Retracted: Transactivation of CCL20 gene by Epstein–Barr virus latent membrane protein 1
Taeko OkudairaKazuo YamamotoHirochika KawakamiJun‐Nosuke UchiharaMariko TomitaMasato MasudaTakehiro MatsudaTakeshi SairenjiHidekatsu IhaKuan‐Teh JeangT. MatsuyamaNobuyuki TakasuNaoki Mori
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Abstract:
Summary CCL20 is expected to play a crucial role in the initiation of immune responses and tumour growth. However, expression of CCL20 in Epstein–Barr virus (EBV)‐associated diseases has not been studied. We examined the contribution of EBV infection and EBV‐encoded latent membrane protein (LMP)‐1 to CCL20 expression. EBV infection and LMP‐1 induced CCL20 mRNA expression in the EBV‐negative Burkitt lymphoma (BL) cell lines and the embryonic kidney cell line. Histone deacetylase inhibitor‐stimulated endogenous LMP‐1 also induced CCL20 expression in an EBV‐positive BL cell line. Analysis of the CCL20 promoter showed that it was activated by LMP‐1 C‐terminal activation region (CTAR)‐1 and CTAR‐2. Co‐expression of I κ B α , I κ B β , I κ B kinase (IKK) α , IKK β , IKK γ , nuclear factor (NF)‐ κ B‐inducing kinase and tumour necrosis factor receptor‐associated factor 2 dominant‐negative constructs with LMP‐1 inhibited the activation of the CCL20 promoter by LMP‐1, suggesting that LMP‐1 induces CCL20 via NF‐ κ B signalling. The requirement for the NF‐ κ B‐binding site in the CCL20 promoter in LMP‐1 responsiveness was established. Our results indicate that activation of the NF‐ κ B pathway by LMP‐1 is required for the activation of CCL20 expression.Keywords:
CCL20
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Objective:To establish a multi-drug resistance(MDR) cell line Jurkat/Doxorubicin(DOX) derived from human T lymphocyte leukemia cell line Jurkat,and to compare the biological characteristics between Jurkat/DOX and Jurkat cell line.Methods:Jurkat cells were induced with long-term,singular,and dose-increasing DOX addition to MDR cell line Jurkat/DOX.The biological characteristics and the spectrum of MDR of Jurkat/DOX were evaluated by MTT assay.The intracellular accumulation of DOX was detected by flow cytometer and laser scanning confocal microscope.The expression of mdr1 was analyzed by RT-PCR.Western blot was also performed to detect the expression of P-gp.Results:MDR cell line Jurkat/DOX was established by long-term,intermittent,singular and dose-increasing DOX induction.The growth of Jurkat/DOX was slower than that in Jurkat cell line.Jurkat/DOX cells were resistant not only to the original DOX,but also to many other anticancer drugs such as CTX,VCR,and DNR.Intracellular accumulation of DOX was significantly less than that in parent cells.The expression of mdr1 gene and P-gp protein were up-regulated in Jurkat/DOX cell line.Conclusion:The MDR cell line Jurkat/DOX was successfully established,it has the property of resistant to many anticancer drugs,and the resistant maybe closely related to the over-expression of P-gp.
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To investigate the role of Tal1 gene, which is aberrantly expressed in 40%-60% of patients with T lymphocytic leukemia (T-ALL), in the proliferation of T-ALL cells.We established stable Jurkat-siTal1 and Jurkat-T1 cell lines by trasnfecting T-ALL Jurkat cells with lentiviral vectors to knock-down or overexpress Tal1. Jurkat cells transfected with negative control siRNAs for Tal1 knock-down (Jurkat-mock1) and over-expression(Jurkat-mock2) served as the control cells. The proliferation of the cells lines was assessed using CCK-8 assay, and the cell cycle distribution was determined by flow cytometry. The mRNA and protein expressions of cyclin-dependent kinase inhibitor 2 (CDKN2A) and cyclin-dependent kinase inhibitor 1 (CDKN2B) were measured by real-time RT-PCR and Western blotting, respectively.Jurkat-T1 cells showed more active proliferation in vitro than Jurkat-mock2 cells, while Jurkat-siTal1 cells showed slower growth than Jurkat-mock1 cells. In Jurkat-T1 cells, G0/G1 phase cells were decreased and S phase cells increased compared with Jurkat-mock2 cells, and Jurkat-siTal1 cells showed increased G0/G1 phase cells and decreased S phase cells compared with Jurkat-mock1 cells. Real-time RT-PCR and Western blotting showed that Tal1 inhibited the cellular expression of CDKN2A and CDKN2B at both mRNA and protein levels.Tal1 promotes the growth and the transition from G0/G1 phase to S phase in T-ALL cells Jurkat by inhibiting the expressions of G0/G1 and S phase negative regulatory proteins CDKN2A and CDKN2B.
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Abstract The control of IL‐2 gene expression in T cells by multiple transcriptional factors has been extensively explored, however, the role of the NF‐κB signaling pathway in TCR‐dependent IL‐2 production still remains unclear. In this study, we used a somatic cell genetics approach to address this question. Triggering TCR in mutant Jurkat T cells lacking IKKγ/NEMO failed to induce IL‐2due to a selective loss in I‐κB kinase activity, I‐κBα degradation and NF‐κB DNA‐binding activity. The AP‐1 and NF‐AT binding activities in the IL‐2 promoter were comparable between wild‐type and mutant T cells. These defects in the mutant cell line were rescued by the reintroduction of exogenous IKKγ. Taken together, our data demonstrate that IKKγ plays an essential role in TCR‐induced signaling pathways leading to IL‐2 expression.
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Experiments from flight- and ground-based model systems suggest that unexpected alterations of the human lymphoblastoid cell line Jurkat, as well as effects on cell growth, metabolism, and apoptosis, can occur in altered gravity conditions. Using a desktop random positioning machine (RPM), we investigated the effects of simulated microgravity on Jurkat cells and their multidrug-resistant subline, Jurkat/A4 cells. The viability of Jurkat/A4 cells decreased after simulated microgravity in contrast with the Jurkat cells. At the same time, the viability between the experimental Jurkat cells and control Jurkat cells was not significantly different. Of note, Jurkat cells appeared as less susceptible to apoptosis than their multidrug-resistant clone Jurkat/A4 cells, whereas cell-cycle analysis showed that the percentage of Jurkat/A4 cells in the S-phase was increased after 72 and 96 h of RPM-simulated microgravity relative to their static counterparts. The differences in Jurkat cells at all phases between static and simulated microgravity were not significant. The surface expression of the intercellular adhesion molecule 3 (ICAM-3)—also known as cluster of differentiation (CD)50—protein was changed for Jurkat/A4 cells following exposure to the RPM. Changes in cell morphology were observed in the Jurkat/A4 cells after 96 h of RPM-simulated microgravity. Thus, we concluded that Jurkat/A4 cells are more sensitive to RPM-simulated microgravity as compared with the parental Jurkat cell line. We also suggest that intercellular adhesion molecule 3 may be an important adhesion molecule involved in the induction of leukocyte apoptosis. The Jurkat/A4 cells with an acquired multidrug resistance phenotype could be a useful model for studying the effects of simulated microgravity and testing anticancer drugs.
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Objective:To investigate the effect of Ara-C at low concentrations on extracellular matrix metalloproteinase inducer(CD147) expression in Jurkat cells.Methods:Jurkat cells were divided into six groups:control group and cells treated with different concentrations of Ara-C at 1,5,10,20,and 30 ng/ml.CD147 mRNA and protein expression were assayed by RT-PCR,Western blot and flow cytometry,respectively.The colony formation in Jurkat cells which was stimulated with Ara-C or not was observed by inverted microscope.Results:Different concentrations of Ara-C significantly up-regulated the expressions of CD147 mRNA and protein,and increased the colony formation in Jurkat cells(P0.01).Conclusions:Ara-C at low concentrations can enhance the expression of CD147 and increase the adhesive force in Jurkat cells.
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OBJECTIVE: To study the role of MEKK2 on the production of IL-2 in Jurkat cells stimulated by PHA/anti-CD28 antibody. METHODS: The MEKK2 and JNK kinase activities were measured in both dominant negative MEKK2 Jurkat (dnMEKK2 Jurkat) cells and parental Jurkat cells. The AP(1) and IL-2 promotor activities were measured by luciferase activity assay. The IL-2 mRNA and protein were detected by RT-PCR and Western blot. RESULTS: After stimulation by PHA/anti-CD28, JNK was activated in parental Jurkat cells but not in dnMEKK2 Jurkat cells. The luciferase report gene activities of AP1 and IL-2 promotors were increased by 4- and 5-folds in parental cells whereas only by 1 fold in dnMEKK2 Jurkat cells. The level of IL-2 mRNA and IL-2 protein were increased in parental Jurkat cells but not in dnMEKK2 Jurkat cells. CONCLUSION: MEKK2 plays an important role on the production of IL-2 in Jurkat cell stimulated with PHA/anti-CD28 antibody. It is a potential drug target for the treatment of GVHD and autoimmune disease.
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Exposure to ultraviolet B (UVB) irradiation from sunlight induces the upregulation of VEGF, a potent angiogenic factor that is critical for mediating angiogenesis-associated photodamage. However, the molecular mechanisms related to UVB-induced VEGF expression have not been fully defined. Here, we demonstrate that one of the catalytic subunits of the IκB kinase complex (IKK), IKKα, plays a critical role in mediating UVB-induced VEGF expression in mouse embryonic fibroblasts (MEFs), which requires IKKα kinase activity but is independent of IKKβ, IKKγ and the transactivation of NF-κB. We further show that the transcriptional factor AP-1 functions as the downstream target of IKKα that is responsible for VEGF induction under UVB exposure. Both the accumulation of AP-1 component, c-Fos and the transactivation of AP-1 by UVB require the activated IKKα located within the nucleus. Moreover, nuclear IKKα can associate with c-Fos and recruit to the vegf promoter regions containing AP-1-responsive element and then trigger phosphorylation of the promoter-bound histone H3. Thus, our results have revealed a novel independent role for IKKα in controlling VEGF expression during the cellular UVB response by regulating the induction of the AP-1 component and phosphorylating histone H3 to facilitate AP-1 transactivation. Targeting IKKα shows promise for the prevention of UVB-induced angiogenesis and the associated photodamage.
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Objective:To investigate the expression regulation of PD-L1(Programed Death Ligand-1) in Jurkat cell and correlation between the expression of PD-L1(Programed Death Ligand-1,B7-H1) and the activity and apoptosis of Jurkat cell.Methods:IFN-γ and the siRNA(small interfering RNA) targeting to PD-L1 were used to control the expression of PD-L1 in the Jurkat cell.RT-PCR,PCR were used to measure the expression of IL-2 and flow cytometry was used to detected the apoptosis of Jurkat cell.Results:Semi-quantitative β-actin,according template amount to PCR,was used to choose siRNA-A from three different siRNA(siRNA-A,siRNA-B,siRNA-C) which can efficiently and specifically inhibit the expression of PD-L1 in Jurkat Cell.80nM siRNA-A transfect Jurkat Cell,after 24h,the expression of PD-L1 decrease obviously.2000U/ml IFN-γ induced Jurkat Cell,24h later,PD-L1 up-regulated.The expression of PD-L1 in Jurkat Cell negatively correlates with the level of IL-2 secreted by Jurkat Cell by RT-PCR and PCR.And the result from flow cytometry showed increased expression of PD-L1 can reduce the apoptosis of Jurkat Cell.Conclusions:Synthesized siRNA for PD-L1,after transfecting Jurkat cell,could specifically inhibit the expression of PD-L1.IFN-γ in vitro could stimulate Jurkat cell and increase the expression of PD-L1.PD-L1 signal negatively correlates with IL-2 secreted by Jurkat cell,and PD-L1 could inhibit the apoptosis of Jurkat cell.
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To investigate the effect of oridonin on the human acute lymphocytic leukemia cell line Jurkat and its mechanism.Jurkat cells were cultured in vitro and treated with various concentrations (0, 1.25, 2.5, 5, and 10 μmol/L) of oridonin for different lengths of time (24, 48, and 72 hours). The proliferation of Jurkat cells was analyzed by MTT assay. The changes in nuclear morphology were evaluated by fluorescence microscopy at 12 hours after treatment with various concentrations of oridonin. The expression levels of Brg1, P53, and C-myc were determined by semi-quantitative Western blot in Jurkat cells treated with various concentrations of oridonin for 24 hours or 5 μmol/L oridonin for various lengths of time (0, 2, 6, 12, and 24 hours). The expression levels of P53 and C-myc and proliferation of Jurkat cells were evaluated after Brg1 expression was knocked down by Brg1-specific siRNA.Compared with the control group, the proliferation of oridonin-treated Jurkat cells was significantly inhibited in a concentration- and time-dependent manner (P<0.05). According to the florescence microscopic analysis, oridonin treatment led to nuclear pyknosis in Jurkat cells. Compared with the control group, Jurkat cells treated with 5 μmol/L oridonin had reduced expression of Brg1 and C-myc but elevated expression of P53. Brg1 knock-down led to a significant reduction in proliferation of Jurkat cells (P<0.05), up-regulated expression of P53, and down-regulated expression of C-myc.Oridonin can inhibit the proliferation of Jurkat cells, probably via the Brg1 signaling pathway.
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