabricated by PCB technology can provide a more uniformly distributed electric field compared to 2D planar electrodes, which is beneficial for reducing the electrolysis of water and improving cell transfection efficiency. The chip constructed here is composed of 18 individually addressable wells for high throughput cell electroporation. HeLa, MCF7, COS7, Jurkat, and 3T3-L1 cells were efficiently transfected with the pEGFP-N1 plasmid using individually optimal electroporation parameters. This work provides a novel method for convenient and rapid cell transfection and thus holds promise for use as a low-cost disposable device in biomedical research.

  10. The interaction of the carbon nanoparticles with human cell plasma membrane

    NASA Astrophysics Data System (ADS)

    Overchuk, M.; Prylutska, S.; Bilyy, Rostyslav; Prylutsky, Yu.; Ritter, U.

    2013-09-01

    The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or photosensitizers. In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells). We have shown, that carbon nanoparticles do not have any hemolytic effects, if judged by the dynamics of acidic hemolysis, although they are capable of permeabilizating the cells and facilitating the internalization of propidium iodide into the nuclei.

  11. The Effects of Ionizing Radiation on Mammalian Cells.

    ERIC Educational Resources Information Center

    Biaglow, John E.

    1981-01-01

    Discusses the effects of radiation on dividing cells and factors influencing these effects; also briefly reviews the radical mechanism for radiation damage. Emphasizes the importance of oxygen in radiation effects. (CS)

  12. Effect of cell hydration on metabolism.

    PubMed

    Lang, Florian

    2011-01-01

    Prerequisites for cell survival include avoidance of excessive cell volume alterations. Cell membranes are highly permeable to water, which follows osmotic gradients. Thus, cell volume constancy requires osmotic equilibrium across cell membranes. Cells accumulate osmotically active organic substances and compensate their osmolarity by lowering cytosolic Cl(-) concentrations. Following cell shrinkage, regulatory cell volume increase is accomplished by ion uptake (activation of Na(+), K(+), 2Cl(-) cotransport, Na(+)/H(+) exchange in - parallel to Cl(-)/HCO(-)(3) exchange and Na(+) channels), by cellular accumulation of organic osmolytes (e.g. myoinositol, betaine, phosphorylcholine, taurine) as well as by proteolysis leading to generation of amino acids and glycogenolysis generating glucose phosphate. Following cell swelling, cell volume is restored by ion exit (activation of K(+) channels and/ - or anion channels, KCl cotransport, parallel activation of K(+)/H(+) exchange and Cl(-)/HCO(-)(3) exchange), release or degradation of organic osmolytes as well as stimulation of protein synthesis and of glycogen synthesis. The activity of cell volume regulatory mechanisms is modified by hormones, transmitters and drugs, which thus influence protein and glycogen metabolism. Moreover, alterations of cell volume modify generation of oxidants and the sensitivity to oxidative stress. Deranged cell volume regulation significantly contributes to the pathophysiology of several disorders such as liver insufficiency, diabetic ketoacidosis, hypercatabolism, ischemia, and fibrosing disease. PMID:22301839

  13. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16).

    PubMed

    Spruyt, L L; Glennie, M J; Beyers, A D; Williams, A F

    1991-12-01

    Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.

  14. Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16)

    PubMed Central

    1991-01-01

    Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i. PMID:1683892

  15. Differential effects of bisphosphonates on breast cancer cell lines.

    PubMed

    Verdijk, R; Franke, H R; Wolbers, F; Vermes, I

    2007-02-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential.

  16. Effect of spaceflight on natural killer cell activity

    NASA Technical Reports Server (NTRS)

    Rykova, Marina P.; Sonnenfeld, Gerald; Lesniak, A. T.; Taylor, Gerald R.; Meshkov, Dimitrii O.; Mandel, Adrian D.; Medvedev, Andrei E.; Berry, Wallace D.; Fuchs, Boris B.; Konstantinova, Irina V.

    1992-01-01

    The effects of spaceflight on immune cell function were determined in rats flown on Cosmos 2044. Control groups included vivarium, synchronous, and antiorthostatically suspended rats. The ability of natural killer cells to lyse two different target cell lines was determined. Spleen and bone marrow cells obtained from flight rats showed significantly inhibited cytotoxicity for YAC-1 target cells compared with cells from synchronous control rats. This could have been due to exposure of the rats to microgravity. Antiorthostatic suspension did not affect the level of cytotoxicity from spleen cells of suspended rats for YAC-1 cells. On the other hand, cells from rats flown in space showed no significant differences from vivarium and synchronous control rats in cytotoxicity for K-562 target cells. Binding of natural killer cells to K-562 target cells was unaffected by spaceflight. Antiorthostatic suspension resulted in higher levels of cytotoxicity from spleen cells for Cr-51-labeled K-562 cells. The results indicate differential effects of spaceflight on function of natural killer cells. This shows that spaceflight has selective effects on the immune response.

  17. Prooxidant and antioxidant behaviour of usnic acid from lichens under UVB-light irradiation--studies on human cells.

    PubMed

    Kohlhardt-Floehr, Cornelia; Boehm, Fritz; Troppens, Stefan; Lademann, Jürgen; Truscott, T George

    2010-10-01

    Natural compounds which can behave as antioxidants and protect against UV-radiation may well have medical and cosmetic value. Usnic acid, which can be obtained from lichens offer such a potential. The latter is one of the best known and reviewed compounds present in lichens and exhibits many properties of value such as antibiotic, antitumor and UV-filter-effects. We report studies of the potential antioxidant and prooxidant activity of usnic acid extracted from Xanthoparmelia farinosa (Vainio) using a human lymphocyte cell line (Jurkat-cells) under UV-B-irradiation. Cell survival and cell metabolism were determined using different conditions such as usnic acid concentration and UVB dose. Compared to the controls the cells incubated with usnic acid in concentrations of 1 x 10(-8) and 1 x 10(-6) M showed a higher cell survival and a normal metabolism under low doses of UVB-light up to 0.1 J/cm(2). When both higher UVB doses (up to 14 J/cm(2)) and higher concentrations of usnic acid (1 x 10(-4) M) where used, the opposite effect was observed. It is concluded that these effects are due to bifunctional (a switch of) anti-oxidative-pro-oxidative behaviour of usnic acid under UV-B-irradiation.

  18. Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis

    PubMed Central

    Lu, Tsung Chi; Zhao, Guan- Hao; Chen, Yao Yun; Chien, Chia-Ying; Huang, Chi-Hung; Lin, Kwang Hui; Chen, Shen Liang

    2016-01-01

    Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors. PMID:27390612

  19. The effects of aspartame on mast cells and basophils.

    PubMed

    Szucs, E F; Barrett, K E; Metcalfe, D D

    1986-02-01

    The artificial sweetener aspartame was studied to determine whether it had any direct effects on mast cells and basophils. Aspartame was not shown to be a direct mast cell or basophil secretagogue in vitro, or in vivo as assessed by skin testing. During an acute incubation, aspartame did not affect IgE-mediated histamine release from mast cells. However, mast cells cultured in aspartame for periods of up to 9 days showed enhanced rates of proliferation and decreased responsiveness to releasing stimuli. The effect of aspartame on proliferation of cells in culture could be ascribed to a non-specific enhancing effect of its constituent amino acids.

  20. Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells

    SciTech Connect

    Duan, Lincan; Shen, Hongmei; Zhao, Guangqiang; Yang, Runxiang; Cai, Xinyi; Zhang, Lijuan; Jin, Congguo; Huang, Yunchao

    2014-04-18

    Highlights: • Disulfiram and copper synergistically inhibit lung cancer cell proliferation. • Lung cancer cell colony formation ability is inhibited by Disulfiram/copper. • Disulfiram/copper increases the sensitivity of cisplatin to lung cancer cells. • Lung cancer stem cells are specifically targeted by Disulfiram/copper complex. - Abstract: Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition of cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients.

  1. Tritium effects on germ cells and fertility

    SciTech Connect

    Dobson, R.L.; Kwan, T.C.; Straume, T.

    1982-11-19

    Primordial oocytes in juvenile mice show acute gamma-ray LD/sub 50/ as low as 6 rad. This provides opportunities for determining dose-response relations at low doses and chronic exposure in the intact animal - conditions of particular interest for hazard evaluation. Examined in this way, /sup 3/HOH in body water is found to kill murine oocytes exponentially with dose, the LD/sub 50/ level for chronic exposure being only 2..mu..Ci/ml (delivering 0.4 rad/day). At very low doses and dose rates, where comparisons between tritium and other radiations are of special significance for radiological protection, the RBE of tritium compared with /sup 60/Co gamma radiation reaches approximately 3. Effects on murine fertility from tritium-induced oocyte loss have been quantified by reproductive capacity measurements. Chronic low-level exposure has been examined also in three primate species - squirrel, rhesus, and bonnet monkeys. In squirrel monkeys the ovarian germ-cell supply is 99% destroyed by the time of birth from prenatal exposure to body-water levels of /sup 3/HOH (administered in maternal drinking water) of only 3 ..mu..Ci/ml, the LD/sub 50/ level being 0.5 ..mu..Ci/ml (giving 0.1 rad/day), one fourth that in mice. Though not completely ruled out, similar high sensitivity of female germ cells has not been found in macaques; and it probably does not occur in man. The exquisite radiosensitivity of primordial oocytes in mice is apparently due to vulnerability of the plasma membrane (or something of similar geometry and location), not DNA. Evidence for this comes from tritium data as well as neutron studies. Tritium administered as /sup 3/HOH, and therefore generally distributed, is much more effective in killing murine oocytes than is tritium administered as /sup 3/H-TdR, localized in the nucleus. This situation in the mouse may have implications for estimating radiation genetic risk in the human female.

  2. Antiproliferative Effects of Mesenchymal Stem Cells and Epithelial Cells on Lymphocytes.

    PubMed

    Svirshchevskaya, E V; Poltavtseva, R A; Beletskii, I P; Selezneva, I I; Sukhikh, G T

    2016-08-01

    We analyzed the interactions between peripheral blood lymphocytes from heterologous donors with mesenchymal stem cells obtained from the tooth pulp and trophoblast. In mixed cultures, proliferation of both lymphocytes and mesenchymal stem cells was suppressed. Similar suppressive effects were observed in lymphocyte cultures mixed with epithelial cells (hepatocytes HeG2 and renal epithelial cells HEK293). This suppression can be determined by impairment of normal adhesion contacts between cells of different origin. PMID:27590756

  3. Localized electroporation effect on adherent cells in modified electric cell-substrate impedance sensing circuits

    NASA Astrophysics Data System (ADS)

    Kim, Yu Jin; Ram Song, Ka; Kim, Hee-Dae; Park, Bum Chul; Kim, Young Keun; Kang, Chi Jung

    2016-10-01

    Electroporation is a physical transfection method for introducing foreign genes or drugs into cells. It does not require toxic reagents or transfection vectors. However, its applications have been limited because of cell damage and nonspecific transport. Here, we present an effective method for selective and localized electroporation using atomic force microscopy. This electroporation method is applied to adherent cells on substrates, instead of conventionally used suspended cells, and offers relatively effective cell transfection. Moreover, this method enables localized transfection into targeted areas at the single-cell level.

  4. Effects of space flight exposure on cell growth, tumorigenicity and gene expression in cancer cells

    NASA Astrophysics Data System (ADS)

    Yang, Cheng; Li, Yuehui; Zhang, Zhijie; Luo, Chen; Tong, Yongqing; Zhou, Guohua; Xie, Pingli; Hu, Jinyue; Li, Guancheng

    2008-12-01

    It is well recognized that harsh outer space environment, consisting of microgravity and radiation, poses significant health risks for human cells. To investigate potential effects of the space environment exposure on cancer cells we examined the biological changes in Caski cells carried by the "Shen Zhou IV" spaceship. After exposure for 7 days in spaceflight, 1440 survival subclonal cell lines were established and 4 cell lines were screened. 44F10 and 17E3 were selected because of their increased cell proliferation and tumorigenesis, while 48A9 and 31F2 had slower cytological events. Experiments with cell proliferation assay, flow cytometry, soft agar assay, tumorigenesis assay and DNA microarray analysis have shown that selected cell lines presented multiple biological changes in cell morphology, cell growth, tumorigenicity and gene expression. These results suggest that space environment exposure can make significant biological impact on cancer cells and provide an entry point to find the immunological target of tumorigenesis.

  5. Curcumin induces apoptotic cell death of activated human CD4+ T cells via increasing endoplasmic reticulum stress and mitochondrial dysfunction.

    PubMed

    Zheng, Min; Zhang, Qinggao; Joe, Yeonsoo; Lee, Bong Hee; Ryu, Do Gon; Kwon, Kang Beom; Ryter, Stefan W; Chung, Hun Taeg

    2013-03-01

    Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases. PMID:23415873

  6. Force Microscopy of Nonadherent Cells: A Comparison of Leukemia Cell Deformability

    PubMed Central

    Rosenbluth, Michael J.; Lam, Wilbur A.; Fletcher, Daniel A.

    2006-01-01

    Atomic force microscopy (AFM) has become an important tool for quantifying mechanical properties of biological materials ranging from single molecules to cells and tissues. Current AFM techniques for measuring elastic and viscoelastic properties of whole cells are based on indentation of cells firmly adhered to a substrate, but these techniques are not appropriate for probing nonadherent cells, such as passive human leukocytes, due to a lateral instability of the cells under load. Here we present a method for characterizing nonadherent cells with AFM by mechanically immobilizing them in microfabricated wells. We apply this technique to compare the deformability of human myeloid and lymphoid leukemia cells and neutrophils at low deformation rates, and we find that the cells are well described by an elastic model based on Hertzian mechanics. Myeloid (HL60) cells were measured to be a factor of 18 times stiffer than lymphoid (Jurkat) cells and six times stiffer than human neutrophils on average (E∞ = 855 ± 670 Pa for HL60 cells, E∞ = 48 ± 35 Pa for Jurkat cells, E∞ = 156 ± 87 for neutrophils, mean ± SD). This work demonstrates a simple method for extending AFM mechanical property measurements to nonadherent cells and characterizes properties of human leukemia cells that may contribute to leukostasis, a complication associated with acute leukemia. PMID:16443660

  7. Dicyanogold Effects on Lymphokine Production

    PubMed Central

    Roy, Pamela W.; Moloney, Brian F.; Elder, R. C.

    1999-01-01

    Having identified dicyanogold(I) as a common metabolite of gold-based antiarthritis drugs, we are investigating the effects of the compound on the production of lymphokines. Handel, et al. 1 suggested that the transcription factor AP-1, critical to the production of a number of cytokines, might be the target for gold compounds because of a critical cysteine within its DNA binding region. Using Jurkat cells, an established cell line as a model for CD4+ lymphocytes, we have shown that dicyanogold inhibits the binding of AP-1 to DNA and inhibits the synthesis of IL-2 mRNA and protein. In a macrophage line, THP-1, which synthesizes IL-1β in response to mitogen, we have shown that dicyanogold inhibits the binding of a second transcription factor, CREB to DNA. Incubation of THP-1 cells with dicyanogold inhibits the production of IL-1β mRNA. These results suggest that the mechanism of action of gold drugs may be through their interaction with transcription factors necessary for the immune activation seen in Rheumatoid Arthritis. PMID:18475905

  8. Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells

    PubMed Central

    Soo, Chin Y; Song, Yaqiong; Zheng, Ying; Campbell, Elaine C; Riches, Andrew C; Gunn-Moore, Frank; Powis, Simon J

    2012-01-01

    Nanoparticle tracking analysis permits the determination of both the size distribution and relative concentration of microvesicles, including exosomes, in the supernatants of cultured cells and biological fluids. We have studied the release of microvesicles from the human lymphoblastoid T-cell lines Jurkat and CEM. Unstimulated, both cell lines release microvesicles in the size range 70–90 nm, which can be depleted from the supernatant by ultracentrifugation at 100 000 g, and by anti-CD45 magnetic beads, and which by immunoblotting also contain the exosome-associated proteins Alix and Tsg101. Incubation with known potentiators of exosome release, the ionophores monensin and A23187, resulted in a significant increase in microvesicle release that was both time and concentration dependent. Mass spectrometric analysis of proteins isolated from ultracentrifuged supernatants of A23187-treated cells revealed the presence of exosome-associated proteins including heat-shock protein 90, tubulin, elongation factor α1, actin and glyceraldehyde 3-phosphate dehydrogenase. Additionally, treatment of peripheral blood monocyte-derived dendritic cells with bacterial lipopolysaccharide displayed an increase in secreted microvesicles. Consequently, nanoparticle tracking analysis can be effectively applied to monitor microvesicle release from cells of the immune system. PMID:22348503

  9. Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion.

    PubMed

    Nath, Shubhankar; Christian, Laura; Tan, Sarah Youngsun; Ki, Sanghee; Ehrlich, Lauren I R; Poenie, Martin

    2016-09-15

    Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150(Glued)) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1-enhanced GFP fusion) were activated by Staphylococcus enterotoxin E-coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150(Glued), which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B-containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150(Glued) in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell-focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively. PMID:27534551

  10. Src-like Adaptor Protein (Slap) Is a Negative Regulator of T Cell Receptor Signaling

    PubMed Central

    Sosinowski, Tomasz; Pandey, Akhilesh; Dixit, Vishva M.; Weiss, Arthur

    2000-01-01

    Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)–, and interleukin 2–dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3ζ, ZAP-70, SH2 domain–containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling. PMID:10662792

  11. Src-like adaptor protein (SLAP) is a negative regulator of T cell receptor signaling.

    PubMed

    Sosinowski, T; Pandey, A; Dixit, V M; Weiss, A

    2000-02-01

    Initiation of T cell antigen receptor (TCR) signaling is dependent on Lck, a Src family kinase. The Src-like adaptor protein (SLAP) contains Src homology (SH)3 and SH2 domains, which are highly homologous to those of Lck and other Src family members. Because of the structural similarity between Lck and SLAP, we studied its potential role in TCR signaling. Here, we show that SLAP is expressed in T cells, and that when expressed in Jurkat T cells it can specifically inhibit TCR signaling leading to nuclear factor of activated T cells (NFAT)-, activator protein 1 (AP-1)-, and interleukin 2-dependent transcription. The SH3 and SH2 domains of SLAP are required for maximal attenuation of TCR signaling. This inhibitory activity can be bypassed by the combination of phorbol myristate acetate (PMA) and ionomycin, suggesting that SLAP acts proximally in the TCR signaling pathway. SLAP colocalizes with endosomes in Jurkat and in HeLa cells, and is insoluble in mild detergents. In stimulated Jurkat cells, SLAP associates with a molecular signaling complex containing CD3zeta, ZAP-70, SH2 domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possibly linker for activation of T cells (LAT). These results suggest that SLAP is a negative regulator of TCR signaling.

  12. Baculovirus Stimulates Antiviral Effects in Mammalian Cells

    PubMed Central

    Gronowski, Ann M.; Hilbert, David M.; Sheehan, Kathleen C. F.; Garotta, Gianni; Schreiber, Robert D.

    1999-01-01

    Herein, we report that Autographa californica nucleopolyhedrovirus, a member of the Baculoviridae family, is capable of stimulating antiviral activity in mammalian cells. Baculoviruses are not pathogenic to mammalian cells. Nevertheless, live baculovirus is shown here to induce interferons (IFN) from murine and human cell lines and induces in vivo protection of mice from encephalomyocarditis virus infection. Monoclonal antibodies specific for the baculovirus envelope gp67 neutralize baculovirus-dependent IFN production. Moreover, UV treatment of baculovirus eliminates both infectivity and IFN-inducing activity. In contrast, the IFN-inducing activity of the baculovirus was unaffected by DNase or RNase treatment. These data demonstrate that IFN production can be induced in mammalian cells by baculovirus even though the cells fail to serve as a natural host for an active viral infection. Baculoviruses, therefore, provide a novel model in which to study at least one alternative mechanism for IFN induction in mammalian cells. PMID:10559307

  13. WNT5A expression is regulated by the status of its promoter methylation in leukaemia and can inhibit leukemic cell malignant proliferation.

    PubMed

    Deng, Gang; Li, Zhao Quan; Zhao, Chen; Yuan, Yuan; Niu, Chang Chun; Zhao, Chen; Pan, Jing; Si, Wei Ke

    2011-02-01

    Although down-regulation of WNT5A expression has been reported in some types of leukaemias, the level of WNT5A expression has not been assessed in leukaemia complete remission (CR) cases, the relationship among WNT5A expression level, the status of its promoter methylation, and the curative effect of leukaemia has not been reported, and the effect of WNT5A on cell proliferation has not been assessed. In this study, we analyzed WNT5A expression in various kinds of leukaemia cases, leukaemia CR cases, non-malignant hematopoietic (NMH) cases, as well as in leukemic cell lines and CD34+ cells. The methylation status of the WNT5A promoter and the levels of the Wnt5a protein were also studied. We also investigated the effect of Wnt5a on leukemic cell proliferation. WNT5A expression level was higher in NMH but lower in leukaemia cases compared to that in CR-cases (P<0.01), and was expressed at low level in leukemic cell lines K562, U937 and Jurkat. Wnt5a protein was positive in NMH, CR cases and CD34+, but negative in leukaemia cases. WNT5A promoter was methylated in leukaemia cases and all leukemic cell lines, but not in NMH and CR cases. WNT5A expression was up-regulated after exposure to the demethylating agent 5-Aza-2'-deoxycytidine (Aza) in the K562, U937, Jurkat leukemic cell lines and in 83.3% (10/12) of CR patients after cure, respectively. The increased Wnt5a protein can inhibit K562 malignant proliferation and arrest cell cycle at the G2/M phase after exposure to Aza. These results indicate that WNT5A expression was restored in complete remission cases due to demethylation, and Wnt5a can inhibit leukaemic cell proliferation. We propose that WNT5A can act as a suppressor factor in leukemogenesis and can be used as a potential marker for curative effect assessment in leukaemia.

  14. Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells.

    PubMed

    Haarmann, Axel; Nehen, Mathias; Deiß, Annika; Buttmann, Mathias

    2015-01-01

    Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1β-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1β-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1β-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells. PMID:26287168

  15. Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells.

    PubMed

    Haarmann, Axel; Nehen, Mathias; Deiß, Annika; Buttmann, Mathias

    2015-08-13

    Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1β-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1β-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1β-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.

  16. Effect of dopamine on viability of BHK-21 cells.

    PubMed

    Moshkov, D A; Abramova, M B; Shubina, V S; Lavrovskaya, V P; Pavlik, L L; Lezhnev, E I

    2010-09-01

    We studied the effects of dopamine added to culture medium on survival of floating or adherent BHK-21 cells differing by organization of actin cytoskeleton. The viability of floating cells more drastically decreased with increasing dopamine concentration and duration of exposure than that of adherent cells. The cells worse adhered to the substrate and formed a monolayer. The formed monolayer degrades, cell borders become blurred, cells, polygonal in the control, are rounded. Preliminary blockade of dopamine receptors with haloperidol, inessential for cell survival and morphology, does not prevent the destructive effect of dopamine on the cells. Ultrastructural study revealed increased density of filamentous actin threads in deep compartments of cell cytoplasm after dopamine treatment, this increase being more pronounced in cells grown in suspension. Bearing in mind the polymerizing effect of dopamine on globular actin in vitro and the fact that the content of this protein in floating cells is higher than in adherent cells, we can conclude that the decrease in viability of BHK-21 cells is caused by interaction of dopamine with cytoplasmic globular actin. PMID:21246101

  17. Effects of thyroid hormones on human breast cancer cell proliferation.

    PubMed

    Hall, Linda C; Salazar, Eddie P; Kane, Staci R; Liu, Nan

    2008-03-01

    The involvement of estrogens in breast cancer development and growth has been well established. However, the effects of thyroid hormones and their combined effects with estrogens are not well studied. We investigated the response of human breast cancer cells to thyroid hormone, particularly the role of T3 in mediating cell proliferation and gene expression. We demonstrated that 17beta-estradiol (E2) or triiodothyronine (T3) promoted cell proliferation in a dose-dependent manner in both MCF-7 and T47-D cell lines. The E2- or T3-dependent cell proliferation was suppressed by co-administration of the ER antagonist ICI. We also demonstrated that T3 could enhance the effect of E2 on cell proliferation in T47-D cells. Using an estrogen response element (ERE)-mediated luciferase assay, we determined that T3 was able to induce the activation of ERE-mediated gene expression in MCF-7 cells, although the effects were much weaker than that induced by E2. These results suggest that T3 can promote breast cancer cell proliferation and increase the effect of E2 on cell proliferation in some breast cancer cell lines and thus that T3 may play a role in breast cancer development and progression. PMID:18328691

  18. Non-cell-autonomous effects of vector-expressed regulatory RNAs in mammalian heart cells.

    PubMed

    Kizana, E; Cingolani, E; Marbán, E

    2009-09-01

    In mammalian cells, small regulatory RNA molecules are able to modulate gene expression in a cell-autonomous manner. In contrast, this mechanism of gene regulation can occur systemically in plants and nematodes. The existence of similar cell-to-cell transmission in mammalian cells has been explored, but generalizibilty and mechanistic insights have remained elusive. Here, we show that small regulatory RNA molecules are capable of a non-cell-autonomous effect between primary cardiac myocytes through a gap-junction-dependent mechanism. Co-culture experiments showed that both Dicer-processed small-interfering RNAs (siRNAs) and Drosha-processed microRNAs (miRNAs) were capable of target gene knockdown and physiological effects in a non-cell-autonomous manner. Target gene siRNA molecules were detected in recipient cells, indicating transfer of the primary effector molecule. All of these effects were abrogated by dominant-negative molecular suppression of gap junction function. Our results show that both siRNAs and miRNAs are capable of a non-cell-autonomous effect between mammalian cells through gap junctions. The recognition of this biological process raises the novel therapeutic prospect of a bystander effect after gene transfer to tissues bearing gap junctions and for cell engineering with a view to creating regulatory RNA donor cells that exert their influence throughout a syncytium. PMID:19516277

  19. Suppressive effects of tumor cell-derived 5'-deoxy-5'-methylthioadenosine on human T cells.

    PubMed

    Henrich, Frederik C; Singer, Katrin; Poller, Kerstin; Bernhardt, Luise; Strobl, Carolin D; Limm, Katharina; Ritter, Axel P; Gottfried, Eva; Völkl, Simon; Jacobs, Benedikt; Peter, Katrin; Mougiakakos, Dimitrios; Dettmer, Katja; Oefner, Peter J; Bosserhoff, Anja-Katrin; Kreutz, Marina P; Aigner, Michael; Mackensen, Andreas

    2016-08-01

    The immunosuppressive tumor microenvironment represents one of the main obstacles for immunotherapy of cancer. The tumor milieu is among others shaped by tumor metabolites such as 5'-deoxy-5'-methylthioadenosine (MTA). Increased intratumoral MTA levels result from a lack of the MTA-catabolizing enzyme methylthioadenosine phosphorylase (MTAP) in tumor cells and are found in various tumor entities. Here, we demonstrate that MTA suppresses proliferation, activation, differentiation, and effector function of antigen-specific T cells without eliciting cell death. Conversely, if MTA is added to highly activated T cells, MTA exerts cytotoxic effects on T cells. We identified the Akt pathway, a critical signal pathway for T cell activation, as a target of MTA, while, for example, p38 remained unaffected. Next, we provide evidence that MTA exerts its immunosuppressive effects by interfering with protein methylation in T cells. To confirm the relevance of the suppressive effects of exogenously added MTA on human T cells, we used an MTAP-deficient tumor cell-line that was stably transfected with the MTAP-coding sequence. We observed that T cells stimulated with MTAP-transfected tumor cells revealed a higher proliferative capacity compared to T cells stimulated with Mock-transfected cells. In conclusion, our findings reveal a novel immune evasion strategy of human tumor cells that could be of interest for therapeutic targeting. PMID:27622058

  20. The Effect of Shape Memory on Red Blood Cell Motions

    NASA Astrophysics Data System (ADS)

    Niu, Xiting; Shi, Lingling; Pan, Tsorng-Whay; Glowinski, Roland

    2013-11-01

    An elastic spring model is applied to study the effect of the shape memory on the motion of red blood cell in flows. In shear flow, shape memory also plays an important role to obtain all three motions: tumbling, swinging, and tank-treading. In Poiseuille flow, cell has an equilibrium shape as a slipper or parachute depending on capillary number. To ensure the tank-treading motion while in slippery shape, a modified model is proposed by introducing a shape memory coefficient which describes the degree of shape memory in cells. The effect of the coefficient on the cell motion of red blood cell will be presented.

  1. Modulation of both activator protein-1 and nuclear factor-kappa B signal transduction of human T cells by amiodarone

    PubMed Central

    Cheng, Shu-Meng; Lin, Wei-Hsiang; Lin, Chin-Sheng; Ho, Ling-Jun; Tsai, Tsung-Neng; Wu, Chun-Hsien; Lai, Jenn-Haung

    2015-01-01

    Amiodarone, a common and effective antiarrhythmic drug, has been reported to have anti-inflammatory effects such as reducing the activation and movement of neutrophils. However, its effects on human T cells remain unclear. The aim of this study was to elucidate the effects and possible underlying mechanisms of amiodarone on human T cells. We isolated human primary T cells from the peripheral blood of healthy volunteers and performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, electrophoretic mobility shift assay, luciferase assay, and Western blotting to evaluate the modulatory effects of amiodarone on human T cells. We found that amiodarone dose dependently inhibited the production of cytokines, including interleukin-2 (IL-2), IL-4, tumor necrosis factor-alpha, and interferon-gamma in activated human T cells. By flow cytometry, we demonstrated that amiodarone suppressed the expression of IL-2 receptor-alpha (CD25) and CD69, the cell surface markers of activated T cells. Moreover, molecular investigations revealed that amiodarone down-regulated activator protein-1 (AP-1) and nuclear factor kappa-B (NF-κB) DNA-binding activities in activated human T cells and also inhibited DNA binding and transcriptional activities of both AP-1 and NF-κB in Jurkat cells. Finally, by Western blotting, we showed that amiodarone reduced the activation of c-Jun NH2-terminal protein kinase and P38 mitogen-activated protein kinase, and suppressed stimuli-induced I-kappa B-alpha degradation in activated human T cells. Through regulation of AP-1 and NF-κB signaling, amiodarone inhibits cytokine production and T cell activation. These results show the pleiotropic effects of amiodarone on human T cells and suggest its therapeutic potential in inflammation-related cardiovascular disorders. PMID:25073960

  2. Differential effect of p7 inhibitors on hepatitis C virus cell-to-cell transmission☆

    PubMed Central

    Meredith, L.W.; Zitzmann, N.; McKeating, J.A.

    2013-01-01

    Inhibitors targeting the hepatitis C virus (HCV) encoded viroporin, p7 prevent virus release in vitro. HCV can transmit by cell-free particle infection of new target cells and via cell-to-cell dependent contact with limited exposure to the extracellular environment. The role of assembly inhibitors in preventing HCV transmission via these pathways has not been studied. We compared the efficacy of three published p7 inhibitors to inhibit cell-free and cell-to-cell transmission of two chimeric HCV strains encoding genotype 2 (GT2) or 5 (GT5) p7 using a recently developed single cycle co-culture assay. The inhibitors reduced the infectivity of extracellular GT2 and GT5 virus by 80–90% and GT2 virus cell-to-cell transmission by 50%. However, all of the p7 inhibitors had minimal effect on GT5 cell contact dependent transmission. Screening a wider panel of diverse viral genotypes demonstrated that p7 viroporin inhibitors were significantly more effective at blocking cell-free virus than cell-to-cell transmission. These results suggest an altered assembly or trafficking of cell-to-cell transmitted compared to secreted virus. These observations have important implications for the validation, therapeutic design and testing of HCV assembly inhibitors. PMID:24157306

  3. The Adapter Molecule Sin Regulates T-Cell-Receptor-Mediated Signal Transduction by Modulating Signaling Substrate Availability

    PubMed Central

    Xing, Luzhou; Donlin, Laura T.; Miller, Rebecca H.; Alexandropoulos, Konstantina

    2004-01-01

    Engagement of the T-cell receptor (TCR) results in the activation of a multitude of signaling events that regulate the function of T lymphocytes. These signaling events are in turn modulated by adapter molecules, which control the final functional output through the formation of multiprotein complexes. In this report, we identified the adapter molecule Sin as a new regulator of T-cell activation. We found that the expression of Sin in transgenic T lymphocytes and Jurkat T cells inhibited interleukin-2 expression and T-cell proliferation. This inhibitory effect was specific and was due to defective phospholipase C-γ (PLC-γ) phosphorylation and activation. In contrast to other adapters that become phosphorylated upon TCR stimulation, Sin was constitutively phosphorylated in resting cells by the Src kinase Fyn and bound to signaling intermediates, including PLC-γ. In stimulated cells, Sin was transiently dephosphorylated, which coincided with transient dissociation of Fyn and PLC-γ. Downregulation of Sin expression using Sin-specific short interfering RNA oligonucleotides inhibited transcriptional activation in response to TCR stimulation. Our results suggest that endogenous Sin influences T-lymphocyte signaling by sequestering signaling substrates and regulating their availability and/or activity in resting cells, while Sin is required for targeting these intermediates to the TCR for fast signal transmission during stimulation. PMID:15121874

  4. Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

    SciTech Connect

    Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia . E-mail: lombardi@pharm.unipmn.it

    2005-12-30

    The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10 U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.

  5. 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

    2014-01-01

    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. PMID:24873950

  6. The effect of internal stresses on solar cell efficiency

    NASA Technical Reports Server (NTRS)

    Weizer, Victor G.

    1987-01-01

    Diffusion induced stresses in silicon are shown to result in large localized changes in the minority carrier mobility which in turn have a significant effect on cell output. Evidence is given that both compressive and tensile stresses can be generated in either the emitter or the base region. Tensile stresses appear to be much more effective in altering cell performance. While most stress related effects appear to degrade cell efficiency, this is not always the case. Evidence is presented showing that arsenic induced stresses can result in emitter characteristics comparable to those found in the MINP cell without requiring a high degree of surface passivation.

  7. Plasma membrane depolarization and Na,K-ATPase impairment induced by mitochondrial toxins augment leukemia cell apoptosis via a novel mitochondrial amplification mechanism.

    PubMed

    Yin, Wu; Li, Xiang; Feng, Su; Cheng, Wei; Tang, Bo; Shi, Yi-Lin; Hua, Zi-Chun

    2009-07-15

    Na,K-ATPase is a ubiquitous transmembrane protein that regulates and maintains the intracellular Na(+) and K(+) gradient necessary for cell homeostasis. Recently, the importance of this pump in external stimuli-induced leukemia cell apoptosis has been increasingly appreciated, however, the exact role of Na,K-ATPase in mitochondrial apoptotic pathway still remains little understood. In this study, we found mitochondrial toxin rotenone caused a rapid mitochondrial membrane potential (MMP) collapse in Jurkat cells followed by plasma membrane depolarization (PMP). Similar results were also obtained in human U937 cells and non-cancerous mouse primary T cells. Rotenone-induced PMP depolarization occurred before apoptosis and well correlated with Na,K-ATPase impairment. To understand the mechanisms, Jurkat cells with mtDNA depletion and catalase overexpression were used. The results demonstrated that both PMP depolarization and Na,K-ATPase impairment induced by rotenone were regulated by mitochondrial H(2)O(2) and Bcl-2. Finally, Na,K-ATPase suppression by ouabain greatly accelerated and enhanced mitochondrial toxins-induced cells apoptosis in Jurkat, U937 and primary T cells. In sum, by using leukemia cells and mouse primary T cells, we confirmed that mitochondria-to-Na,K-ATPase and PMP depolarization might represent a novel mechanism for mitochondria to amplify death signals in the initiation stage of cells apoptosis induced by mitochondrial toxins.

  8. Evaluation of effect of triterpenes and limonoids on cell growth, cell cycle and apoptosis in human tumor cell line.

    PubMed

    Cazal, Cristiane M; Choosang, Kantima; Severino, Vanessa Gisele P; Soares, Marcio S; Sarria, Andre Lucio F; Fernandes, Joao B; Silva, Maria Fatima G F; Vieira, Paulo Cezar; Pakkong, Pannee; Almeida, Gabriela M; Vasconcelos, M Helena; Nascimento, Maria S J; Pinto, Madalena M M

    2010-12-01

    Six triterpenes and eight limonoids were evaluated for their capacity to inhibit the growth of three human tumour cell lines, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and melanoma (A375-C5). The mechanisms involved in the observed cell growth arrest of the four most potent compounds were carried out by studying their effect in cell cycle profile and programmed cell death. The results showed that one triterpene (odoratol) and two limonoids (gedunin and cedrelone) caused cell cycle arrest while only the limonoids gedunin and cedrelone were found to be very potent inducers of apoptosis. PMID:21269253

  9. Cytotoxicity of water-soluble fraction from biodiesel and its diesel blends to human cell lines.

    PubMed

    Leme, Daniela Morais; Grummt, Tamara; Heinze, Rita; Sehr, Andrea; Skerswetat, Matthias; de Marchi, Mary Rosa Rodrigues; Machado, Marcos Canto; de Oliveira, Danielle Palma; Marin-Morales, Maria Aparecida

    2011-11-01

    The designation of biodiesel as a green fuel has increased its commercialization and use, making its fate in the environment a matter of concern. Fuel spills constitute a major source of aquatic pollution and, like diesel spills, biodiesel can produce adverse effects on aquatic environments, animals and humans. The present study assessed cytotoxic effects of water systems contaminated with neat biodiesel and its diesel blends by means of different procedures on human T cell leukemia (Jurkat) and human hepatocellular carcinoma (HepG2) cells [detection of changes in mitochondrial membrane potential (ΔΨ(m)) using tetramethylrhodamine ethyl ester (TMRE), apoptosis recognition by Annexin V and impedance real-time cell analyzer (xCELLigence™ system)]. The data obtained showed concordance across the different bioassays, with cytotoxic effects observed as a dose-dependent response only for waters contaminated with pure diesel (D100) and B5 blend, which is characterized by a mixture of 95% diesel and 5% biodiesel. The data can also lead us to hypothesize that diesel accounts for the harmful effects observed, and that biodiesel does not worsen the impacts caused by diesel pollution.

  10. Anti-apoptotic effect of clusterin on cisplatin-induced cell death of retinoblastoma cells.

    PubMed

    Song, Hyun Beom; Jun, Hyoung-Oh; Kim, Jin Hyoung; Yu, Young Suk; Kim, Kyu-Won; Min, Bon Hong; Kim, Jeong Hun

    2013-12-01

    Clusterin is a cytoprotective chaperone protein that is known to protect various retinal cells. It was also reported to be overexpressed in several types of malignant tumors, whose chemoresistance correlates with the expression of clusterin. Herein, we investigated the effect of clusterin on cisplatin-induced cell death of retinoblastoma cells. Firstly, evaluation of clusterin expression demonstrated that it was highly expressed in human retinoblastoma tissues and cell lines (SNUOT-Rb1 and Y79) particularly in the area between viable cells around vessels and necrotic zones in the relatively avascular area in human retinoblastoma tissues. Furthermore, the effects of cisplatin on retinoblastoma cells were evaluated. Cisplatin (1 µg/ml) significantly affected cell viability of SNUOT-Rb1 cells by inducing caspase-3-dependent apoptosis. Notably, the cell death due to cisplatin was prevented by 5 µg/ml of clusterin administered 4 h prior to cisplatin treatment by inhibiting cisplatin-induced apoptosis. Furthermore, overexpression of clusterin exerted its anti-apoptotic effect on cisplatin-induced apoptosis, and effectively prevented cisplatin-induced cell death. These data suggest that clusterin, found to be expressed in human retinoblastoma, may exert anti-apoptotic effects on cisplatin-induced apoptosis and prevent cell death. Therefore, clusterin can contribute to cisplatin resistance of retinoblastoma.

  11. Anti-apoptotic effect of clusterin on cisplatin-induced cell death of retinoblastoma cells.

    PubMed

    Song, Hyun Beom; Jun, Hyoung-Oh; Kim, Jin Hyoung; Yu, Young Suk; Kim, Kyu-Won; Min, Bon Hong; Kim, Jeong Hun

    2013-12-01

    Clusterin is a cytoprotective chaperone protein that is known to protect various retinal cells. It was also reported to be overexpressed in several types of malignant tumors, whose chemoresistance correlates with the expression of clusterin. Herein, we investigated the effect of clusterin on cisplatin-induced cell death of retinoblastoma cells. Firstly, evaluation of clusterin expression demonstrated that it was highly expressed in human retinoblastoma tissues and cell lines (SNUOT-Rb1 and Y79) particularly in the area between viable cells around vessels and necrotic zones in the relatively avascular area in human retinoblastoma tissues. Furthermore, the effects of cisplatin on retinoblastoma cells were evaluated. Cisplatin (1 µg/ml) significantly affected cell viability of SNUOT-Rb1 cells by inducing caspase-3-dependent apoptosis. Notably, the cell death due to cisplatin was prevented by 5 µg/ml of clusterin administered 4 h prior to cisplatin treatment by inhibiting cisplatin-induced apoptosis. Furthermore, overexpression of clusterin exerted its anti-apoptotic effect on cisplatin-induced apoptosis, and effectively prevented cisplatin-induced cell death. These data suggest that clusterin, found to be expressed in human retinoblastoma, may exert anti-apoptotic effects on cisplatin-induced apoptosis and prevent cell death. Therefore, clusterin can contribute to cisplatin resistance of retinoblastoma. PMID:24085287

  12. Mutagenic Effects of Iron Oxide Nanoparticles on Biological Cells

    PubMed Central

    Dissanayake, Niluka M.; Current, Kelley M.; Obare, Sherine O.

    2015-01-01

    In recent years, there has been an increased interest in the design and use of iron oxide materials with nanoscale dimensions for magnetic, catalytic, biomedical, and electronic applications. The increased manufacture and use of iron oxide nanoparticles (IONPs) in consumer products as well as industrial processes is expected to lead to the unintentional release of IONPs into the environment. The impact of IONPs on the environment and on biological species is not well understood but remains a concern due to the increased chemical reactivity of nanoparticles relative to their bulk counterparts. This review article describes the impact of IONPs on cellular genetic components. The mutagenic impact of IONPs may damage an organism’s ability to develop or reproduce. To date, there has been experimental evidence of IONPs having mutagenic interactions on human cell lines including lymphoblastoids, fibroblasts, microvascular endothelial cells, bone marrow cells, lung epithelial cells, alveolar type II like epithelial cells, bronchial fibroblasts, skin epithelial cells, hepatocytes, cerebral endothelial cells, fibrosarcoma cells, breast carcinoma cells, lung carcinoma cells, and cervix carcinoma cells. Other cell lines including the Chinese hamster ovary cells, mouse fibroblast cells, murine fibroblast cells, Mytilus galloprovincialis sperm cells, mice lung cells, murine alveolar macrophages, mice hepatic and renal tissue cells, and vero cells have also shown mutagenic effects upon exposure to IONPs. We further show the influence of IONPs on microorganisms in the presence and absence of dissolved organic carbon. The results shed light on the transformations IONPs undergo in the environment and the nature of the potential mutagenic impact on biological cells. PMID:26437397

  13. Microgravity-induced alterations in signal transduction in cells of the immune system

    NASA Astrophysics Data System (ADS)

    Paulsen, Katrin; Thiel, Cora; Timm, Johanna; Schmidt, Peter M.; Huber, Kathrin; Tauber, Svantje; Hemmersbach, Ruth; Seibt, Dieter; Kroll, Hartmut; Grote, Karl-Heinrich; Zipp, Frauke; Schneider-Stock, Regine; Cogoli, Augusto; Hilliger, Andre; Engelmann, Frank; Ullrich, Oliver

    2010-11-01

    Since decades it is known that the activity of cells of the immune system is severely dysregulated in microgravity, however, the underlying molecular aspects have not been elucidated yet. The identification of gravity-sensitive molecular mechanisms in cells of the immune system is an important and indispensable prerequisite for the development of counteractive measures to prevent or treat disturbed immune cell function of astronauts during long-term space missions. Moreover, their sensitivity to altered gravity renders immune cells an ideal model system to understand if and how gravity on Earth is required for normal mammalian cell function and signal transduction. We investigated the effect of simulated weightlessness (2D clinostat) and of real microgravity (parabolic flights) on key signal pathways in a human monocytic and a T lymphocyte cell line. We found that cellular responses to microgravity strongly depend on the cell-type and the conditions in which the cells are subjected to microgravity. In Jurkat T cells, enhanced phosphorylation of the MAP kinases ERK-1/2, MEK and p38 and inhibition of nuclear translocation of NF-kB were the predominant responses to simulated weightlessness, in either stimulated or non-stimulated cells. In contrast, non-stimulated monocytic U937 cells responded to simulated weightlessness with enhanced overall tyrosine-phosphorylation and activation of c-jun, whereas PMA-stimulated U937 cells responded the opposite way with reduced tyrosine-phosphorylation and reduced activation of c-jun, compared with PMA-stimulated 1 g controls. P53 protein was phosphorylated rapidly in microgravity. The identification of gravi-sensitive mechanisms in cells of the immune system will not only enable us to understand and prevent the negative effects of long time exposure to microgravity on Astronauts, but could also lead to novel therapeutic targets in general.

  14. Time between onset of apoptosis and release of nucleosomes from apoptotic cells: putative implications for systemic lupus erythematosus

    PubMed Central

    van Nieuwenhuijze, A E M; van Lopik, T; Smeenk, R; Aarden, L

    2003-01-01

    Objective: To investigate the kinetics of nucleosome leakage from apoptotic cells in an in vitro system and extrapolate the results to autoimmune disease, in particular systemic lupus erythematosus. Methods: A sensitive nucleosome enzyme linked immunosorbent assay (ELISA) was developed, using a monoclonal antibody (mAb) against histone 3 and an mAb against nucleosomes. Nucleosome release during apoptotic cell death was studied in Jurkat cells. AnnexinV binding (early apoptosis) and propidium iodide positivity (late apoptosis) of the cells were compared with nucleosome release at different times after apoptosis induction. Results: Nucleosomes appeared in culture supernatant of Jurkat cells 24 to 48 hours after apoptosis induction, when the cells had been late apoptotic for more than 12 hours. Conclusion: Nucleosomes are released from late apoptotic Jurkat cells, with a 12 hour delay from the appearance of AnnexinV binding cells. This result suggests that in vivo scavenger mechanisms have 12 hours to remove apoptotic material from the circulation. PMID:12480662

  15. The effect of TRAIL molecule on cell viability in in vitro beta cell culture.

    PubMed

    Tekmen, I; Ozyurt, D; Pekçetin, C; Buldan, Z

    2007-06-01

    Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disorder triggered by autoreactive T cells directed to pancreas beta-cell antigens. In this disorder, more than 90% of beta cells are destroyed. Cell death may be mediated via soluble or membrane-bound cell death ligands. One of these ligands may be tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-alpha superfamily. In the present study, we examined whether TRAIL had cytotoxic effects on adult rat pancreas beta cell cultures and INS1-E rat insulinoma cell line cultures or not. In this study, cell destruction models were built with TRAIL concentrations of 10, 100 and 1000 ng. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used for evaluating cell viability. It was detected that cell cultures with TRAIL added showed no differences statistically when compared with control cultures containing no toxic additions. These results showed that TRAIL did not have significant cytotoxic effects on pancreas beta cell culture and INS-1E rat insulinoma cell line cultures. Detection of the expression of TRAIL receptors and natural apoptosis inhibitor proteins will be favourable to investigate the resistance mechanisms to TRAIL-induced cell death in this cell culture system.

  16. Effects of cell cycle on the uptake of water soluble quantum dots by cells

    NASA Astrophysics Data System (ADS)

    Zheng, Shen; Chen, Ji-Yao; Wang, Jun-Yong; Zhou, Lu-Wei; Peng, Qian

    2011-12-01

    Quantum dots (QDs) with excellent optical properties have become powerful candidates for cell imaging. Although numerous reports have studied the uptake of QDs by cells, little information exists on the effects of cell cycle on the cellular QD uptake. In this report, the effects of cell cycle on the uptake of water soluble thiol-capped CdTe QDs by the human cervical carcinoma Hela cell line, human hepatocellular carcinoma QGY7701 cell line, and human embryonic kidney 293T cell line were studied by means of laser scanning confocal microscopy and flow cytometry. All three cell lines show to take up CdTe QDs via endocytosis. After arresting cells at specific phases with pharmacological agents, the cells in G2/M phase take up the most CdTe QDs, probably due to an increased membrane expansion during mitosis; whereas the cells in G1 phase do the least. A mathematical physics model was built to calculate the relative uptake rates of CdTe QDs by cells in different phases of the cell cycle, with the result as the uptake rate in G2/M phase is 2-4 times higher than that in G1 phase for these three cell lines. The results obtained from this study may provide the information useful for intracellular delivery of QDs.

  17. Effects of metformin on cell kinetic parameters of MCF-7 breast cancer cells in vitro.

    PubMed

    Topcul, Mehmet; Cetin, Idil

    2015-01-01

    In this study, the antiproliferative effects of the metformin was evaluated on MCF-7 Cells (human breast adenocarcinoma cell line). For this purpose cell kinetic parameters including cell proliferation assay, mitotic index and labelling index analysis were used. 30 μM, 65 μM and 130 μM Metformin doses were applied to cells for 24, 48 and 72 hours. The results showed that there was a significant decrease in cell proliferation, mitotic index and labelling index for all experimental groups (p<0.05) for all applications. PMID:25824763

  18. Tamoxifen induces oxidative stress and apoptosis in oestrogen receptor-negative human cancer cell lines

    PubMed Central

    Ferlini, C; Scambia, G; Marone, M; Distefano, M; Gaggini, C; Ferrandina, G; Fattorossi, A; Isola, G; Panici, P Benedetti; Mancuso, S

    1999-01-01

    Recent data have demonstrated that the anti-oestrogen tamoxifen (TAM) is able to facilitate apoptosis in cancer cells not expressing oestrogen receptor (ER). In an attempt to identify the biochemical pathway for this phenomenon, we investigated the role of TAM as an oxidative stress agent. In two ER-negative human cancer cell lines, namely T-leukaemic Jurkat and ovarian A2780 cancer cells, we have demonstrated that TAM is able to generate oxidative stress, thereby causing thiol depletion and activation of the transcriptional factor NF-κB. As described for other oxidative agents, TAM was able to induce either cell proliferation or apoptosis depending on the dose. When used at the lowest dose tested (0.1 μM), a slight proliferative effect of TAM was noticed in terms of cell counts and DNA synthesis rate, whereas at higher doses (10 μM) a consistent occurrence of apoptosis was detected. Importantly, the induction of apoptosis by TAM is not linked to down-regulation or functional inactivation by phosphorylation of the antiapoptotic bcl-2 protein. © 1999 Cancer Research Campaign PMID:9888466

  19. Evaluation of the use of Sr2+ in alginate immobilization of cells

    NASA Astrophysics Data System (ADS)

    Widerøe, Hege; Danielsen, Signe

    2001-05-01

    In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wide range of applications. In all cases high chemical stability of the immobilization material and mild conditions for the cells are prerequisites. However, in long-term experiments that may last for several days Ca-alginate may dissolve due to an exchange of Ca2+ with Na+, forming fluid Na-alginate. As well as Ca-alginate, the more chemically stable Sr-alginate and Ba-alginate are materials that have been used for the immobilization of living cells. In this study, the effects of Ca2+, Sr2+ and Ba2+ on growth, viability and intracellular free calcium concentration in a human leukemic T cell line (Jurkat) were investigated. The findings in this study, and the fact that Sr-alginate has a considerably higher chemical stability than Ca-alginate, led to the conclusion that Sr-alginate is a more suitable material for use in the entrapment of living cells in long-term studies.

  20. Evaluation of the use of Sr2+ in alginate immobilization of cells.

    PubMed

    Widerøe, H; Danielsen, S

    2001-05-01

    In recent years the method of immobilization of living cells in Ca-alginate beads has gained a wide range of applications. In all cases high chemical stability of the immobilization material and mild conditions for the cells are prerequisites. However, in long-term experiments that may last for several days Ca-alginate may dissolve due to an exchange of Ca2+ with Na+, forming fluid Na-alginate. As well as Ca-alginate, the more chemically stable Sr-alginate and Ba-alginate are materials that have been used for the immobilization of living cells. In this study, the effects of Ca2+, Sr2+ and Ba2+ on growth, viability and intracellular free calcium concentration in a human leukemic T cell line (Jurkat) were investigated. The findings in this study, and the fact that Sr-alginate has a considerably higher chemical stability than Ca-alginate, led to the conclusion that Sr-alginate is a more suitable material for use in the entrapment of living cells in long-term studies.

  1. Inhibitory effect of Disulfiram/copper complex on non-small cell lung cancer cells.

    PubMed

    Duan, Lincan; Shen, Hongmei; Zhao, Guangqiang; Yang, Runxiang; Cai, Xinyi; Zhang, Lijuan; Jin, Congguo; Huang, Yunchao

    2014-04-18

    Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in both men and women worldwide. Recently, Disulfiram has been reported to be able to inhibit glioblastoma, prostate, or breast cancer cell proliferation. In this study, the synergistic effect of Disulfiram and copper on NSCLC cell growth was investigated. Inhibition of cancer cell proliferation was detected by 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay and cell cycle analysis. Liquid colony formation and tumor spheroid formation assays were used to evaluate their effect on cancer cell clonogenicity. Real-time PCR was performed to test the mRNA level of cancer stem cell related genes. We found that Disulfiram or copper alone did not potently inhibit NSCLC cell proliferation in vitro. However, the presence of copper significantly enhanced inhibitory effect of Disulfiram on NSCLC cell growth, indicating a synergistic effect between Disulfiram and copper. Cell cycle analysis showed that Disulfiram/copper complex caused NSCLC cell cycle arrest in G2/M phase. Furthermore, Disulfiram/copper significantly increased the sensitivity of cisplatin in NSCLC cells tested by MTT assay. Liquid colony formation assay revealed that copper dramatically increased the inhibitory effect of Disulfiram on NSCLC cell colony forming ability. Disulfiram combined with copper significantly attenuated NSCLC cell spheroid formation and recuded the mRNA expression of lung cancer stem cell related genes. Our data suggest that Disulfiram/copper complex alone or combined with other chemotherapy is a potential therapeutic strategy for NSCLC patients.

  2. Effects of verteporfin-mediated photodynamic therapy on endothelial cells

    NASA Astrophysics Data System (ADS)

    Kraus, Daniel; Chen, Bin

    2015-03-01

    Photodynamic therapy (PDT) is a treatment modality in which cytotoxic reactive oxygen species are generated from oxygen and other biological molecules when a photosensitizer is activated by light. PDT has been approved for the treatment of cancers and age-related macular degeneration (AMD) due to its effectiveness in cell killing and manageable normal tissue complications. In this study, we characterized the effects of verteporfin-PDT on SVEC mouse endothelial cells and determined its underlying cell death mechanisms. We found that verteporfin was primarily localized in mitochondria and endoplasmic reticulum (ER) in SVEC cells. Light treatment of photosensitized SVEC cells induced a rapid onset of cell apoptosis. In addition to significant structural damages to mitochondria and ER, verteporfin-PDT caused substantial degradation of ER signaling molecules, suggesting ER stress. These results demonstrate that verteporfin-PDT triggered SVEC cell apoptosis by both mitochondrial and ER stress pathways. Results from this study may lead to novel therapeutic approaches to enhance PDT outcome.

  3. HIV cell-to-cell transmission: effects on pathogenesis and antiretroviral therapy

    PubMed Central

    Agosto, Luis M.; Uchil, Pradeep D.; Mothes, Walther

    2015-01-01

    The human immunodeficiency virus (HIV) spreads more efficiently in vitro when infected cells directly contact uninfected cells to form virological synapses. A hallmark of virological synapses is that viruses can be transmitted at a higher multiplicity of infection (MOI) that, in vitro, results in a higher number of proviruses. Whether HIV also spreads by cell-cell contact in vivo is a matter of debate. Here we discuss recent data that suggest that contact-mediated transmission largely manifests itself in vivo as CD4+ T cell depletion. The assault of a cell by a large number of incoming particles is likely efficiently sensed by the innate cellular surveillance to trigger cell death. The large number of particles transferred across virological synapses has also been implicated in reduced efficacy of antiretroviral therapies. Thus, antiretroviral therapies must remain effective against the high MOI observed during cell-to-cell transmission to inhibit both viral replication and the pathogenesis associated with HIV infection. PMID:25766144

  4. Effects of vibrations and shocks on lithium-ion cells

    NASA Astrophysics Data System (ADS)

    Brand, Martin J.; Schuster, Simon F.; Bach, Tobias; Fleder, Elena; Stelz, Manfred; Gläser, Simon; Müller, Jana; Sextl, Gerhard; Jossen, Andreas

    2015-08-01

    Lithium-ion batteries are increasingly used in mobile applications where mechanical vibrations and shocks are a constant companion. This work shows how these mechanical loads affect lithium-ion cells. Therefore pouch and cylindrical cells are stressed with vibrational and shock profiles according to the UN 38.3 standard. Additionally, a vibration test is set up to reflect stress in real-world applications and is carried out for 186 days. The effects of the load profiles on the tested cells are investigated by capacity measurement, impedance spectroscopy, micro-X-ray computed tomography and post mortem analyses. The mechanical stress has no effect on the investigated pouch cells. Although all tested cylindrical cells would pass the standard tests, in certain cells stressed in a vertical position the mandrel dispatched itself and struck against internal components. This caused bruised active materials, short circuits, a damaged current collector and current interrupt device. The investigations are not directly transferrable to all pouch or cylindrical cells but show that the mechanical cell design, especially the fixation of the internal components, determines whether a cell withstands vibrations and shocks. Depending on the cell design and the loading direction, long-term vibrational loads can have additional detrimental effects on lithium-ion cells compared to standard tests.

  5. Effects of metal ions on fibroblasts and spiral ganglion cells.

    PubMed

    Paasche, G; Ceschi, P; Löbler, M; Rösl, C; Gomes, P; Hahn, A; Rohm, H W; Sternberg, K; Lenarz, T; Schmitz, K-P; Barcikowski, S; Stöver, T

    2011-04-01

    Degeneration of spiral ganglion cells (SGC) after deafness and fibrous tissue growth around the electrode carrier after cochlear implantation are two of the major challenges in current cochlear implant research. Metal ions are known to possess antimicrobial and antiproliferative potential. The use of metal ions could therefore provide a way to reduce tissue growth around the electrode array after cochlear implantation. Here, we report on in vitro experiments with different concentrations of metal salts with antiproliferative and toxic effects on fibroblasts, PC-12 cells, and freshly isolated spiral ganglion cells, the target cells for electrical stimulation by a cochlear implant. Standard cell lines (NIH/3T3 and L-929 fibroblasts and PC-12 cells) and freshly isolated SGC were incubated with concentrations of metal ions between 0.3 μmol/liter and 10 mmol/liter for 48 hr. Cell survival was investigated by neutral red uptake, CellQuantiBlue assay, or counting of stained surviving neurons. Silver ions exhibited distinct thresholds for proliferating and confluent cells. For zinc ions, the effective concentration was lower for fibroblasts than for PC-12 cells. SGC showed comparable thresholds for reduced cell survival not only for silver and zinc ions but also for copper(II) ions, indicating that these ions might be promising for reducing tissue growth on the surface of CI electrode arrays. These effects were also observed when combinations of two of these ions were investigated. PMID:21312225

  6. Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium

    PubMed Central

    Chinthamani, Sreedevi; Odusanwo, Olutayo; Mondal, Nandini; Nelson, Joel; Neelamegham, Sriram

    2012-01-01

    Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A4 (LXA4) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA4 inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA4 thus appears to serve as an endogenous “stop signal” for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101–137, 2007). The role of LXA4 has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA4 decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA4 blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA4. Furthermore, LXA4 preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sjögren's syndrome (SS). PMID:22205391

  7. Effects of glucocorticoids on the interaction of lymphoblastoid cells with human endothelial cells in vitro.

    PubMed

    Maca, R D; Fry, G L; Hakes, A D

    1978-08-01

    The adhesive characteristics of cultured acute lymphocytic leukemia cells (CCRF-CEM), lymphoma cells (Raji), and freshly isolated acute lymphocytic leukemia cells to human cultured endothelial cells were studied. An assay system was used whereby these neoplastic cells were allowed to interact with endothelial cells while being continuously agitated on a rocking platform. All cell lines adhered significantly to the endothelium monolayers. This process appeared not to be dependent upon intact microtubular or microfilament function. Likewise, removing surface sialic acid from either cell type did not alter this process. In contrast incubating the endothelial cells for 24 or 48 hr with dexamethasone decreased adhesiveness of either CCRF-CEM or Raji cells to the endothelial cells by approximately 40%. Incubating these cells with hydrocortisone instead of dexamethasone for 48 hr was equally as effective in altering the endothelial cell adhesiveness. The decreased adhesiveness could be blocked by cycloheximide, indicating that this altered adhesiveness of the endothelial cells involves protein synthesis, presumably of a surface protein. We suggest that this assay system may provide a means to evaluate other agents that can alter the surface characteristics of endothelial cells, which may have important implications in various disease states such as inflammation, thrombogenesis, and metastatic disease. PMID:276420

  8. The effect of cell phones on human health

    NASA Astrophysics Data System (ADS)

    Abu-Isbeih, Ibrahim N.; Saad, Dina

    2011-10-01

    The effect of cell phone radiation on human health is the subject of recent interest and study, as a result of the enormous increase in cell phone usage throughout the world. Cell phones use electromagnetic radiation in the microwave range, which some believe may be harmful to human health. Other digital wireless systems, such as data communication networks, produce similar radiation. The objective of this survey is to review the effects of cell phones on human health: A large body of research exists, both epidemiological and experimental, in non-human animals and in humans, of which the majority shows no definite causative relationship between exposure to cell phones and harmful biological effects in humans. This is often paraphrased simply as the balance of evidence showing no harm to humans from cell phones, although a significant number of individual studies do suggest such a relationship, or are inconclusive.

  9. The Effect of Polybutadiene Polymer on Cell Aging In Vitro.

    NASA Astrophysics Data System (ADS)

    Liu, Ying; Collazo, Lourdes; Rafailovich, Miriam; Sokolov, Jonathan

    2006-03-01

    Cell experimentation often undergoes several weeks of culturing. The most common problem that scientists face is the variability of cell behavior due to subculturing. Most cells have a limited lifespan in vitro, changing their cell characteristic after just a few passages. Here we focus on the changes in cell function with passage. We used human CRF31 dermal fibroblasts initially cultured from lower passages (P11) to higher passages (P20) at a density of 5000/cm2. We first generated a series of cell growth curves for the different passages. We observed that as cell passage number increased, cell proliferation decreased significantly. Western Blot analysis indicated that the composition of the extracellular matrix proteins changed with increasing passage. The effect of these changes on migration and actin production will be presented.

  10. Effects of Polyhydroxybutyrate Production on Cell Division

    NASA Technical Reports Server (NTRS)

    Miller, Kathleen; Rahman, Asif; Hadi, Masood Z.

    2015-01-01

    Synthetic biological engineering can be utilized to aide the advancement of improved long-term space flight. The potential to use synthetic biology as a platform to biomanufacture desired equipment on demand using the three dimensional (3D) printer on the International Space Station (ISS) gives long-term NASA missions the flexibility to produce materials as needed on site. Polyhydroxybutyrates (PHBs) are biodegradable, have properties similar to plastics, and can be produced in Escherichia coli using genetic engineering. Using PHBs during space flight could assist mission success by providing a valuable source of biomaterials that can have many potential applications, particularly through 3D printing. It is well documented that during PHB production E. coli cells can become significantly elongated. The elongation of cells reduces the ability of the cells to divide and thus to produce PHB. I aim to better understand cell division during PHB production, through the design, building, and testing of synthetic biological circuits, and identify how to potentially increase yields of PHB with FtsZ overexpression, the gene responsible for cell division. Ultimately, an increase in the yield will allow more products to be created using the 3D printer on the ISS and beyond, thus aiding astronauts in their missions.

  11. Effects of maternal diabetes on trophoblast cells.

    PubMed

    Aires, Marlúcia Bastos; Dos Santos, Anne Carolline Veríssimo

    2015-03-15

    Diabetes mellitus (DM) is a health condition characterized by hyperglycemia over a prolonged period. There are three main types of DM: DM type 1 (DM1), DM2 and gestational DM (GDM). Maternal diabetes, which includes the occurrence of DM1 and DM2 during pregnancy or GDM, increases the occurrence of gesttional complications and adverse fetal outcomes. The hyperglycemic intrauterine environment affects not only the fetus but also the placental development and function in humans and experimental rodents. The underlying mechanisms are still unclear, but some evidence indicates alterations in trophoblast proliferation, apoptosis and cell cycle control in diabetes. A proper coordination of trophoblast proliferation, differentiation and invasion is required for placental development. Initially, increased expression of proliferative markers in junctional and labyrinth zones of rat placentas and villous cytotrophoblast, syncytiotrophoblast, stromal cells and fetal endothelial cells in human placentas is reported among diabetics. Moreover, reduced apoptotic index and expression of some apoptotic genes are described in placentas of GDM women. In addition, cell cycle regulators including cyclins and cyclin-dependent kinase inhibitors seem to be affected by the hyperglycemic environment. More studies are necessary to check the balance between proliferation, apoptosis and differentiation in trophoblast cells during maternal diabetes. PMID:25789116

  12. Physiological effects of microgravity on bone cells.

    PubMed

    Arfat, Yasir; Xiao, Wei-Zhong; Iftikhar, Salman; Zhao, Fan; Li, Di-Jie; Sun, Yu-Long; Zhang, Ge; Shang, Peng; Qian, Ai-Rong

    2014-06-01

    Life on Earth developed under the influence of normal gravity (1g). With evidence from previous studies, scientists have suggested that normal physiological processes, such as the functional integrity of muscles and bone mass, can be affected by microgravity during spaceflight. During the life span, bone not only develops as a structure designed specifically for mechanical tasks but also adapts for efficiency. The lack of weight-bearing forces makes microgravity an ideal physical stimulus to evaluate bone cell responses. One of the most serious problems induced by long-term weightlessness is bone mineral loss. Results from in vitro studies that entailed the use of bone cells in spaceflights showed modification in cell attachment structures and cytoskeletal reorganization, which may be involved in bone loss. Humans exposed to microgravity conditions experience various physiological changes, including loss of bone mass, muscle deterioration, and immunodeficiency. In vitro models can be used to extract valuable information about changes in mechanical stress to ultimately identify the different pathways of mechanotransduction in bone cells. Despite many in vivo and in vitro studies under both real microgravity and simulated conditions, the mechanism of bone loss is still not well defined. The objective of this review is to summarize the recent research on bone cells under microgravity conditions based on advances in the field.

  13. Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells.

    PubMed

    Hiwarkar, Prashant; Qasim, Waseem; Ricciardelli, Ida; Gilmour, Kimberly; Quezada, Sergio; Saudemont, Aurore; Amrolia, Persis; Veys, Paul

    2015-12-24

    Unrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naïve CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rg(null) mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P < .0003). Comparison of CB T cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7(+)CD8(+) T cells and prompt induction of cytotoxic CD8(+) and CD4(+) T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.

  14. Electron irradiation effects in epitaxial InP solar cells

    NASA Technical Reports Server (NTRS)

    Pearsall, N. M.; Robson, N.; Sambell, A. J.; Anspaugh, B.; Cross, T. A.

    1991-01-01

    Performance data for InP-based solar cells after irradiation with 1-MeV electrons up to a fluence of 1 x 1016 e/cm2 are presented. Three InP cell structures are considered. Two of these have epitaxially grown active regions, these being a homojunction design and in ITO/InP structure. These are compared with ITO/InP cells without the epitaxial base region. The cell parameter variations, the influence of illumination during irradiation, and the effect on cell spectral response and capacitance measurements are discussed. Substantial performance recovery after thermal annealing at 90 C is reported.

  15. The anticancer effects of hispolon on lung cancer cells.

    PubMed

    Wu, Qiuge; Kang, Yan; Zhang, Hui; Wang, Hongmin; Liu, Yuanhua; Wang, Jing

    2014-10-24

    Hispolon is isolated from Phellinus igniarius and exhibits antitumor activity. Here, we explored the effects of hispolon on the lung cancer A549 and H661 cells. Cells were incubated with various concentrations of hispolon (0, 5, 10, 20, 40, 80 or 160μM) for 12, 24, 48 or 72h. Cell viability was examined by MTT assay. Cell cycle and apoptosis assay were assessed by flow cytometry. Hispolon decreased cell viability in a dose- and time-dependent manner. The cell cycle distribution showed that hispolon enhanced the accumulations of the cells in G0/G1 phase. Mechanically, hispolon decreased the expression of G1-S transition-related proteins: Cyclin D1, cyclin E, CDK2, CDK4 and CDK6, but increased the expression of CDK inhibitor p21(CIP1) and p27(KIP1). Moreover, hispolon induced cell apoptosis through activation of the mitochondrial pathway, evidenced by the loss of mitochondrial membrane potential, the release of cytochrome c into cytosol, and the cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) in hispolon-treated cells. Additionally, hispolon enhanced the expression of p53, specific silencing of which almost completely reversed hispolon-mediated antitumor activity. Moreover, hispolon treatment was more effective on H661 cells than on A549 cells in inhibiting cell viability and inducing cell apoptosis. Our results indicate that hispolon inhibits the cell viability, induces G0/G1 cell cycle arrest and apoptosis in lung cancer cells and p53 plays a critical role in hispolon-mediated antitumor activity. PMID:25268766

  16. Cytotoxic activity of novel palladium-based compounds on leukemia cell lines.

    PubMed

    Antunovic, Maja; Kriznik, Bojana; Ulukaya, Engin; Yilmaz, Veysel T; Mihalic, Katarina C; Madunic, Josip; Marijanovic, Inga

    2015-02-01

    Effective treatment methods for human leukemia are under development, but so far none of them have been found to be completely satisfactory. It was recently reported that palladium complexes have significant anticancer activity as well as lower toxicity compared with some clinically used chemotherapeutics. The anticancer activities of two novel palladium(II) complexes, [Pd(sac)(terpy)](sac)·4H2O and [PdCl(terpy)](sac)·2H2O, were tested against three human leukemia cell lines, Jurkat, MOLT-4, and THP-1, in comparison with cisplatin and adriamycin. The cytotoxic effect of the drugs was determined using the MTT assay. Cell death was assessed using fluorescein isothiocyanate-annexin/propidium iodide staining for flow cytometry. Furthermore, p53 phosphorylation, poly(ADP-ribose) polymerase cleavage, and Bax and Bcl-2 mRNA levels were examined to elucidate the mechanism of cell death induction. Both complexes exhibited a significant dose-dependent antigrowth effect in vitro. The complexes predominately induced apoptosis, but necrosis was also observed. In-vitro results have shown that palladium(II) complexes may be regarded as potential anticancer agents for treating human leukemia. Therefore, further analysis to determine the putative mechanism of action and in-vivo studies on animal models are warranted.

  17. Effects of citric acid on cultured human osteoblastic cells.

    PubMed

    Guimarães, Lizandra Ferrari; Fidalgo, Tatiana Kelly da Silva; Menezes, Gustavo Conde; Primo, Laura Guimarães; Costa e Silva-Filho, Fernando

    2010-11-01

    We investigated the effects of citric acid (CA) on cultured human osteoblastic (HOB) cells by evaluating cell adhesion, proliferation, and cytotoxicity. (3)H-Thymidine-labeled HOB cells were incubated in culture medium supplemented or not with 4%, 6%, 8%, or 10% CA for 1 minute. After incubation, cell morphology was evaluated by Nomarski interferential light microscopy, cell proliferation was accessed by measurements of (3)H-thymidine associated to the cells, and cell lysis was monitored by measuring the amount of (3)H-thymidine released by cells. We observed that most of the CA-treated cells presented numerous atypical vacuoles, and such cells were also highly polymorphic, exhibiting round-shaped cells. Nonetheless, CA at all concentrations assayed did not yield cytotoxicity as measured by (3)H-containing DNA release, although significant decrease in cell proliferation was observed (P > .05). Furthermore, cells which were treated with CA at the lowest concentration assayed (4%) restored normal proliferation rates 3 days after treatment.

  18. Antiretroviral Agents Effectively Block HIV Replication after Cell-to-Cell Transfer

    PubMed Central

    Permanyer, Marc; Ballana, Ester; Ruiz, Alba; Badia, Roger; Riveira-Munoz, Eva; Gonzalo, Encarna; Clotet, Bonaventura

    2012-01-01

    Cell-to-cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4+ T cells or lymphoid cells were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (RTIs) zidovudine (AZT) and tenofovir, but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency to cell-free virus infections. Cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when HIV-1 long terminal repeat (LTR)-driven green fluorescent protein (GFP) expression in target cells was measured. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication-independent Tat-mediated LTR transactivation as a consequence of cell-to-cell events that did not occur in short-term (48-h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell-to-cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell-to-cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo. PMID:22696642

  19. Cell phone radiation effects on cytogenetic abnormalities of oral mucosal cells.

    PubMed

    Daroit, Natália Batista; Visioli, Fernanda; Magnusson, Alessandra Selinger; Vieira, Geila Radunz; Rados, Pantelis Varvaki

    2015-01-01

    The aim of this study was to evaluate the effects of exposure to cell phone electromagnetic radiation on the frequency of micronuclei, broken eggs cells, binucleated cells, and karyorrhexis in epithelial cells of the oral mucosa. The sample was composed of 60 cell phone users, who were non-smokers and non-drinkers, and had no clinically visible oral lesions. Cells were obtained from anatomical sites with the highest incidence of oral cancer: lower lip, border of the tongue, and floor of the mouth. The Feulgen reaction was used for quantification of nuclear anomalies in 1,000 cells/slide. A slightly increase in the number of micronucleated cells in the lower lip and in binucleated cells on the floor of the mouth was observed in individuals who used their phones > 60 minutes/week. The analysis also revealed an increased number of broken eggs in the tongue of individuals owning a cell phone for over eight years. Results suggest that exposure to electromagnetic waves emitted by cell phones can increase nuclear abnormalities in individuals who use a cell phone for more than 60 minutes per week and for over eight years. Based on the present findings, we suggest that exposure to electromagnetic radiation emitted by cell phones may interfere with the development of metanuclear anomalies. Therefore, it is demonstrated that, despite a significant increase in these anomalies, the radiation emitted by cell phones among frequent users is within acceptable physiological limits.

  20. Cell phone radiation effects on cytogenetic abnormalities of oral mucosal cells.

    PubMed

    Daroit, Natália Batista; Visioli, Fernanda; Magnusson, Alessandra Selinger; Vieira, Geila Radunz; Rados, Pantelis Varvaki

    2015-01-01

    The aim of this study was to evaluate the effects of exposure to cell phone electromagnetic radiation on the frequency of micronuclei, broken eggs cells, binucleated cells, and karyorrhexis in epithelial cells of the oral mucosa. The sample was composed of 60 cell phone users, who were non-smokers and non-drinkers, and had no clinically visible oral lesions. Cells were obtained from anatomical sites with the highest incidence of oral cancer: lower lip, border of the tongue, and floor of the mouth. The Feulgen reaction was used for quantification of nuclear anomalies in 1,000 cells/slide. A slightly increase in the number of micronucleated cells in the lower lip and in binucleated cells on the floor of the mouth was observed in individuals who used their phones > 60 minutes/week. The analysis also revealed an increased number of broken eggs in the tongue of individuals owning a cell phone for over eight years. Results suggest that exposure to electromagnetic waves emitted by cell phones can increase nuclear abnormalities in individuals who use a cell phone for more than 60 minutes per week and for over eight years. Based on the present findings, we suggest that exposure to electromagnetic radiation emitted by cell phones may interfere with the development of metanuclear anomalies. Therefore, it is demonstrated that, despite a significant increase in these anomalies, the radiation emitted by cell phones among frequent users is within acceptable physiological limits. PMID:26486771

  1. PTD-hFOXP3 protein acts as an immune regulator to convert human CD4(+)CD25(-) T cells to regulatory T-like cells.

    PubMed

    Liu, Xia; Xu, Xun; Lin, Xin; Tian, Yuxiang; Ji, Baoju; Xia, Sheng; Xu, Sanrong; Yin, Qing; Zhang, Miaomiao; Jiao, Zhijun; Wang, Shengjun; Xu, Huaxi; Shao, Qixiang

    2012-12-01

    Regulatory T cells (Tregs) are critical for maintaining self-tolerance and homeostasis, and have potential application in clinical disease therapy, such as autoimmune diseases and transplant rejection, but their numbers are limited. FOXP3 is a key transcription factor controlling Tregs development and function. Although transfection of CD4(+)CD25(-) lymphocytes with the FOXP3 gene can convert them to Treg-like cells, there is the risk of insertional mutagenesis and thus an alternative to genetic intervention is sought. The protein transduction domain (PTD) from the HIV transactivator of transcription is a useful tool to deliver protein to the cytoplasm and nucleus. In this study, we generated a fusion protein linking the human FOXP3 to PTD (PTD-hFOXP3), and explored its function in T cells. The results showed that the PTD rapidly and effectively delivered the hFOXP3 protein into cells where it localized not only in the cytoplasm, but also to the nucleus. PTD-hFOXP3-transduced Jurkat cells (human T lymphoma cell line) and CD4(+)CD25(-) T cells failed to proliferate and produce IL-2 and IFN-γ, but produced large amounts of the cytokines IL-4, IL-10, and TGF-β, in response to TCR stimulation in vitro. PTD-hFOXP3-transduced CD4(+)CD25(-) T cells also expressed high levels of CTLA-4 and low levels of CD25 after stimulation. Most importantly, PTD-hFOXP3-transduced T cells inhibited the proliferation of activated CD4(+)CD25(-) T cells. Furthermore, chromatin immunoprecipitation assays demonstrated that PTD-hFOXP3 can bind with the IL-2 gene promoter and repress the expression of IL-2. These results indicate that PTD-hFOXP3 has the capability to convert conventional T cells to Treg-like cells.

  2. The effects of T cells and their products on in vitro healing of epitenon cell microwounds.

    PubMed Central

    Wòjciak, B; Crossan, J F

    1994-01-01

    The purpose of the present investigation was to study the activity and behaviour of rat epitenon cells cultured in the presence of activated helper/inducer T lymphocytes and T-cell-derived cytokines interleukin-1 (IL-1), IL-2, transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma). We measured the speed of healing of microwounded monolayers of epitenon cells as well as intercellular adhesion, cell proliferation and fibronectin production. The speed of monolayer healing was increased in the presence of activated CD4+ T lymphocytes and cytokines: TGF-beta > IL-2 > IL-1 > IFN-gamma. TGF-beta and IL-2 were also found to promote cell-cell adhesion; other cytokines had a minor effect. IL-2, IL-1 and IFN-gamma promoted [3H]thymidine incorporation in epitenon cells whereas TGF-beta had an inhibitory effect. Fibronectin production was studied with immunofluorescence staining methods. Activated CD4+ T lymphocytes and TGF-beta stimulated the deposition of fibronectin whereas other cytokines did not have a significant effect. Our results suggest that activated T lymphocytes and T-cell-derived cytokines, especially IL-2 and TGF-beta play a crucial role in the regulation of epitenon cell proliferation, adhesion and extracellular matrix production during in vitro microwound healing. As epitenon cells are the main cell type participating in tendon repair, factors regulating their activity may find application in clinical practice. Images Figure 1 PMID:7821974

  3. Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture.

    PubMed

    Beauchesne, Pascal R; Bruce, Katherine J; Bowen, Bruce D; Piret, James M

    2010-04-15

    Recombinant retroviruses are effective vectors able to integrate transgenes into the target cell's genome to achieve longer-term expression. This study investigates the effect of cell lysis products, a common cell culture by-product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates derived from human and murine suspension cell lines significantly increased the transduction of human TF-1 and K-562 cell lines by gibbon ape leukemia virus-pseudotyped retroviral vectors without altering tropism. The transduction efficiency of TF-1 cells increased as a function of lysate concentration and decreased with increasing target cell concentrations. This was adequately predicted using a saturation equation based on the lysed-to-target cell concentration ratio, R, where: Fold increase = 1+Fold_(Max) (R/(K_(L)+R)). Lysate completely masked the effects of fibronectin when the two were added in combination. With protamine sulfate, the transduction efficiency was increased by lysate to 58% from 20% for protamine sulfate alone. Overall, the presence of cell lysate significantly influenced the outcome of the transduction process, either alone or in the presence of protamine sulfate or fibronectin. PMID:20014140

  4. Immunosuppressive Effects of Erythropoietin on Human Alloreactive T Cells

    PubMed Central

    Cravedi, Paolo; Manrique, Joaquin; Hanlon, Katherine E.; Reid-Adam, Jessica; Brody, Joshua; Prathuangsuk, Praeophayom; Mehrotra, Anita

    2014-01-01

    Correction of anemia with erythropoietin (EPO) is associated with improved kidney transplant outcomes. Emerging evidence, predominantly from animal models, indicates that these observations may be erythropoiesis-independent and that EPO exhibits immunosuppressive properties. We examined the effects of EPO on human T-cell alloimmunity by first documenting that CD4+ and CD8+ T cells express EPO receptor (EPO-R) on their surfaces. In mixed lymphocyte reactions, EPO induced a dose-dependent decrease in allogeneic CD4+ T-cell proliferation (EPO 1000 U/ml: 44.6%±22.9% of vehicle, P<0.05; 2000 U/ml: 11.1%±4% of vehicle, P<0.001) without inducing cell death. The effects required signals transmitted directly through the EPO-R expressed on T cells, resulting in diminished Th1 differentiation without effects on regulatory T-cell induction. Mechanistic studies revealed that EPO prevented IL-2–induced proliferation by uncoupling IL-2 receptor signaling, inhibiting phosphorylation of the intracellular intermediaries AKT and extracellular signal-regulated kinase that are known to mediate T-cell expansion. EPO treatment reduced expansion of human naïve CD4+ T cells after adoptive transfer into NOD scid γcnull mouse recipients, verifying the effects in vivo. Although activated T cells expressed CD131, an alternative EPO receptor, addition of a specific CD131 agonist peptide, ARA290, did not alter T-cell proliferation or cytokine production. Our findings link EPO-R signaling on T cells to inhibition of T-cell immunity, providing one mechanism that could explain the observed protective effects of EPO in kidney transplant recipients. PMID:24676641

  5. Immunosuppressive effects of erythropoietin on human alloreactive T cells.

    PubMed

    Cravedi, Paolo; Manrique, Joaquin; Hanlon, Katherine E; Reid-Adam, Jessica; Brody, Joshua; Prathuangsuk, Praeophayom; Mehrotra, Anita; Heeger, Peter S

    2014-09-01

    Correction of anemia with erythropoietin (EPO) is associated with improved kidney transplant outcomes. Emerging evidence, predominantly from animal models, indicates that these observations may be erythropoiesis-independent and that EPO exhibits immunosuppressive properties. We examined the effects of EPO on human T-cell alloimmunity by first documenting that CD4(+) and CD8(+) T cells express EPO receptor (EPO-R) on their surfaces. In mixed lymphocyte reactions, EPO induced a dose-dependent decrease in allogeneic CD4(+) T-cell proliferation (EPO 1000 U/ml: 44.6%±22.9% of vehicle, P<0.05; 2000 U/ml: 11.1%±4% of vehicle, P<0.001) without inducing cell death. The effects required signals transmitted directly through the EPO-R expressed on T cells, resulting in diminished Th1 differentiation without effects on regulatory T-cell induction. Mechanistic studies revealed that EPO prevented IL-2-induced proliferation by uncoupling IL-2 receptor signaling, inhibiting phosphorylation of the intracellular intermediaries AKT and extracellular signal-regulated kinase that are known to mediate T-cell expansion. EPO treatment reduced expansion of human naïve CD4(+) T cells after adoptive transfer into NOD scid γc(null) mouse recipients, verifying the effects in vivo. Although activated T cells expressed CD131, an alternative EPO receptor, addition of a specific CD131 agonist peptide, ARA290, did not alter T-cell proliferation or cytokine production. Our findings link EPO-R signaling on T cells to inhibition of T-cell immunity, providing one mechanism that could explain the observed protective effects of EPO in kidney transplant recipients.

  6. The effects of glucocorticoids on cultured human endothelial cells.

    PubMed

    Maca, R D; Fry, G L; Hoak, J C

    1978-04-01

    The effects of hydrocortisone, dexamethasone and prednisone on the morphology, replication, DNA synthesis, cell protein content and protein synthesis of cultured, human endothelial cells were evaluated. After culturing the cells with these glucocorticoids for 24-48 h, the cells covered a greater portion of the culture surface area. The mean surface area of the individual endothelial cell treated with glucocorticoids was 1.53 times greater than that of the untreated control endothelial cell. When compared with controls, the endothelial cover provided by the cells treated with glucocorticoids was more extensive and in many instances covered the entire culture surface. The change in morphology was associated with an increase in protein synthesis and protein content of the cells without an increase in DNA synthesis or cellular replication. Dexamethasone was approximately 10-fold more effective than hydrocortisone, while prednisone was the least effective. Aldosterone, DOCA, testosterone, progesterone, oestradiol and oestriol were ineffective. These studies indicate that glucocorticoids can alter the morphology and biochemistry of cultured endothelial cells and may have implications for the effects of steroids in the treatment of thrombocytopenic states and vascular disorders in man. PMID:646949

  7. Do cell-autonomous and non-cell-autonomous effects drive the structure of tumor ecosystems?

    PubMed

    Tissot, Tazzio; Ujvari, Beata; Solary, Eric; Lassus, Patrice; Roche, Benjamin; Thomas, Frédéric

    2016-04-01

    By definition, a driver mutation confers a growth advantage to the cancer cell in which it occurs, while a passenger mutation does not: the former is usually considered as the engine of cancer progression, while the latter is not. Actually, the effects of a given mutation depend on the genetic background of the cell in which it appears, thus can differ in the subclones that form a tumor. In addition to cell-autonomous effects generated by the mutations, non-cell-autonomous effects shape the phenotype of a cancer cell. Here, we review the evidence that a network of biological interactions between subclones drives cancer cell adaptation and amplifies intra-tumor heterogeneity. Integrating the role of mutations in tumor ecosystems generates innovative strategies targeting the tumor ecosystem's weaknesses to improve cancer treatment. PMID:26845682

  8. Induction of Cell Death Mechanisms and Apoptosis by Nanosecond Pulsed Electric Fields (nsPEFs)

    PubMed Central

    Beebe, Stephen J.; Sain, Nova M.; Ren, Wei

    2013-01-01

    Pulse power technology using nanosecond pulsed electric fields (nsPEFs) offers a new stimulus to modulate cell functions or induce cell death for cancer cell ablation. New data and a literature review demonstrate fundamental and basic cellular mechanisms when nsPEFs interact with cellular targets. NsPEFs supra-electroporate cells creating large numbers of nanopores in all cell membranes. While nsPEFs have multiple cellular targets, these studies show that nsPEF-induced dissipation of ΔΨm closely parallels deterioration in cell viability. Increases in intracellular Ca2+ alone were not sufficient for cell death; however, cell death depended of the presence of Ca2+. When both events occur, cell death ensues. Further, direct evidence supports the hypothesis that pulse rise-fall times or high frequency components of nsPEFs are important for decreasing ΔΨm and cell viability. Evidence indicates in Jurkat cells that cytochrome c release from mitochondria is caspase-independent indicating an absence of extrinsic apoptosis and that cell death can be caspase-dependent and –independent. The Ca2+ dependence of nsPEF-induced dissipation of ΔΨm suggests that nanoporation of inner mitochondria membranes is less likely and effects on a Ca2+-dependent protein(s) or the membrane in which it is embedded are more likely a target for nsPEF-induced cell death. The mitochondria permeability transition pore (mPTP) complex is a likely candidate. Data demonstrate that nsPEFs can bypass cancer mutations that evade apoptosis through mechanisms at either the DISC or the apoptosome. PMID:24709649

  9. The effects of acoustic vibration on fibroblast cell migration.

    PubMed

    Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

    2016-12-01

    Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. PMID:27612824

  10. The effects of IL-17 upon human natural killer cells.

    PubMed

    Al Omar, Suliman; Flanagan, Brian F; Almehmadi, Mazen; Christmas, Stephen E

    2013-04-01

    These experiments were designed to investigate the effects of IL-17 upon the phenotype and function of human Natural Killer (NK) cells. Peripheral blood mononuclear cells from healthy subjects were cultured in the presence or absence of different combinations of IL-17s and changes in relative numbers and cell surface phenotype of NK cells and CD56+CD3+ cells measured by flow cytometry. Real time PCR was used to measure changes in expression of the cytotoxicity-related genes perforin A and granzymes A and B and IL-17 receptors. A chromium release assay was used to measure cytotoxic function against K562 tumour cells. IL-17D, IL-17A, IL-17F or the combination of both of the latter had little effect upon NK cell surface expression of Killer Immunoglobulin-like Receptors, although IL-17A modestly increased NK cell numbers. Slight but not significant increases in expression of perforin and granzymes were induced by IL-17A and/or IL-17F. Both IL-17A and D significantly increased cytotoxic function of NK cells at some E:T ratios. Similarly, numbers of NK cells induced to express CD107a after interaction with K562 cells were increased, but not significantly, by all combinations of IL-17s tested. IL-17RC was not found at the NK cell surface but was expressed at the message level and the protein detected intracellularly. NK cells are known to produce IL-17 but here we report that there is little response to this cytokine although some isoforms may moderately enhance cytotoxic function. There may therefore be some enhancement of NK cell function resulting from Th17 cell activation.

  11. Cell adhesion: The effect of a surprising cohesive force

    NASA Astrophysics Data System (ADS)

    Vasseur, H.

    2009-10-01

    When an experimentalist or a biological mechanism applies an external force onto a cell chemically sticking to its substrate, a reacting “suction” force, due to the slow penetration of the surrounding fluid between the cell and the substrate, opposes to the dissociation. This force can overcome other known adhesive forces when the process is sufficiently violent (typically 105pN ). Its maximal contribution to the total adhesive energy of the cell can then be estimated to 2×10-3J/m2 . The physical origin of this effect is quite simple and it may be compared to that leaning a “suction cup” against a bathroom wall. We address the consequences of this effect on (i) the separation energy, (ii) the motion of the fluid surrounding the cell, and more especially on the pumping of the fluid by moving cells, and (iii) the inhibition of cell motion.

  12. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  13. Effects of vibration on differentiation of cultured PC12 cells.

    PubMed

    Ito, Yukiko; Kimura, Tsuyoshi; Nam, Kwangwoo; Katoh, Ayako; Masuzawa, Toru; Kishida, Akio

    2011-03-01

    Different types of physiological-mechanical stress, such as shear stress in vascular endothelial cells or hydrostatic pressure in chondrocytes are well known as regulators of cell function. In this study, the effects of vibration, a type of non-physiological mechanical stimulation, on differentiation of rat pheochromocytoma (PC12) cells are reported. A nano-vibration system was designed to produce nanometer-scale vibration. The frequency and amplitude of the nano-vibrations were monitored by a capacitance displacement sensor connected to an oscilloscope. When PC12 cells exposed to nerve growth factor were subjected to vibration at 10 kHz, differentiation and elongation of their neurites were promoted earlier in the culture. Vibration promoted differentiation of PC12 cells. This approach could therefore also be promising for determining of the effects of the physical environment on cell differentiation.

  14. 3,3′-Diindolylmethane Induces G1 Arrest and Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia Cells

    PubMed Central

    Shorey, Lyndsey E.; Hagman, Amanda M.; Williams, David E.; Ho, Emily; Dashwood, Roderick H.; Benninghoff, Abby D.

    2012-01-01

    Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL. PMID:22514694

  15. Effect of gyromagnetic fields on human prostatic adenocarcinoma cells

    PubMed Central

    Lei, Hongen; Xu, Yongde; Guan, Ruili; Li, Meng; Hui, Yu; Gao, Zhezhu; Yang, Bicheng; Xin, Zhongcheng

    2015-01-01

    Purpose To investigate the biological effect of gyromagnetic fields (GMFs) on cell proliferation and apoptosis of human prostatic adenocarcinoma cells and explore the underlying mechanisms. Methods PC-3 cells were grouped into normal control (NC) and GMF treatment groups. Cell proliferation was analyzed with kit-8 and Ki67 immunofluorescence staining, while cell apoptosis was analyzed with flow cytometry double staining of Annexin V-PE/7-AAD. The Akt and p38 MAPK/Caspase signaling pathways were analyzed by western blotting and immunofluorescence staining, and cell polarization was analyzed with PARD3. Results Cell proliferation and activity of the Akt pathway were significantly decreased by the GMF, while cell apoptosis, activity of p38 MAPK, and PARD3-positive cell number were significantly increased in the GMF group compared to the NC group. Conclusion GMFs inhibit cell proliferation, induce apoptosis, and regulate tumor cell polarity conditions, potentially through down-regulating Akt, activating the p38 MAPK/Caspase pathway, and promoting PARD3 expression in PC-3 cells. PMID:26648740

  16. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    PubMed

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity. PMID:27262873

  17. An Investigation of the Growth Inhibitory Capacity of Several Medicinal Plants From Iran on Tumor Cell Lines

    PubMed Central

    Esmaeilbeig, Maryam; Kouhpayeh, Seyed Amin; Amirghofran, Zahra

    2015-01-01

    Background: Traditional herbal medicine is a valuable resource that provides new drugs for cancer treatment. Objectives: In this study we aim to screen and investigate the in vitro anti-tumor activities of ten species of plants commonly grown in Southern Iran. Materials and Methods: We used the MTT colorimetric assay to evaluate the cytotoxic activities of the methanol extracts of these plants on various tumor cell lines. The IC50 was calculated as a scale for this evaluation. Results: Satureja bachtiarica, Satureja hortensis, Thymus vulgaris, Thymus daenensis and Mentha lonigfolia showed the inhibitoriest effects on Jurkat cells with > 80% inhibition at 200 µg/mL. Satureja hortensis (IC50: 66.7 µg/mL) was the most effective. These plants also strongly inhibited K562 cell growth; Satureja bachtiarica (IC50: 28.3 µg/mL), Satureja hortensis (IC50: 52 µg/mL) and Thymus vulgaris (IC50: 87 µg/mL) were the most effective extracts. Cichorium intybus, Rheum ribes, Alhagi pseudalhagi and Glycyrrihza glabra also showed notable effects on the leukemia cell lines. The Raji cell line was mostly inhibited by Satureja bachtiarica and Thymus vulgaris with approximately 40% inhibition at 200µg/ml. The influence of these extracts on solid tumor cell lines was not strong. Fen cells were mostly affected by Glycyrrihza glabra (IC50: 182 µg/mL) and HeLa cells by Satureja hortensis (31.6% growth inhibitory effect at 200 µg/mL). Conclusions: Leukemic cell lines were more sensitive to the extracts than the solid tumor cell lines; Satureja hortensis, Satureja bachtiarica, Thymus vulgaris, Thymus daenensis and Mentha lonigfolia showed remarkable inhibitory potential. PMID:26634114

  18. The Effect of Titanium Dioxide Nanoparticles on Keratinocyte Cell (KC) and Squamous Cell Carcinoma (SCC-13)

    NASA Astrophysics Data System (ADS)

    Lin, Chienhsiu; Simon, Marcia; Jurukovski, Vladimir; Lee, Wilson; Rafailovich, Miriam

    2009-03-01

    We have studied the effects of TiO2 nanoparticles on cell keratinocyte and SCC (Squamous Cell Carcinoma) cells. We found that the concentration of particles required to adversely affect the cells was many times higher for keratinocyte than SCC cells. Confocal microscope shows that the particles in keratinocyte culture are sequestered in membranes between the cell colonies. The particles penetrated into the cells in the case of the SCC cells. TEM images revealed very few particles in the keratinocyte, many more particles were observed sequestered in vacuole of the SCC cells. These results indicate that the keratinocyte layer behaves very different from the fibroblast layers which are much more sensate to TiO2 nanoparticle damage and may suggest a protection mechanism of the dermal tissue. The effect of UV exposure in the presence of DNA was also investigated. We found that adsorbed proteins, as well as grafted polymer provided a measure of protection against free radical formation. The effects of low level UV exposure when the particles are near in-vitro cell culture will be presented.

  19. Effects of chronic cocaine in rat C6 astroglial cells

    PubMed Central

    BADISA, RAMESH B.; GOODMAN, CARL B.

    2012-01-01

    Investigations with astroglial cells carry equal importance as those with neurons in drug abuse studies. The present study was aimed to investigate the effect of chronic cocaine administration on cell viability, nitric oxide (NO) production, general respiratory status of mitochondria and total protein levels in rat astroglioma cells after 24 h of treatment. In addition, the effect of cocaine was assessed for 24 h on brine shrimp larvae in order to study their sensitivity to the drug. It was observed that cocaine caused a significant dose-dependent decrease in astroglial cell viability with an LC50 of 4.717 mM. It was found that cocaine did not induce or inhibit NO production in the cells. Evaluation of mitochondrial dehydrogenase activity in terms of formazan production in astroglial cells indicated that cocaine significantly interfered with the general respiratory status of mitochondria with an ED50 of 6.153 mM. Furthermore, cocaine was shown to deplete the total protein levels in the cells with an ED50 of 5.435 mM. In vivo study with brine shrimp larvae showed that these larvae were highly sensitive to cocaine with an ED50 of 2.41 mM. In summary, our findings suggest that cocaine-induced cytotoxicity in the cells was non-specific. The cumulative effect arising from the significant loss of respiration and total cellular proteins is the cause of astroglial cell death. PMID:22735768

  20. Effects of chronic cocaine in rat C6 astroglial cells.

    PubMed

    Badisa, Ramesh B; Goodman, Carl B

    2012-09-01

    Investigations with astroglial cells carry equal importance as those with neurons in drug abuse studies. The present study was aimed to investigate the effect of chronic cocaine administration on cell viability, nitric oxide (NO) production, general respiratory status of mitochondria and total protein levels in rat astroglioma cells after 24 h of treatment. In addition, the effect of cocaine was assessed for 24 h on brine shrimp larvae in order to study their sensitivity to the drug. It was observed that cocaine caused a significant dose-dependent decrease in astroglial cell viability with an LC(50) of 4.717 mM. It was found that cocaine did not induce or inhibit NO production in the cells. Evaluation of mitochondrial dehydrogenase activity in terms of formazan production in astroglial cells indicated that cocaine significantly interfered with the general respiratory status of mitochondria with an ED(50) of 6.153 mM. Furthermore, cocaine was shown to deplete the total protein levels in the cells with an ED(50) of 5.435 mM. In vivo study with brine shrimp larvae showed that these larvae were highly sensitive to cocaine with an ED(50) of 2.41 mM. In summary, our findings suggest that cocaine-induced cytotoxicity in the cells was non-specific. The cumulative effect arising from the significant loss of respiration and total cellular proteins is the cause of astroglial cell death. PMID:22735768

  1. Growth-stimulatory effect of resveratrol in human cancer cells.

    PubMed

    Fukui, Masayuki; Yamabe, Noriko; Kang, Ki Sung; Zhu, Bao Ting

    2010-08-01

    Earlier studies have shown that resveratrol could induce death in several human cancer cell lines in culture. Here we report our observation that resveratrol can also promote the growth of certain human cancer cells when they are grown either in culture or in athymic nude mice as xenografts. At relatively low concentrations (effect in the MDA-MB-435s human cancer cells, but this effect was not observed in several other human cell lines tested. Analysis of cell signaling molecules showed that resveratrol induced the activation of JNK, p38, Akt, and NF-kappaB signaling pathways in these cells. Further analysis using pharmacological inhibitors showed that only the NF-kappaB inhibitor (BAY11-7082) abrogated the growth-stimulatory effect of resveratrol in cultured cells. In athymic nude mice, resveratrol at 16.5 mg/kg body weight enhanced the growth of MDA-MB-435s xenografts compared to the control group, while resveratrol at the 33 mg/kg body weight dose did not have a similar effect. Additional analyses confirmed that resveratrol stimulated cancer cell growth in vivo through activation of the NF-kappaB signaling pathway. Taken together, these observations suggest that resveratrol at low concentrations could stimulate the growth of certain types of human cancer cells in vivo. This cell type-specific mitogenic effect of resveratrol may also partly contribute to the procarcinogenic effect of alcohol consumption (rich in resveratrol) in the development of certain human cancers.

  2. Antitumoral Effect of Hibiscus sabdariffa on Human Squamous Cell Carcinoma and Multiple Myeloma Cells.

    PubMed

    Malacrida, Alessio; Maggioni, Daniele; Cassetti, Arianna; Nicolini, Gabriella; Cavaletti, Guido; Miloso, Mariarosaria

    2016-10-01

    Cancer is a leading cause of death worldwide. Despite therapeutic improvements, some cancers are still untreatable. Recently there has been an increasing interest in the use of natural substances for cancer prevention and treatment. Hibiscus sabdariffa (HS) is a plant, belonging to Malvaceae family, widespread in South Asia and Central Africa. HS extract (HSE) used in folk medicine, gained researchers' interest thanks to its antioxidant, anti-inflammatory, and chemopreventive properties. In the present study, we initially assessed HSE effect on a panel of human tumor cell lines. Then we focused our study on the following that are most sensitive to HSE action cell lines: Multiple Myeloma (MM) cells (RPMI 8226) and Oral Squamous Cell Carcinoma (OSCC) cells (SCC-25). In both RPMI 8226 and SCC-25 cells, HSE impaired cell growth, exerted a reversible cytostatic effect, and reduced cell motility and invasiveness. We evaluated the involvement of MAPKs ERK1/2 and p38 in HSE effects by using specific inhibitors, U0126 and SB203580, respectively. For both SCC-25 and RPMI 8226, HSE cytostatic effect depends on p38 activation, whereas ERK1/2 modulation is crucial for cell motility and invasiveness. Our results suggest that HSE may be a potential therapeutic agent against MM and OSCC.

  3. Antitumoral Effect of Hibiscus sabdariffa on Human Squamous Cell Carcinoma and Multiple Myeloma Cells.

    PubMed

    Malacrida, Alessio; Maggioni, Daniele; Cassetti, Arianna; Nicolini, Gabriella; Cavaletti, Guido; Miloso, Mariarosaria

    2016-10-01

    Cancer is a leading cause of death worldwide. Despite therapeutic improvements, some cancers are still untreatable. Recently there has been an increasing interest in the use of natural substances for cancer prevention and treatment. Hibiscus sabdariffa (HS) is a plant, belonging to Malvaceae family, widespread in South Asia and Central Africa. HS extract (HSE) used in folk medicine, gained researchers' interest thanks to its antioxidant, anti-inflammatory, and chemopreventive properties. In the present study, we initially assessed HSE effect on a panel of human tumor cell lines. Then we focused our study on the following that are most sensitive to HSE action cell lines: Multiple Myeloma (MM) cells (RPMI 8226) and Oral Squamous Cell Carcinoma (OSCC) cells (SCC-25). In both RPMI 8226 and SCC-25 cells, HSE impaired cell growth, exerted a reversible cytostatic effect, and reduced cell motility and invasiveness. We evaluated the involvement of MAPKs ERK1/2 and p38 in HSE effects by using specific inhibitors, U0126 and SB203580, respectively. For both SCC-25 and RPMI 8226, HSE cytostatic effect depends on p38 activation, whereas ERK1/2 modulation is crucial for cell motility and invasiveness. Our results suggest that HSE may be a potential therapeutic agent against MM and OSCC. PMID:27618152

  4. Effects of Benzodiazepines on Acinar and Myoepithelial Cells

    PubMed Central

    Mattioli, Tatiana M. F.; Alanis, Luciana R. A.; Sapelli, Silvana da Silva; de Lima, Antonio A. S.; de Noronha, Lucia; Rosa, Edvaldo A. R.; Althobaiti, Yusuf S.; Almalki, Atiah H.; Sari, Youssef; Ignacio, Sergio A.; Johann, Aline C. B. R.; Gregio, Ana M. T.

    2016-01-01

    Background: Benzodiazepines (BZDs), the most commonly prescribed psychotropic drugs with anxiolytic action, may cause hyposalivation. It has been previously shown that BZDs can cause hypertrophy and decrease the acini cell number. In this study, we investigated the effects of BZDs and pilocarpine on rat parotid glands, specifically on acinar, ductal, and myoepithelial cells. Methods: Ninety male Wistar rats were divided into nine groups. Control groups received a saline solution for 30 days (C30) and 60 days (C60), and pilocarpine (PILO) for 60 days. Experimental groups received lorazepam (L30) and midazolam (M30) for 30 days. Another group (LS60 or MS60) received lorazepam or midazolam for 30 days, respectively, and saline for additional 30 days. Finally, other groups (LP60 or MP60) received either lorazepam or midazolam for 30 days, respectively, and pilocarpine for additional 30 days. The expression of calponin in myoepithelial cells and the proliferating cell nuclear antigen (PCNA) in acinar and ductal cells were evaluated. Results: Animals treated with lorazepam showed an increase in the number of positive staining cells for calponin as compared to control animals (p < 0.05). Midazolam administered with pilocarpine (MP60) induced an increase in the proliferation of acinar and ductal cells and a decrease in the positive staining cells for calponin as compared to midazolam administered with saline (MS60). Conclusion: We found that myoepithelial cells might be more sensitive to the effects of BZD than acinar and ductal cells in rat parotid glands. PMID:27445812

  5. Modeling circadian clock-cell cycle interaction effects on cell population growth rates.

    PubMed

    El Cheikh, R; Bernard, S; El Khatib, N

    2014-12-21

    The circadian clock and the cell cycle are two tightly coupled oscillators. Recent analytical studies have shown counter-intuitive effects of circadian gating of the cell cycle on growth rates of proliferating cells which cannot be explained by a molecular model or a population model alone. In this work, we present a combined molecular-population model that studies how coupling the circadian clock to the cell cycle, through the protein WEE1, affects a proliferating cell population. We show that the cell cycle can entrain to the circadian clock with different rational period ratios and characterize multiple domains of entrainment. We show that coupling increases the growth rate for autonomous periods of the cell cycle around 24 h and above 48 h. We study the effect of mutation of circadian genes on the growth rate of cells and show that disruption of the circadian clock can lead to abnormal proliferation. Particularly, we show that Cry 1, Cry 2 mutations decrease the growth rate of cells, Per 2 mutation enhances it and Bmal 1 knockout increases it for autonomous periods of the cell cycle less than 21 h and decreases it elsewhere. Combining a molecular model to a population model offers new insight on the influence of the circadian clock on the growth of a cell population. This can help chronotherapy which takes benefits of physiological rhythms to improve anti-cancer efficacy and tolerance to drugs by administering treatments at a specific time of the day.

  6. Effects of LG268 on Cell Proliferation and Apoptosis of NB4 Cells

    PubMed Central

    Xu, Ting; Zhong, Liang; Gan, Liu-Gen; Xiao, Chun-Lan; Shan, Zhi-Ling; Yang, Rong; Song, Hao; Li, Liu; Liu, Bei-Zhong

    2016-01-01

    Aims: To investigate the effect of LG100268 (LG268) on cell proliferation and apoptosis in NB4 cells. Methods: NB4 cells were treated with LG268 for 24 h or 48 h. The effect of LG268 on cell proliferation was assessed by the CCK-8 assay and colony-forming assay. Apoptosis and cell cycle were evaluated by flow cytometry. The protein expression levels of Survivin, PARP, c-Myc, cyclin D1, ERK, p-ERK, p38 MAPK, and p- p38 MAPK were detected by western blot. Results: We found that LG268 inhibited the proliferation of NB4 cells in a dose-dependent manner. Flow cytometry analysis showed that LG268 accelerated apoptosis in NB4 cells in a time- dependent manner and that LG268 treatment led to cell cycle arrest at G0/G1 phase. Moreover, LG268 significantly decreased the protein levels of Survivin, c-Myc, and cyclinD1. Cleaved PARP was observed in the LG268 treatment group but not in the control group. In addition, LG268 increased the phosphorylation level of p38 MAPK and decreased the phosphorylation level of ERK. Conclusions: LG268 inhibited cell proliferation and promoted cell apoptosis in NB4 cells. PMID:27429588

  7. Effect of EMP fields on cell membrane potentials

    SciTech Connect

    Gailey, P.C.; Easterly, C.E.

    1993-06-01

    A simple model is presented for cell membrane potentials induced during exposure to electromagnetic pulse (EMP). Using calculated values of internal electric field strength induced during EMP exposure, the model predicts that cell membrane potentials of about 100 mV may be induced for time frames on the order of 10 ns. Possible biological effects of these potentials including electroporation area discussed.

  8. Pockels-effect cell for gas-flow simulation

    NASA Technical Reports Server (NTRS)

    Weimer, D.

    1982-01-01

    A Pockels effect cell using a 75 cu cm DK*P crystal was developed and used as a gas flow simulator. Index of refraction gradients were produced in the cell by the fringing fields of parallel plate electrodes. Calibration curves for the device were obtained for index of refraction gradients in excess of .00025 m.

  9. Thymoquinone causes multiple effects, including cell death, on dividing plant cells.

    PubMed

    Hassanien, Sameh E; Ramadan, Ahmed M; Azeiz, Ahmed Z Abdel; Mohammed, Rasha A; Hassan, Sabah M; Shokry, Ahmed M; Atef, Ahmed; Kamal, Khalid B H; Rabah, Samar; Sabir, Jamal S M; Abuzinadah, Osama A; El-Domyati, Fotouh M; Martin, Gregory B; Bahieldin, Ahmed

    2013-01-01

    Thymoquinone (TQ) is a major constituent of Nigella sativa oil with reported anti-oxidative activity and anti-inflammatory activity in animal cells. It also inhibits proliferation and induces programmed cell death (apoptosis) in human skin cancer cells. The present study sought to detect the influence of TQ on dividing cells of three plant systems and on expression of Bcl2-associated athanogene-like (BAG-like) genes that might be involved during the process of cell death. BAG genes are known for the regulation of diverse physiological processes in animals, including apoptosis, tumorigenesis, stress responses, and cell division. Synthetic TQ at 0.1mg/mL greatly reduced wheat seed germination rate, whereas 0.2mg/mL completely inhibited germination. An Evans blue assay revealed moderate cell death in the meristematic zone of Glycine max roots after 1h of TQ treatment (0.2mg/mL), with severe cell death occurring in this zone after 2h of treatment. Light microscopy of TQ-treated (0.2mg/mL) onion hairy root tips for 1h revealed anti-mitotic activity and also cell death-associated changes, including nuclear membrane disruption and nuclear fragmentation. Transmission electron microscopy of TQ-treated cells (0.2mg/mL) for 1h revealed shrinkage of the plasma membrane, leakage of cell lysate, degradation of cell walls, enlargement of vacuoles and condensation of nuclei. Expression of one BAG-like gene, previously associated with cell death, was induced 20 min after TQ treatment in Glycine max root tip cells. Thus, TQ has multiple effects, including cell death, on dividing plant cells and plants may serve as a useful system to further investigate the mechanisms underlying the response of eukaryotic cells to TQ.

  10. Effect of antigen-induced suppressor B cells on development of memory B cells, carrier-specific helper T cells, and antibody-forming cells

    SciTech Connect

    Kalinkovich, A.G.; Luganskaya, E.L.; Pinegin, B.V.

    1986-12-01

    This paper studies the presence of an Fc receptor on the surface of antigen-induced suppressor B cells (AISB), their sensitivity to antiproliferative agents, their interaction with other immunoregulator cells and with the precursors of antibody-forming cells (AFC). Mice were used in the experiments and were irradiated in a dose of 8.5 Gy (/sup 137/Cs). The effect of AISB on memory B cells is shown as is the effect of AISB on development of carrier-specific helper T cells. Also presented is the effect of AISB on development of AFC.

  11. Effects of simulated microgravity on cell cycle in human endothelial cells

    NASA Astrophysics Data System (ADS)

    Sokolovskaya, Alisa A.; Ignashkova, Tatiana I.; Bochenkova, Anna V.; Moskovtsev, Aleksey A.; Baranov, Victor M.; Kubatiev, Aslan A.

    2014-06-01

    The aim of the current study is to investigate effects of simulated microgravity on the cell cycle of endothelial cells. We analyze changes in the cell cycle after exposure of endothelial-like EA.hy 926 cells to simulated microgravity using a Desktop random positioning machine (RPM). Cell cycle profiles determined by flow cytometry show, that the percentage of the cells in the G0/G1 phase after 24 and 96 h of RPM-simulated microgravity is significantly increased as compared to the control group. However, no significant difference is observed after 120 h of RPM-simulated microgravity. In regard to S phase, the percentage of cells is significantly decreased after 24 and 96 h of RPM, respectively; whereas 120 h later, the number of S-phase cells is comparable to the control group. Thus, we show that simulated microgravity inhibits cell cycle progression of human EA.hy 926 cells from the G0/G1 phase to the S phase. We observe an effect of a hibernation-like state, when the growth of the cells in the RPM group slows down, but does not stop. Our results further show that simulated microgravity can affect adhesion of endothelial cells, and alpha-tubulin expression, as most cells begin to detach from the surface of OptiCell unit after 24 h, form aggregates after 48 h, and exhibit accumulation of alpha-tubulin around the nucleus after 48 h of exposure to simulated microgravity conditions. Our results demonstrate a chance in the cell cycle in a low gravitational field.

  12. Identifying and removing the cell-cycle effect from single-cell RNA-Sequencing data

    PubMed Central

    Barron, Martin; Li, Jun

    2016-01-01

    Single-cell RNA-Sequencing (scRNA-Seq) is a revolutionary technique for discovering and describing cell types in heterogeneous tissues, yet its measurement of expression often suffers from large systematic bias. A major source of this bias is the cell cycle, which introduces large within-cell-type heterogeneity that can obscure the differences in expression between cell types. The current method for removing the cell-cycle effect is unable to effectively identify this effect and has a high risk of removing other biological components of interest, compromising downstream analysis. We present ccRemover, a new method that reliably identifies the cell-cycle effect and removes it. ccRemover preserves other biological signals of interest in the data and thus can serve as an important pre-processing step for many scRNA-Seq data analyses. The effectiveness of ccRemover is demonstrated using simulation data and three real scRNA-Seq datasets, where it boosts the performance of existing clustering algorithms in distinguishing between cell types. PMID:27670849

  13. Inhibitory effect of maple syrup on the cell growth and invasion of human colorectal cancer cells.

    PubMed

    Yamamoto, Tetsushi; Uemura, Kentaro; Moriyama, Kaho; Mitamura, Kuniko; Taga, Atsushi

    2015-04-01

    Maple syrup is a natural sweetener consumed by individuals of all ages throughout the world. Maple syrup contains not only carbohydrates such as sucrose but also various components such as organic acids, amino acids, vitamins and phenolic compounds. Recent studies have shown that these phenolic compounds in maple syrup may possess various activities such as decreasing the blood glucose level and an anticancer effect. In this study, we examined the effect of three types of maple syrup, classified by color, on the cell proliferation, migration and invasion of colorectal cancer (CRC) cells in order to investigate whether the maple syrup is suitable as a phytomedicine for cancer treatment. CRC cells that were administered maple syrup showed significantly lower growth rates than cells that were administered sucrose. In addition, administration of maple syrup to CRC cells caused inhibition of cell invasion, while there was no effect on cell migration. Administration of maple syrup clearly inhibited AKT phosphorylation, while there was no effect on ERK phosphorylation. These data suggest that maple syrup might inhibit cell proliferation and invasion through suppression of AKT activation and be suitable as a phytomedicine for CRC treatment, with fewer adverse effects than traditional chemotherapy.

  14. Stromal Cells from Human Decidua Exert a Strong Inhibitory Effect on NK Cell Function and Dendritic Cell Differentiation

    PubMed Central

    Canegallo, Francesca; Conte, Romana; Venturini, Pier Luigi; Moretta, Lorenzo; Mingari, Maria Cristina

    2014-01-01

    Stromal cells (SC) are an important component of decidual tissues where they are in strict proximity with both NK and CD14+ myelomonocytic cells that play a role in the maintenance of pregnancy. In this study we analyzed whether decidual SC (DSC) could exert a regulatory role on NK and CD14+ cells that migrate from peripheral blood (PB) to decidua during pregnancy. We show that DSCs inhibit the IL15-mediated up-regulation of major activating NK receptors in PB-derived NK cells. In addition, the IL15-induced NK cell proliferation, cytolytic activity and IFN-γ production were severely impaired. DSCs sharply inhibited dendritic cells differentiation and their ability to induce allogeneic T cell proliferation. Indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) mediated the inhibitory effect of DSCs. Our results strongly suggest an important role of DSCs in preventing potentially dangerous immune response, thus contributing to maintenance of pregnancy. PMID:24586479

  15. Growth modulating effects of chlorinated oleic acid in cell cultures.

    PubMed

    Høstmark, A T; Lystad, E; Jebens, E; Skramstad, J; Frøyen, P

    1998-07-01

    Chlorinated fatty acids represent a major fraction of extractable, organically bound chlorine in fish. After dietary intake such fatty acids may be transferred from the mother to the foetus through the placenta, and via breast milk to the child. In the present work we have studied the effect of chlorinated oleic acid on the growth of three widely differing types of cells in culture. Chlorinated oleic acid inhibited growth of Human Microvascular Endothelial Cells (HMVEC), Immortilized Human Kidney Epithelial (IHKE) cells, and human Hepatoma cells (HepG2). The order of potency was: HMVEC > IHKE > HepG2. Vitamin E counteracted the inhibitory effect of chlorinated oleic acid on HepG2 cells, but did not significantly affect the fatty acid effect on HMVEC or IHKE. Defatted serum albumin stimulated the growth of HMVEC and IHKE. With HMVEC there was no major interaction between the effect of albumin and chlorinated oleic acid on cell growth. In contrast, with IHKE albumin at low concentration abolished the growth inhibiting effect of chlorinated oleic acid and appreciably counteracted growth inhibition by the fatty acid of HepG2. We conclude that the growth modulation by chlorinated oleic acid and its interaction with vitamin E and albumin are cell specific.

  16. Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

    PubMed Central

    2010-01-01

    Background Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. Results The study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ≤5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. Conclusion The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy. PMID:20925932

  17. The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells

    PubMed Central

    Ruiz-Magaña, María J.; Martínez-Aguilar, Rocío; Lucendo, Estefanía; Campillo-Davo, Diana; Schulze-Osthoff, Klaus; Ruiz-Ruiz, Carmen

    2016-01-01

    Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect. PMID:26942461

  18. The antihypertensive drug hydralazine activates the intrinsic pathway of apoptosis and causes DNA damage in leukemic T cells.

    PubMed

    Ruiz-Magaña, María J; Martínez-Aguilar, Rocío; Lucendo, Estefanía; Campillo-Davo, Diana; Schulze-Osthoff, Klaus; Ruiz-Ruiz, Carmen

    2016-04-19

    Epigenetic therapies have emerged as promising anticancer approaches, since epigenetic modifications play a major role in tumor initiation and progression. Hydralazine, an approved vasodilator and antihypertensive drug, has been recently shown to act as a DNA methylation inhibitor. Even though hydralazine is already tested in clinical cancer trials, its mechanism of antitumor action remains undefined. Here, we show that hydralazine induced caspase-dependent apoptotic cell death in human p53-mutant leukemic T cells. Moreover, we demonstrate that hydralazine triggered the mitochondrial pathway of apoptosis by inducing Bak activation and loss of the mitochondrial membrane potential. Hydralazine treatment further resulted in the accumulation of reactive oxygen species, whereas a superoxide dismutase mimetic inhibited hydralazine-induced cell death. Interestingly, caspase-9-deficient Jurkat cells or Bcl-2- and Bcl-xL-overexpressing cells were strongly resistant to hydralazine treatment, thereby demonstrating the dependence of hydralazine-induced apoptosis on the mitochondrial death pathway. Furthermore, we demonstrate that hydralazine treatment triggered DNA damage which might contribute to its antitumor effect.

  19. Volume and ion effects of adenosine on MDCK cells

    SciTech Connect

    Coffey, A.K.; Allen, J.C.; Coutermarsh, B.A.; Mills, J.W.

    1986-03-01

    Adenosine (ADO) receptors, both internal inhibitory (P-site) and external stimulatory (A2) and inhibitory (Al) have been shown to modulate adenylate cyclase levels. Since the authors have previously shown that cAMP modulates ion content and volume of confluent monolayers of MDCK epithelial cells, the authors investigated the effects of ADO on these parameters. Exposure of MDCK cells to 0.1 mM ADO significantly increased cell volumes (2.35 +/- .10 pL/cell vs. control 2.08 +/- .01; p < .001) as determined by /sup 14/C-urea distribution space, and increased Na content (133 +/- 13 vs. 91 +/- 11 nmol/10/sup 6/ cells; p < .0002). Cl/sup -/ content also increased while K did not change. These effects were completely inhibited by treatment with the Na/sup +//H/sup +/ exchange inhibitor amiloride (0.1 mM) and by incubation in Na-free media. Together these results suggest that cell volume increased due to Na/sup +/ entry via the Na/sup +//H/sup +/ exchanger. However, 2-chloroadenosine (2CA), a potent analogue of ADO, caused a significant decrease in cell volume (1.99 +/- .86 vs. 2.21 +/- 0.13 pL/cell; p < .02) and Na (97 +/- 22 vs. 141 +/- 21 nmol/10/sup 6/ cells; p < .005). Measurement of cell cAMP by radioimmunoassay showed an increase in response to 2CA but not to ADO. Since the effects of 2CA mimic those of exogenous cAMP, MDCK cells appear to have a stimulatory (A2) receptor. However, ADO itself did not interact with this receptor and may have produced its effects by binding to a higher affinity, inhibitory receptor.

  20. Potentiation of Anticancer Drugs: Effects of Pentoxifylline on Neoplastic Cells

    PubMed Central

    Barancik, Miroslav; Bohacova, Viera; Gibalova, Lenka; Sedlak, Jan; Sulova, Zdena; Breier, Albert

    2012-01-01

    The drug efflux activity of P-glycoprotein (P-gp, a product of the mdr1 gene, ABCB1 member of ABC transporter family) represents a mechanism by which tumor cells escape death induced by chemotherapeutics. In this study, we investigated the mechanisms involved in the effects of pentoxifylline (PTX) on P-gp-mediated multidrug resistance (MDR) in mouse leukemia L1210/VCR cells. Parental sensitive mouse leukemia cells L1210, and multidrug-resistant cells, L1210/VCR, which are characterized by the overexpression of P-gp, were used as experimental models. The cells were exposed to 100 μmol/L PTX in the presence or absence of 1.2 μmol/L vincristine (VCR). Western blot analysis indicated a downregulation of P-gp protein expression when multidrug-resistant L1210/VCR cells were exposed to PTX. The effects of PTX on the sensitization of L1210/VCR cells to VCR correlate with the stimulation of apoptosis detected by Annexin V/propidium iodide apoptosis necrosis kit and proteolytic activation of both caspase-3 and caspase-9 monitored by Western blot analysis. Higher release of matrix metalloproteinases (MMPs), especially MMP-2, which could be attenuated by PTX, was found in L1210/VCR than in L1210 cells by gelatin zymography in electrophoretic gel. Exposure of resistant cells to PTX increased the content of phosphorylated Akt kinase. In contrast, the presence of VCR eliminated the effects of PTX on Akt kinase phosphorylation. Taken together, we conclude that PTX induces the sensitization of multidrug-resistant cells to VCR via downregulation of P-gp, stimulation of apoptosis and reduction of MMPs released from drug-resistant L1210/VCR cells. These facts bring new insights into the mechanisms of PTX action on cancer cells. PMID:22312258

  1. Gravity effect on lymphocyte deformation through cell shape change.

    PubMed

    Hung, R J; Tsao, Y D; Spauling, G F

    1995-01-01

    The effects on human cells (lymphocyte) immersed in a culture liquid under microgravity environment has been investigated. The study was based on the numerical simulation of the Morphology of human cells affected by the time dependent variation of gravity acceleration ranging from 10(-3) to 2 g(o) (g(o) = 9.81 m/s2) in 15 s. Both the free floating cells and the cells which came into contact with the upper and lower inclined walls imposed by the time-dependent reduced gravity acceleration were considered in this study. The results show that, when the gravity acceleration increased, the cell morphology changed from spherical to horizontally elongated ellipsoid for both the free floating cells and the stationary cells on the lower inclined wall while the cell morphology varied from spherical to vertically-elongated ellipsoid for the cells hanging on the upper inclined wall. A test of the deformation of human cells exposed to the variation of gravity levels, carried out in the KC-135 free fall aircraft, show that the results of experimental observations agree exactly with the theoretical model computation described in this paper. These results will be useful for study of the behavior and morphology of cells in space.

  2. Cell-density dependent effects of low-dose ionizing radiation on E. coli cells.

    PubMed

    Alipov, E D; Shcheglov, V S; Sarimov, R M; Belyaev, I Ya

    2003-01-01

    The changes in genome conformational state (GCS) induced by low-dose ionizing radiation in E. coli cells were measured by the method of anomalous viscosity time dependence (AVTD) in cellular lysates. Effects of X-rays at doses 0.1 cGy--1 Gy depended on post-irradiation time. Significant relaxation of DNA loops followed by a decrease in AVTD. The time of maximum relaxation was between 5-80 min depending on the dose of irradiation. U-shaped dose response was observed with increase of AVTD in the range of 0.1-4 Gy and decrease in AVTD at higher doses. No such increase in AVTD was seen upon irradiation of cells at the beginning of cell lysis while the AVTD decrease was the same. Significant differences in the effects of X-rays and gamma-rays at the same doses were observed suggesting a strong dependence of low-dose effects on LET. Effects of 0.01 cGy gamma-rays were studied at different cell densities during irradiation. We show that the radiation-induced changes in GCS lasted longer at higher cell density as compared to lower cell density. Only small amount of cells were hit at this dose and the data suggest cell-to-cell communication in response to low-dose ionizing radiation. This prolonged effect was also observed when cells were irradiated at high cell density and diluted to low cell density immediately after irradiation. These data suggest that cell-to-cell communication occur during irradiation or within 3 min post-irradiation. The cell-density dependent response to low-dose ionizing radiation was compared with previously reported data on exposure of E. coli cells to electromagnetic fields of extremely low frequency and extremely high frequency (millimeter waves). The body of our data show that cells can communicate in response to electromagnetic fields and ionizing radiation, presumably by reemission of secondary photons in infrared-submillimeter frequency range.

  3. Neutron exposures in human cells: bystander effect and relative biological effectiveness.

    PubMed

    Seth, Isheeta; Schwartz, Jeffrey L; Stewart, Robert D; Emery, Robert; Joiner, Michael C; Tucker, James D

    2014-01-01

    Bystander effects have been observed repeatedly in mammalian cells following photon and alpha particle irradiation. However, few studies have been performed to investigate bystander effects arising from neutron irradiation. Here we asked whether neutrons also induce a bystander effect in two normal human lymphoblastoid cell lines. These cells were exposed to fast neutrons produced by targeting a near-monoenergetic 50.5 MeV proton beam at a Be target (17 MeV average neutron energy), and irradiated-cell conditioned media (ICCM) was transferred to unirradiated cells. The cytokinesis-block micronucleus assay was used to quantify genetic damage in radiation-naïve cells exposed to ICCM from cultures that received 0 (control), 0.5, 1, 1.5, 2, 3 or 4 Gy neutrons. Cells grown in ICCM from irradiated cells showed no significant increase in the frequencies of micronuclei or nucleoplasmic bridges compared to cells grown in ICCM from sham irradiated cells for either cell line. However, the neutron beam has a photon dose-contamination of 5%, which may modulate a neutron-induced bystander effect. To determine whether these low doses of contaminating photons can induce a bystander effect, cells were irradiated with cobalt-60 at doses equivalent to the percent contamination for each neutron dose. No significant increase in the frequencies of micronuclei or bridges was observed at these doses of photons for either cell line when cultured in ICCM. As expected, high doses of photons induced a clear bystander effect in both cell lines for micronuclei and bridges (p<0.0001). These data indicate that neutrons do not induce a bystander effect in these cells. Finally, neutrons had a relative biological effectiveness of 2.0 ± 0.13 for micronuclei and 5.8 ± 2.9 for bridges compared to cobalt-60. These results may be relevant to radiation therapy with fast neutrons and for regulatory agencies setting standards for neutron radiation protection and safety.

  4. Quantifying the effect of electric current on cell adhesion studied by single-cell force spectroscopy.

    PubMed

    Jaatinen, Leena; Young, Eleanore; Hyttinen, Jari; Vörös, János; Zambelli, Tomaso; Demkó, László

    2016-03-20

    This study presents the effect of external electric current on the cell adhesive and mechanical properties of the C2C12 mouse myoblast cell line. Changes in cell morphology, viability, cytoskeleton, and focal adhesion structure were studied by standard staining protocols, while single-cell force spectroscopy based on the fluidic force microscopy technology provided a rapid, serial quantification and detailed analysis of cell adhesion and its dynamics. The setup allowed measurements of adhesion forces up to the μN range, and total detachment distances over 40 μm. Force-distance curves have been fitted with a simple elastic model including a cell detachment protocol in order to estimate the Young's modulus of the cells, as well as to reveal changes in the dynamic properties as functions of the applied current dose. While the cell spreading area decreased monotonously with increasing current doses, small current doses resulted only in differences related to cell elasticity. Current doses above 11 As/m(2), however, initiated more drastic changes in cell morphology, viability, cellular structure, as well as in properties related to cell adhesion. The observed differences, eventually leading to cell death toward higher doses, might originate from both the decrease in pH and the generation of reactive oxygen species.

  5. The effect of food chemicals on cell aging of human diploid cells in in vitro culture.

    PubMed

    Kasamaki, A; Urasawa, S

    1993-08-01

    The potency of food chemicals to induce cell aging was evaluated in human diploid fibroblast cells HAIN-55 having a finite replicative potential by using in vitro aging markers, i.e., decreases of maximum proliferative potential (lifespan) of cells, saturation density in monolayer culture (SD), plating efficiency (PE) and mitotic index (MI), and an increase of cells with polyploid karyotypes. By treatment twice with low concentration of genotoxic chemicals aflatoxin B1, allylisothiocyanate or trans-cinnamaldehyde (severe clastogenic flavoring agent; Kasamaki et al., 1982), lifespan (expressed by the number of cumulative cell population doubling (CPD)) of the treated cells was reduced by 8-12 CPDs accompanied by change of the other aging markers. By successive treatment (29 or 25 times) with non-genotoxic chemical aspartame (N-L-aspartyl-L-phenylalanine) or L-canavanine (structural analogue of L-arginine), lifespan of the treated cells was also slightly shortened (by 2-6 CPDs) compared with the untreated control cells. In the process of cell aging, Mitochondrial activity (MTT activity) decreased almost in parallel with the decrease of SD and MI. On the basis of these results, a variety of genotoxic and non-genotoxic chemicals were examined by using MTT activity as the aging marker for their effects on the aging of HAIN-55 cells and bovine artery endothelial cells which also had a finite replicative potential. The results showed that seven genotoxic and nine non-genotoxic chemicals promoted cell aging.

  6. Cancer Stem Cells Protect Non-Stem Cells From Anoikis: Bystander Effects.

    PubMed

    Kim, Seog-Young; Hong, Se-Hoon; Basse, Per H; Wu, Chuanyue; Bartlett, David L; Kwon, Yong Tae; Lee, Yong J

    2016-10-01

    Cancer stem cells (CSCs) are capable of initiation and metastasis of tumors. Therefore, understanding the biology of CSCs and the interaction between CSCs and their counterpart non-stem cells is crucial for developing a novel cancer therapy. We used CSC-like and non-stem breast cancer MDA-MB-231 and MDA-MB-453 cells to investigate mammosphere formation. We investigated the role of the epithelial cadherin (E-cadherin)-extracellular signal-regulated kinase (Erk) axis in anoikis. Data from E-cadherin small hairpin RNA assay and mitogen-activated protein kinase kinase (MEK) inhibitor study show that activation of Erk, but not modulation of E-cadherin level, may play an important role in anoikis resistance. Next, the two cell subtypes were mixed and the interaction between them during mammosphere culture and xenograft tumor formation was investigated. Unlike CSC-like cells, increased secretion of interleukin-6 (IL-6) and growth-related oncogene (Gro) chemokines was detected during mammosphere culture in non-stem cells. Similar results were observed in mixed cells. Interestingly, CSC-like cells protected non-stem cells from anoikis and promoted tumor growth. Our results suggest bystander effects between CSC-like cells and non-stem cells. J. Cell. Biochem. 117: 2289-2301, 2016. © 2016 Wiley Periodicals, Inc. PMID:26918647

  7. Effects of mimosine on Wolbachia in mosquito cells: cell cycle suppression reduces bacterial abundance

    PubMed Central

    Fallon, Ann M.

    2016-01-01

    The plant allelochemical l-mimosine (β-[N-(3-hydroxy-4-pyridone)]-α-aminopropionic acid; leucenol) resembles the nonessential amino acid, tyrosine. Because the obligate intracellular alphaproteobacterium, Wolbachia pipientis, metabolizes amino acids derived from host cells, the effects of mimosine on infected and uninfected mosquito cells were investigated. The EC50 for mimosine was 6–7 μM with Aedes albopictus C7-10 and C/wStr cell lines, and was not influenced by infection status. Mosquito cells responded to concentrations of mimosine substantially lower than those used to synchronize the mammalian cell cycle; at concentrations of 30–35 μM, mimosine reversibly arrested the mosquito cell cycle at the G1/S boundary and inhibited growth of Wolbachia strain wStr. Although lower concentrations of mimosine slightly increased wStr abundance, concentrations that suppressed the cell cycle reduced Wolbachia levels. PMID:26019119

  8. Effects of cell area on the performance of dye sensitized solar cell

    SciTech Connect

    Khatani, Mehboob E-mail: noranimuti-mohamed@petronas.com.my E-mail: azclement@yahoo.com Mohamed, Norani Muti E-mail: noranimuti-mohamed@petronas.com.my E-mail: azclement@yahoo.com Hamid, Nor Hisham E-mail: noranimuti-mohamed@petronas.com.my E-mail: azclement@yahoo.com Sahmer, Ahmad Zahrin E-mail: noranimuti-mohamed@petronas.com.my E-mail: azclement@yahoo.com Samsudin, Adel E-mail: noranimuti-mohamed@petronas.com.my E-mail: azclement@yahoo.com

    2014-10-24

    Dye sensitized solar cells (DSCs) have significant advantage over the current silicon cells by having low manufacturing cost and potentially high conversion efficiency. Therefore, DSCs are expected to be used as the next generation solar cell device that covers wide range of new applications. In order to achieve highly efficient DSCs for practical application, study on the effect of increasing the cell’s area on the performance of dye sensitized solar need to be carried out. Three different DSC cell areas namely, 1, 12.96 and 93.5 cm{sup 2} respectively were fabricated and analyzed through solar simulator and electrochemical impedance spectroscopy (EIS). From the analysis of electrochemical impedance spectroscopy (EIS), it was observed that the cell’s electron lifetime was influenced significantly by the cell’s area. Although the collection efficiency of all cells recorded to be approximately 100% but higher recombination rate with increased cell area reduced the performance of the cell.

  9. Morphological effect of oscillating magnetic nanoparticles in killing tumor cells

    PubMed Central

    2014-01-01

    Forced oscillation of spherical and rod-shaped iron oxide magnetic nanoparticles (MNPs) via low-power and low-frequency alternating magnetic field (AMF) was firstly used to kill cancer cells in vitro. After being loaded by human cervical cancer cells line (HeLa) and then exposed to a 35-kHz AMF, MNPs mechanically damaged cell membranes and cytoplasm, decreasing the cell viability. It was found that the concentration and morphology of the MNPs significantly influenced the cell-killing efficiency of oscillating MNPs. In this preliminary study, when HeLa cells were pre-incubated with 100 μg/mL rod-shaped MNPs (rMNP, length of 200 ± 50 nm and diameter of 50 to 120 nm) for 20 h, MTT assay proved that the cell viability decreased by 30.9% after being exposed to AMF for 2 h, while the cell viability decreased by 11.7% if spherical MNPs (sMNP, diameter of 200 ± 50 nm) were used for investigation. Furthermore, the morphological effect of MNPs on cell viability was confirmed by trypan blue assay: 39.5% rMNP-loaded cells and 15.1% sMNP-loaded cells were stained after being exposed to AMF for 2 h. It was also interesting to find that killing tumor cells at either higher (500 μg/mL) or lower (20 μg/mL) concentration of MNPs was less efficient than that achieved at 100 μg/mL concentration. In conclusion, the relatively asymmetric morphological rod-shaped MNPs can kill cancer cells more effectively than spherical MNPs when being exposed to AMF by virtue of their mechanical oscillations. PMID:24872797

  10. The effect of stem cell factor on proliferation of human endometrial CD146+ cells

    PubMed Central

    Fayazi, Mehri; Salehnia, Mojdeh; Ziaei, Saeideh

    2016-01-01

    Background: Stem cell factor (SCF) is a transcriptional factor which plays crucial roles in normal proliferation, differentiation and survival in a range of stem cells. Objective: The aim of the present study was to examine the proliferation effect of different concentrations of SCF on expansion of human endometrial CD146+ cells. Materials and Methods: In this experimental study, total populations of isolated human endometrial suspensions after fourth passage were isolated by magnetic activated cell sorting (MACS) into CD146+ cells. Human endometrial CD146+ cells were karyotyped and tested for the effect of SCF on proliferation of CD146+ cells, then different concentrations of 0, 12.5, 25, 50 and 100 ng/ml was carried out and mitogens-stimulated endometrial CD146+ cells proliferation was assessed by MTT assay. Results: Chromosomal analysis showed a normal metaphase spread and 46XX karyotype. The proliferation rate of endometrial CD146+ cells in the presence of 0, 12.5, 25, 50 and 100 ng/ml SCF were 0.945±0.094, 0.962±0.151, 0.988±0.028, 1.679±0.012 and 1.129±0.145 respectively. There was a significant increase in stem/ stromal cell proliferation following in vitro treatment by 50 ng/ml than other concentrations of SCF (p=0.01). Conclusion: The present study suggests that SCF could have effect on the proliferation and cell survival of human endometrial CD146+ cells and it has important implications for medical sciences and cell therapies. PMID:27525327

  11. Effect of radiofrequency radiation in cultured mammalian cells: A review.

    PubMed

    Manna, Debashri; Ghosh, Rita

    2016-01-01

    The use of mobile phone related technologies will continue to increase in the foreseeable future worldwide. This has drawn attention to the probable interaction of radiofrequency electromagnetic radiation with different biological targets. Studies have been conducted on various organisms to evaluate the alleged ill-effect on health. We have therefore attempted to review those work limited to in vitro cultured cells where irradiation conditions were well controlled. Different investigators have studied varied endpoints like DNA damage, cell cycle arrest, reactive oxygen species (ROS) formation, cellular morphology and viability to weigh the genotoxic effect of such radiation by utilizing different frequencies and dose rates under various irradiation conditions that include continuous or pulsed exposures and also amplitude- or frequency-modulated waves. Cells adapt to change in their intra and extracellular environment from different chemical and physical stimuli through organized alterations in gene or protein expression that result in the induction of stress responses. Many studies have focused on such effects for risk estimations. Though the effects of microwave radiation on cells are often not pronounced, some investigators have therefore combined radiofrequency radiation with other physical or chemical agents to observe whether the effects of such agents were augmented or not. Such reports in cultured cellular systems have also included in this review. The findings from different workers have revealed that, effects were dependent on cell type and the endpoint selection. However, contradictory findings were also observed in same cell types with same assay, in such cases the specific absorption rate (SAR) values were significant.

  12. The effect of enrofloxacin on cell proliferation and proteoglycans in horse tendon cells.

    PubMed

    Yoon, J H; Brooks, R L; Khan, A; Pan, H; Bryan, J; Zhang, J; Budsberg, S C; Mueller, P O E; Halper, J

    2004-02-01

    Fluoroquinolone antibiotics have been used widely in humans and domestic animals, including horses, because of their broad-spectrum bactericidal activity, and relative safety. The use of fluoroquinolones, however, is not without risk. Tendonitis and spontaneous tendon rupture have been reported in people during or following therapy with fluoroquinolones. We have studied the effects of enrofloxacin, a fluoroquinolone antibiotic used commonly in domestic animals, on tendon cell cultures established from equine superficial digital flexor tendons. Effects on cell proliferation and morphology were studied using cell counting and scanning electron microscopy. Monosaccharide content and composition was determined by gas chromatography-mass spectrometry analysis. Western and Northern blot analyses were utilized to evaluate the synthesis and expression of two proteoglycans, biglycan and decorin. Our data demonstrate that enrofloxacin inhibits cell proliferation, induces morphological changes, decreases total monosacharide content and alters small proteoglycan synthesis at the glycosylation level in equine tendon cell cultures. These effects are more pronounced in juvenile tendon cells than in adult equine tendon cells. We hypothesize that morphological changes and inhibition of cell proliferation are a result of impaired production of biglycan and decorin, proteoglycans involved in fibrillogenesis of collagen, the most important structural component of the tendon of enrofloxacin-treated tendon cells. Our findings suggest that fluoroquinolones should be used with caution in horses, especially in foals.

  13. Toxic effects of Karenia mikimotoi extracts on mammalian cells

    NASA Astrophysics Data System (ADS)

    Chen, Yang; Yan, Tian; Yu, Rencheng; Zhou, Mingjiang

    2011-07-01

    Karenia is one of the most harmful and representative red tide genus in a temperate zone. Blooms caused by this genus have resulted in massive fish death in the South China Sea and the East China Sea. However, the potential effects of this dinoflagellate on human health through the transfer of toxins via marine food webs, and the mechanisms of toxicity, are still unknown. Therefore, we examined the toxic effects of a strain of K. mikimotoi (isolated from the South China Sea) on the proliferation and morphology of four mammalian cell lines (two normal cell lines and two cancer cell lines). In addition, we carried out a preliminary investigation on the mechanism of toxicity of the alga. The results show that the polar lipid-soluble component of K. mikimotoi significantly inhibited proliferation of the four cell lines, and resulted in the cells becoming spherical, swollen and damaged. The result of Annexin V and PI double-staining confirmed that cell membranes were disrupted. The malonaldehyde (MDA) contents in the medium of the four cell lines treated with the polar-lipid extracts all increased significantly, which indicates that the polar-lipid toxins produced by K. mikimotoi could adversely affect mammalian cells by inducing lipid peroxidation. We conclude that K. mikimotoi is a potential threat to human health, and the comprehensive effect of this dinoflagellate and its mechanisms should be investigated further.

  14. Effect of microwell chip structure on cell microsphere production of various animal cells.

    PubMed

    Sakai, Yusuke; Yoshida, Shirou; Yoshiura, Yukiko; Mori, Rhuhei; Tamura, Tomoko; Yahiro, Kanji; Mori, Hideki; Kanemura, Yonehiro; Yamasaki, Mami; Nakazawa, Kohji

    2010-08-01

    The formation of three-dimensional cell microspheres such as spheroids, embryoid bodies, and neurospheres has attracted attention as a useful culture technique. In this study, we investigated a technique for effective cell microsphere production by using specially prepared microchip. The basic chip design was a multimicrowell structure in triangular arrangement within a 100-mm(2) region in the center of a polymethylmethacrylate (PMMA) plate (24x24 mm(2)), the surface of which was modified with polyethylene glycol (PEG) to render it nonadhesive to cells. We also designed six similar chips with microwell diameters of 200, 300, 400, 600, 800, and 1000 microm to investigate the effect of the microwell diameter on the cell microsphere diameter. Rat hepatocytes, HepG2 cells, mouse embryonic stem (ES) cells, and mouse neural progenitor/stem (NPS) cells formed hepatocyte spheroids, HepG2 spheroids, embryoid bodies, and neurospheres, respectively, in the microwells within 5 days of culture. For all the cells, a single microsphere was formed in each microwell under all the chip conditions, and such microsphere configurations remained throughout the culture period. Furthermore, the microsphere diameters of each type of cell were strongly positively correlated with the microwell diameters of the chips, suggesting that microsphere diameter can be factitiously controlled by using different chip conditions. Thus, this chip technique is a promising cellular platform for tissue engineering or regenerative medicine research, pharmacological and toxicological studies, and fundamental studies in cell biology.

  15. Selective apoptotic cell death effects of oral cancer cells treated with destruxin B

    PubMed Central

    2014-01-01

    Background Recent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent. Methods Destruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses. Results The results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners. Conclusions This is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications. PMID:24972848

  16. [Effects of decitabine on biological behavior of U266 cells].

    PubMed

    Wang, Mei-Fang; Yang, Lin-Hua; Dong, Chun-Xia; Zhang, Rui-Juan; Zhang, Jian-Hua; Guo, Zhi-Ping; Chen, Jian-Fang; Zhagn, Li; Feng, Da-Wei

    2011-08-01

    This study was aimed to explore the effects of decitabine on the biological behaviour of U266 cells in vitro so as to provide a new thinking and experiment basis, as well as new evidences for the pathogenesis of multiple myeloma. MTT and colony formation assays were used to evaluate the impact of decitabine on the ability of proliferation of U266 cells; flow cytometry was used to analyze the cell distribution in cell cycle; transwell chamber and matrigel assays were used to observe the ability of migration and invasion. The results indicated that decitabine could significantly suppress the proliferation of U266 cells in time-and dose-dependent manners. The flow cytometric analysis demonstrated that the cells in G(0)-G(1) phase significantly increased while the cells in S and G(2)/M phase decreased. The migration and matrigel invading tests showed that the number of cells moving into under chamber of transwell decreased after U266 cells treated with decitabine. It is concluded that decitabine may act as an effective drug for MM by inhibiting the proliferation, migration and invasion ability, and the specific mechanism needs to be deeply explored.

  17. Effect of curcumin on aging retinal pigment epithelial cells.

    PubMed

    Zhu, Wei; Wu, Yan; Meng, Yi-Fang; Wang, Jin-Yu; Xu, Ming; Tao, Jian-Jun; Lu, Jiong

    2015-01-01

    Age-related macular degeneration (AMD) is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE) cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 µM, 40 µM, and 80 µM. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE cell model. Curcumin improved cell viability and decreased apoptosis and oxidative stress. Further, curcumin had a significant influence on expression of apoptosis-associated proteins and oxidative stress biomarkers. In conclusion, treatment with curcumin was able to regulate proliferation, oxidative stress, and apoptosis in aging RPE cells. Accordingly, application of curcumin may be a novel strategy to protect against age-related change in AMD.

  18. LOX-1-Mediated Effects on Vascular Cells in Atherosclerosis.

    PubMed

    Chistiakov, Dimitry A; Orekhov, Alexander N; Bobryshev, Yuri V

    2016-01-01

    In healthy arteries, expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is almost undetectable. However, in proatherogenic conditions, LOX-1 is markedly up-regulated in vascular cells. In atherosclerosis, LOX-1 appears to be the key scavenger receptor for binding oxidized LDL (oxLDL). Notably, a positive feedback exists between LOX-1 and oxLDL. LOX-1 is involved in mediating of proatherosclerotic effects of oxLDL which result in endothelial dysfunction, proinflammatory recruitment of monocytes into the arterial intima, formation of foam cells, apoptosis of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), as well as in plaque destabilization and rupture. In this review, we consider effects of the LOX-1/oxLDL axis on several types of vascular cells such as ECs, VSMCs, and macrophages. PMID:27160316