Proteins In Jurkat Cells Research Articles

Effect of Down-Regulation of LncRNA-HOTAIRM1 to Proliferation, Apoptosis and KIT/AKT Pathway of Jurkat Cells

To observe the effects of down-regulation of long non-coding RNA HOX antisense intergenic RNA myeloid 1 (LncRNA-HOTAIRM1) to the proliferation and apoptosis of Jurkat in human leukemia T lymphocytes, and explore its mechanism. Jurkat cells were cultured in vitro and randomly divided into control group, HOTAIRM1 siRNA-NC group and HOTAIRM1 siRNA group; the expressions of LncRNA-HOTAIRM1 mRNA, KIT receptor tyrosine kinase (KIT) mRNA and serine threonine kinase (AKT) mRNA in Jurkat cells were detected by real-time fluorescence quantification (RT-qPCR); the proliferation of Jurkat cells in each groups was detected by CCK-8 method; the apoptosis of Jurkat cells in each groups was detected by Annexin V-FITC/PI double staining; the expressions of KIT, AKT, p-KIT, p-AKT, B-lymphoma-2 gene (BCL-2) and Caspase-3 were detected by Western blot. Compared with the cells in the control group and HOTAIRM1 siRNA-NC group, the expression level of LncRNA-HOTAIRM1 mRNA, cell survival rate, expression levels of KIT mRNA, AKT mRNA, p-KIT, p-AKT and BCL-2 proteins in Jurkat cells in HOTAIRM1 siRNA group were significantly lower (P<0.05), while the expression level of Cleared Caspase-3 protein and Jurkat cell apoptosis rate were significantly higher (P<0.05). LncRNA-HOTAIRM1 may inhibit Jurkat cell proliferation and induce apoptosis through KIT/AKT signaling pathway.

Pharmacokinetic properties and anti-proliferative mechanisms of vanillin against acute lymphoblastic leukemia (Jurkat) cells

The anti-proliferative potential of vanillin isolated from an ethnomedicine Flacourtia indica bark was evaluated in an acute lymphoblastic leukemia cell line (Jurkat) in our previous study, but its molecular mechanisms of action are not yet revealed. The present work investigated the pharmacokinetic properties & cellular uptake of vanillin, in addition to evaluating the effect of vanillin treatment on cell morphology, mitochondrial membrane potential, DNA fragmentation, and cell survival & pro-apoptotic proteins in Jurkat cells. High absorption, good oral bioavailability, and safety of vanillin were recorded through in silico pharmacokinetic studies. In vitro study results indicate that 24.07% of vanillin was absorbed by Jurkat cells within 120 min. The IC50 dose of vanillin reduced the mitochondrial membrane potential up to 77.05% and also caused 60.94% of DNA fragmentation in Jurkat cells. Further, inhibition of cell survival proteins such as H-RAS, K-RAS, N-RAS, & BCL-2 and activation of pro-apoptotic proteins like p-38, BAX, pro-caspase-9, pro-caspase-3, and caspase-3 by vanillin was confirmed through molecular docking and western blotting studies. Thus, the current study revealed the pharmacokinetic properties and anti-proliferative mechanisms of vanillin in Jurkat cells. Therefore vanillin could be further investigated to develop a safe and alternative phytopharmaceutical to treat acute lymphoblastic leukemia.

The Signature Amino Acid Residue Serine 31 of HIV-1C Tat Potentiates an Activated Phenotype in Endothelial Cells.

The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and in vitro morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.

20(S)-Ginsenoside Rh2 displays efficacy against T-cell acute lymphoblastic leukemia through the PI3K/Akt/mTOR signal pathway

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a kind of aggressive hematological cancer, and the PI3K/Akt/mTOR signaling pathway is activated in most patients with T-ALL and responsible for poor prognosis. 20(S)-Ginsenoside Rh2 (20(S)-GRh2) is a major active compound extracted from ginseng, which exhibits anti-cancer effects. However, the underlying anticancer mechanisms of 20(S)-GRh2 targeting the PI3K/Akt/mTOR pathway in T-ALL have not been explored. MethodsCell growth and cell cycle were determined to investigate the effect of 20(S)-GRh2 on ALL cells. PI3K/Akt/mTOR pathway–related proteins were detected in 20(S)-GRh2–treated Jurkat cells by immunoblotting. Antitumor effect of 20(S)-GRh2 against T-ALL was investigated in xenograft mice. The mechanisms of 20(S)-GRh2 against T-ALL were examined by cell proliferation, apoptosis, and autophagy. ResultsIn the present study, the results showed that 20(S)-GRh2 decreased cell growth and arrested cell cycle at the G1 phase in ALL cells. 20(S)-GRh2 induced apoptosis through enhancing reactive oxygen species generation and upregulating apoptosis-related proteins. 20(S)-GRh2 significantly elevated the levels of pEGFP-LC3 and autophagy-related proteins in Jurkat cells. Furthermore, the PI3K/Akt/mTOR signaling pathway was effectively blocked by 20(S)-GRh2. 20(S)-GRh2 suppressed cell proliferation and promoted apoptosis and autophagy by suppressing the PI3K/Akt/mTOR pathway in Jurkat cells. Finally, 20(S)-GRh2 alleviated symptoms of leukemia and reduced the number of white blood cells and CD3 staining in the spleen of xenograft mice, indicating antitumor effects against T-ALL invivo. ConclusionThese findings indicate that 20(S)-GRh2 exhibits beneficial effects against T-ALL through the PI3K/Akt/mTOR pathway and could be a natural product of novel target for T-ALL therapy.

A single miRNA and miRNA sponge expression system for efficient modulation of miR-223 availability in mammalian cells.

Hundreds of microRNAs (miRNAs), comprising small non-coding RNAs of 20-24nucleotides, have been discovered, although the entirety of their biological functions is poorly understood. Overexpression or suppression approaches are commonly performed to investigate the function of specific miRNAs. In the present study, we focused on generating a lentiviral vector-based strategy that enables hsa-miR-223-3p (miR-223) overexpression and suppression in the target cells for functional analysis of this miRNA easily and rapidly. The sequence that gives rise to miR-223 and the sequence generating the sponge RNA with four binding sites for miR-223 were cloned in pLVX-shRNA2 vector. The functionality of the vector to overexpress miR-223 was evaluated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays, whereas the post-transcriptional regulation exerted by miR-223 was evaluated by luciferase reporter assays in AD-293 cells. The anti-miR-223 sponge activity with one binding site for miR-223 (pmCherry-anti-miR-223) was confirmed by qRT-PCR and the restoration of its target (IKKα) was evaluated by western blot assays in Jurkat cells. The pLVX-miR-223 vector is functional for over-expressing miR-223 and regulates the mRNA of MDR1/ABCB1 at the post-transcriptional level in AD-293 cells. The anti-miR-223 sponge with one miR-223 binding site efficiently modulates the miR-223 availability and not the one with four sites. The over-expression of anti-miR-223 correlated with a decrease in the levels of miR-223 and, consequently, with an increase in the expression level of the IKKα protein in Jurkat cells. This single miRNA and miRNA sponge expression system specifically alters the availability of miR-223 in mammalian cells.

Anthocyanin-rich bilberry extract induces apoptosis in acute lymphoblastic leukemia cells via redox-sensitive epigenetic modifications

Polycomb group (PcG) proteins are epigenetic regulators that reduce tumor suppressor gene expression and promote cancer cell survival. The aim of the present study was to determine whether a bilberry extract (Antho 50) inhibits the expression of PcG proteins in Jurkat cells and, if so, to determine the underlying mechanism. Apoptotic rates and the formation of reactive oxygen species (ROS) were assessed by flow cytometry, and protein expression by Western blotting. Antho 50 induced apoptosis, augmented ROS formation, increased p73, p21 and cleaved caspase-3 expression levels, and decreased those of p-Akt, survivin, PcG proteins, HDACs, DNMT1 and UHRF1. The antioxidant enzyme PEG-catalase prevented the formation of ROS and apoptosis induced by Antho 50 and also by H2O2. These findings indicate that Antho 50 promotes apoptosis in Jurkat cells, in part, by decreasing the expression levels of PcG proteins, and, hence, the subsequent PcG proteins-dependent pro-survival events via a redox-dependent mechanism.

The potential mechanism of extracellular high mobility group box-1 protein mediated p53 expression in immune dysfunction of T lymphocytes.

In the present study, we examined the activity of p53 protein in Jurkat cells treated with high mobility group box-1 protein (HMGB1), thereafter we investigated the mechanism of extracellular HMGB1 mediated p53 expression in immune dysfunction of T lymphocytes. mRNA expression of p53, mdm2, and p21 was determined by Real-time reverse transcription-polymerase chain reaction(RT-PCR). The apoptotic rate of Jurkat cells was analyzed by flow cytometry. Expressions of bcl-2, bax, caspase-3, phosphorylated (p) extracellular signal-regulated kinase (ERK)1/2, ERK1/2, p-p38 mitogen-activated protein kinase (MAPK), p38 MAPK, and p-c-jun amino-terminal kinase (JNK)1/2 and JNK1/2 were simultaneously determined by Western blotting. After treatment with HMGB1 (100 ng/ml or 1000 ng/ml), the proliferative activity of Jurkat cells was significantly decreased, and a low and medium concentration of HMGB1 induced an up-regulation of p53 mRNA, p-p53 and p53 protein expression. Meanwhile, levels of mdm2 and p21 were elevated by incubated with HMGB1 (100 ng/ml) for 24 or 48 hours. Moreover, the proliferation of Jurkat cells in response to HMGB1 (100 ng/ml) in the vector group was significantly depressed. The bax and caspase-3 levels in p53 shRNA-expressed cells treated with HMGB1 (100 ng/ml) was markedly decreased, whereas expression of bcl-2 was obviously enhanced. Among ERK1/2, p38 MAPK and JNK1/2 signaling, only p38 MAPK pathway could be significantly activated by treatment with HMGB1, and the specific inhibitor of p38 MAPK was used, p53 and p-p53 expression induced by HMGB1 were significantly down-regulated. Taken together, our data strongly indicated that HMGB1 might enhance p53 expression, which was associated with both the proliferative activity as well as apoptosis of T cells.

Hox5 Paralogous Genes Modulate Th2 Cell Function during Chronic Allergic Inflammation via Regulation of Gata3.

Allergic asthma is a significant health burden in western countries, and continues to increase in prevalence. Th2 cells contribute to the development of disease through release of the cytokines IL-4, IL-5, and IL-13, resulting in increased airway eosinophils and mucus hypersecretion. The molecular mechanisms behind the disease pathology remain largely unknown. In this study we investigated a potential regulatory role for the Hox5 gene family, Hoxa5, Hoxb5, and Hoxc5, genes known to be important in lung development within mesenchymal cell populations. We found that Hox5-mutant mice show exacerbated pathology compared with wild-type controls in a chronic allergen model, with an increased Th2 response and exacerbated lung tissue pathology. Bone marrow chimera experiments indicated that the observed enhanced pathology was mediated by immune cell function independent of mesenchymal cell Hox5 family function. Examination of T cells grown in Th2 polarizing conditions showed increased proliferation, enhanced Gata3 expression, and elevated production of IL-4, IL-5, and IL-13 in Hox5-deficient T cells compared with wild-type controls. Overexpression of FLAG-tagged HOX5 proteins in Jurkat cells demonstrated HOX5 binding to the Gata3 locus and decreased Gata3 and IL-4 expression, supporting a role for HOX5 proteins in direct transcriptional control of Th2 development. These results reveal a novel role for Hox5 genes as developmental regulators of Th2 immune cell function that demonstrates a redeployment of mesenchyme-associated developmental genes.

Nicotinamidephosphoribosyltransferase (NAMPT) Induces Ubiquitination of LEF-1 Transcription Factor By Deacetylation

We and others demonstrated, that transcription factor lymphoid enhancer-binding factor 1 (LEF-1) is crucial for neutrophil granulocytopoiesis and lymphopoiesis. LEF-1 expression is severely diminished in myeloid progenitors of severe congenital neutropenia (CN) patients, leading to "maturation arrest" of granulopoiesis.In the present study we evaluated the mechanisms of LEF-1 regulation. Previously we identified elevated levels of the enzyme Nicotinamide phosphoribosyltransferase (NAMPT) and its target Sirtuin 1 (SIRT1) in myeloid cells of CN patients. SIRT1 is NAD+-dependent protein deacetylase and in this study we analyzed whether LEF-1 could be regulated by NAMPT/SIRT1-triggered deacetylation. We identified lysine residue in LEF-1 protein, which could be de-/acetylated, generated specific rabbit polyclonal antibody recognizing this acetyl-Lys and confirmed NAMPT/SIRT1-triggered deacetylation of LEF-1 protein on this lysine in T-cell leukemia cell line Jurkat and in AML cell line LW/SO.We first studied the effects of LEF-1 deacetylation by NAMPT/SIRT1 on its expression and functions. We found markedly diminished levels of total LEF-1 protein as well as its nuclear translocation in Jurkat and LW/SO cells treated with NAMPT. At the same time, inhibition of NAMPT using specific NAMPT inhibitor FK866 resulted in elevated mRNA and protein levels of LEF-1 and its target genes survivin, c-myc and cyclin D1.We also performed reporter gene assay using WT or acetyl-Lys LEF-1 mutant and TOP promoter construct, containing six LEF-1/TCFs binding sites. Acetyl-Lys LEF-1 mutant was not able to activate TOP promoter, as compared to its strong activation by WT LEF-1. Moreover, co-transfection with NAMPT- or SIRT1-expression constructs also showed inhibitory effects on LEF-1-triggered activation of TOP promoter. These experiments clearly demonstrated that NAMPT/SIRT1-triggered deacetylation of the LEF-1 protein inhibited its expression and functions.We further evaluated the mechanism of NAMPT-triggered reduction of LEF-1 expression. In silico analysis of LEF-1 protein using CPLM software revealed that lysine residue in LEF-1 protein, which is de-/acetylated, could be also ubiquitinated. We therefore studied the effects of deacetylation on the ubiquitination of LEF-1 protein in Jurkat cells. We evaluated the effects of 26S proteasome inhibitor, bortezomib, on LEF-1 protein levels after treatment with NAMPT. Indeed, upon treatment with bortezomib the level of LEF-1 protein was restored. This confirmed, that deacetylation of LEF-1 by NAMPT led to degradation of LEF-1 by ubiquitination. We assumed, that in CN patients, hyperactivation of NAMPT/SIRT1 pathway might lead to downregulation of LEF-1 by deacetylation and ubiquitination. Indeed, we observed decreased G-CSF-triggered granulocytic differentiation and proliferation of CD34+ cells from healthy individuals transduced with LEF-1 acetyl-lysine mutant, as compared to cells transduced with WT LEF-1. Moreover, treatment of CD34+ cells of CN patients with bortezomib resulted in restoration of defective LEF-1 expression ultimately leading to normalization of granulocytic differentiation.In conclusion, NAMPT/SIRT1-triggered deacetylation of LEF-1 resulted in its degradation by ubiquitination. DisclosuresNo relevant conflicts of interest to declare.

A role for Hox5 genes in T cell function during chronic allergic inflammation (HYP7P.268)

Abstract Allergic asthma is a significant health burden in western countries, and continues to increase in prevalence. T helper 2 (Th2) cells contribute to the development of disease through release of the cytokines interleukin (IL)-4, IL-5 and IL-13, resulting in increased airway eosinophils and mucus hypersecretion. While many factors are involved in the development of allergic asthma, the molecular mechanisms behind the disease remain largely unknown. As the three members of the Hox5 gene family (Hoxa5, Hoxb5 and Hoxc5) have been shown to play a role in lung development, we asked whether these genes also play a role in the adult lung in response to disease. We found that Hox5-deficient mice show exacerbated pathology compared to wild-type controls in a chronic allergen model, with an increased Th2 response as well as changes in lung tissue pathology. Examination of T cells from naïve wild-type or Hox5-deficient mice in Th2 polarizing conditions showed increased proliferation, enhanced Gata3 expression and production of IL-4, IL-5 and IL-13 in Hox5-deficient mice compared to wild-type controls. Overexpression of FLAG-tagged HOX5 proteins in Jurkat cells demonstrated HOX5 binding to the Gata3 locus, supporting a role for HOX5 proteins in direct transcriptional control of Th2 development. These results reveal a novel role for Hox5 genes as developmental regulators of immune cell function and Th2-induced pathology during allergic disease in the lung.

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

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.

Differential 12-O-Tetradecanoylphorbol-13-acetate-induced activation of rat mammary carcinoma susceptibility Fbxo10 variant promoters via a PKC-AP1 pathway.

Rat mammary carcinoma susceptibility 5a1 (Mcs5a1), which is concordant to human MCS5A1 breast cancer risk locus, mediates susceptibility by a non-mammary cell-autonomous mechanism associated with T cell differential expression of F-box protein 10 (Fbxo10). Human FBXO10, an evolutionarily conserved ubiquitin ligase gene, was shown to have a potential role in regulating cell death by controlling the degradation of Bcl-2, a key protein involved in apoptosis. Breast cancer susceptibility is controlled by interactions between environmental and genetic factors; therefore, we sought to determine if breast cancer risk-associated environmental chemicals interact with Mcs5a1 variants using luciferase reporter constructs containing 4.2 kb Fbxo10 promoters based on alleles of mammary cancer susceptible Wistar Furth (WF) and resistant Wistar Kyoto (WKY) rat strains. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced activation of a 4.2 kb WF Fbxo10 promoter region, but lower levels of activation of the homologous WKY Fbxo10 promoter region. Using general and specific protein kinase inhibitors, we identified a protein kinase C (PKC) pathway that mediated TPA activation. We narrowed the possible PKCs to a member of the atypical PKC isoforms, namely PKCµ. We also determined that activator protein 1 (AP1) family member c-Fos mediated TPA activation of the 4.2 kb WF Fbxo10 promoter. TPA was shown to induce endogenous FBXO10 mRNA and FBXO10 protein in Jurkat cells, a human T cell line, with a maximal level of expression from 1.5 to 2.5 h after exposure. These results indicate that FBXO10/Fbxo10 expression is regulated by a PKC-dependent pathway acting through c-Fos, which binds AP1-specific DNA elements in Mcs5a1.

NF-κB Is Constitutively Activated in EBV-Infected T or NK Cells and Can Be a Molecular Target for EBV-Positive T/NK-Cell Lymphoproliferative Disease Treatment

AbstractAbstract 1998Epstein-Barr virus (EBV) can infect not only B cells but also rarely T or NK cells and causes EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), such as extranodal NK/T-cell lymphoma (ENKL), aggressive NK-cell leukemia, and chronic active EBV infection (CAEBV). However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies have not been elucidated to date. Furthermore, optimal chemotherapy for these, especially for CAEBV, has not been established and the prognosis of EBV-T/NK-LPD remains very poor. NF-kB is a transcription factor that mediates anti-apoptotic molecular signaling and promotes proliferation of cancer cells. NF-kB is known to be constitutively activated in some hematological malignancies; the proteasome inhibitor bortezomib suppresses its activity and is used clinically as an anticancer reagent. To clarify the molecular mechanism underlying the development of EBV-T/NK-LPD, we focused on NF-kB in four EBV-positive T- and NK-cell lines, SNT8, SNT15, SNT16, and SNK6, which had been established from primary lesions of ENKL patients (SNT8 and SNK6) and the peripheral blood (PB) of a CAEBV patient (SNT15, 16), respectively. In these cells, it was demonstrated that p50, p52, RelA, and RelB—the components of NF-kB—existed constitutively in the nucleus. On the other hand, the EBV-negative T-cell lines, Jurkat and Molt4, and an NK-cell line, KHYG1, were negative for nuclear localization of these molecules. Elevated nuclear NF- kB–DNA binding activity was demonstrated by electrophoretic mobility shift assay (EMSA), using oligonucleotides encoding an NF-kB-binding sequence as a probe. Supershift EMSA revealed that NF- kB–DNA binding complexes in these cells involved p50, p52, and RelA. In addition, reporter assay using NF-kB-dependent luciferase reporter gene consisting of an NF-kB-binding site as a promoter demonstrated constitutive activation of NF-kB in SNT8 cells, whereas not in Jurkat cells. From these results, we concluded that NF-kB was constitutively activated through the canonical and noncanonical pathways in these EBV-infected T- or NK-cell (EBV-T/NK cell) lines. Next, we investigated NF-kB activation in EBV-T/NK cells derived from CAEBV patients. CAEBV was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in PB, and detection of EBV-infected T or NK cells with clonal proliferation. To detect infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. The EBV DNA of each fraction was quantified using a real-time quantitative polymerase chain reaction assay. Ten patients (aged 8–72 years; 4 male, 6 female; infected cell types CD4:3, CD8:3, γδ:1, and CD56:3) were diagnosed as CAEBV. Then, we examined NF-kB activation in these cells. Similar to the cell lines, immunoblotting and EMSA demonstrated evidence of constitutive activation of NF-kB such as nuclear localization and DNA binding of p50, p52, and RelA in EBV-T/NK cells from CAEBV patients. To investigate the molecular mechanism of NF-kB activation in EBV-T/NK cells, we examined the influence of viral proteins expressed in EBV-T/NK cells, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We performed luciferase reporter assay using expression vectors for these proteins in Jurkat cells. LMP1, most significantly, and LMP2A, to a lesser extent, upregulated NF-kB-dependent reporter gene expression in Jurkat cells, whereas the other viral proteins did not, suggesting that LMP1 and LMP2A mediated NF-kB activation in T/NK cells. Finally we investigated the contribution of NF-kB to the development of EBV-T/NK-LPD. Bortezomib, in a dose-dependent manner, inhibited translocation of p50, p52, and RelA to the nucleus and reduced the expression of Bcl-XL, which was a transcriptional target of NF-kB in SNT8, SNK6, and CAEBV patients' cells. In addition, it was also confirmed that bortezomib (0.5nM -5nM) suppressed the survival of and induced apoptosis of EBV-T/NK cells derived from 5 CAEBV patients. These results suggest that constitutive activation of NF-kB contributes to the development of EBV-T/NK-LPD and that the NF-kB signaling pathway can be an attractive molecular target for treatment. Disclosures:No relevant conflicts of interest to declare.

Changes in the level of apoptosis-related proteins in Jurkat cells infected with HIV-1 versus HIV-2

Human immunodeficiency virus (HIV) infection-induced apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 T cells and other types of cells is a major factor in the pathogenesis of AIDS. Clinically, HIV-2 patients have a higher CD4 cell count at the time of an AIDS diagnosis, and generally have longer survival after development of symptoms. The mortality after an AIDS diagnosis has been reported to be more influenced by CD4 cell count than HIV type. Previous studies have shown significant variations in cytopathic effects following in vitro infection with primary isolates of HIV-1 or HIV-2 subtypes; however, the relative contributions of HIV-1 and HIV-2 infection leading to cell death remain unclear. Using a human cell line, Jurkat, we examined differences in key molecules involved in apoptotic signaling pathways during infection with either HIV-1 or HIV-2. HIV-1 infection generated more reactive oxygen species (ROS), increased the expression of a larger number of molecules involved in cell signaling such as p47, p38alpha, JNK, c-Yes, total PKC, and decreased the expression of molecules such as p38beta, ERK1/2, and XIAP relative to HIV-2 infection. HIV-1 induced a higher degree of cell death through stronger activation of both apoptotic pathways. HIV-1 infection downregulated both Bcl-X(L) and FLIP expressions at later time points postinfection, while HIV-2 infection dramatically upregulated both Bcl-X(L) and FLIP expression. We also found that the expression of Bcl-X(L) or FLIP resulted in significant inhibition of HIV replication in Jurkat cells. These findings suggest that HIV-1 infection with high levels of cytotoxicity results in a higher level of cell death through apoptosis during a short time postinfection. The longer period of infection observed with HIV-2 with a lower degree of cytotoxicity was accompanied by increased Bcl-X(L) and FLIP expression. High protein levels of Bcl-X(L) or FLIP inhibit HIV replication and may be one explanation for the clinical observation that HIV-2 infected patients generally tend to be long-term nonprogressors with high CD4 lymphocyte counts compared with HIV-1 infected persons.

TRPV5 and TRPV6 calcium channels in human T cells

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) provided the molecular base for the studying of new candidate of calcium influx in non-excitable cells. Using RT-PCR technique we obtained endogenous expression of the mRNAs trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in human blood primary T lymphocytes. Additionally, Western blot analysis showed TRPV5 proteins in both the whole lysate and in the crude membrane preparations from Jurkat cells and normal T lymphocytes. The using of the immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes TRPV6 was not detected. The expression pattern and the selective Ca2+ permeation properties of TRPV5 and TRPV6 channels indicate an important role of these channels in the Ca2+ homeostasis and probably in malignant transformation of blood cells.

Characterization of β-N-Acetylglucosaminidase Cleavage by Caspase-3 during Apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.

Evidence for the Involvement of Tyrosine Kinase ZAP 70 in Nuclear Retinoid Receptor-dependent Transactivation in T Lymphocytes

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are transcription factors that control diverse cellular functions during development and homeostasis. The biochemical role of these proteins in T lymphocytes is not well known. Here we have studied the role of protein-tyrosine kinase ZAP 70, a key enzyme involved in the proximal signaling events during T cell activation, in the modulation of RXRE- and RARE-dependent activation in T lymphocytes. Surprisingly, ZAP 70-negative Jurkat T cells showed considerable loss of both RXRE- and RARE-mediated transactivation as compared with wild type Jurkat cells. In addition, ZAP 70-negative cells failed to exhibit normal protein kinase C and calcineurin-induced transcriptional activity. ZAP 70-negative cells that were reconstituted with active ZAP 70 regained the transactivation function, whereas cells expressing kinase-dead form of ZAP 70 failed to do so. Defective transcriptional activation was also observed in actively proliferating human peripheral blood T lymphocytes in which RNA interference was used to induce loss of ZAP 70 expression. In addition, an Lck-deficient Jurkat cell line that cannot efficiently activate ZAP 70 was also found defective in RXRE-mediated transcription. Finally, RNA interference-induced loss of ZAP 70 or Lck protein in Jurkat cells resulted in significant decrease in the RXRE-dependent activation. Together, these results suggest a novel functional role for ZAP 70 in nuclear receptor-driven transactivation in T lymphocytes.

Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells

Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells

Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells

Thiol proteins are important in cellular antioxidant defenses and redox signalling. It is postulated that reactive oxidants cause selective thiol oxidation, but relative sensitivities of different cell proteins and critical targets are not well characterized. We exposed Jurkat cells to H2O2 for 10 min and measured changes in reversibly oxidized proteins by labelling with iodoacetamidofluorescein and two-dimensional electrophoresis. At 200 microM H2O2, which caused activation of the MAP (mitogen-activated protein) kinase ERK (extracellular-signal-regulated kinase), growth arrest and apoptosis, relatively few changes were seen. A total of 28 spots were reversibly oxidized (increased labelling intensity) and 24 decreased. The latter included isoforms of peroxiredoxins 1 and 2, which were irreversibly oxidized. Oxidation of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was striking, and other affected proteins included glutathione S-transferase P1-1, enolase, a regulatory subunit of protein kinase A, annexin VI, the mitotic checkpoint serine/threonine-protein kinase BUB1beta, HSP90beta (heat-shock protein 90beta) and proteosome components. At 20 microM H2O2, changes were fewer, but GAPDH and peroxiredoxin 2 were still modified. Dinitrochlorobenzene treatment, which inhibited cellular thioredoxin reductase and partially depleted GSH, caused reversible oxidation of several proteins, including thioredoxin 1 and peroxiredoxins 1 and 2. Most changes were distinct from those with H2O2, and changes with H2O2 were scarcely enhanced by dinitrochlorobenzene. Relatively few proteins, including deoxycytidine kinase, nucleoside diphosphate kinase and a proteosome activator subunit, responded only to the combined treatment. Thus most of the effects of H2O2 were not linked to thioredoxin oxidation. Our study has identified peroxiredoxin 2 and GAPDH as two of the most oxidant-sensitive cell proteins and has highlighted how readily peroxiredoxins undergo irreversible oxidation.

MIZORIBINE-MEDIATED APOPTOTIC SIGNALING PATHWAY IN HUMAN T CELL LINE

P778 Propose: Mizoribine (MZR), an inhibitor of IMP dehydrogenase which depletes cellular GTP, is used clinically as an immunosuppressive drug. This study was designed to evaluate the mechanism by which MZR exerts the cytotoxic effect on Jurkat T cells. Methods: The data demonstrated that treatment of MZR decreased cell viability in a dose- and time-dependent manner. MZR-induced cell death was confirmed as apoptosis characterized by chromatin condensation and H2AX phosphorylation. Results: MZR increased the catalytic activity of caspase-3 protease -8 protease and -9 proteases. Activation of caspase-3 protease was further confirmed by degradation of polymerase (PARP), a substrate of caspase-3 protease by MZR in Jurkat cells. Furthermore, MZR induced the changes of mitochondrial transmembrane potential (MTP) and the cytosolic release of cytochrome c from mitochondria. In addition, MZR induced the decrease of Bcl-XL expression whereas increase of Bcl-XS, Bak and Bim expression. Guanosine markedly inhibited cell viability and apoptosis with consistent suppression of activity of caspase-8 protease, upstream caspase among caspase family, H2AX phosphorylaion and PARP cleavage in MZR-treated cells. Also, we have screened the expression profile of proteins in Jurkat cells by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in 2-D gels, the comparison of control versus apoptotic cells revealed that signal intensity of 10 spots was decreased and 5 spots was increased. Conclusions: The results suggest that MZR functions as an inhibitor of IMP dehydrogenase in apoptosis of Jurkat cells via activation of intrinsic caspase cascades as well as mitochondrial dysfunction.