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@# Objective: To investigate the reverse effect and mechanism of IL-12 on chemotherapeutic medicine suppressing the immune function of NK cells. Methods: Purified NK cells were stimulated with PMAplus Ionomycin in the presence or absent of Cisplatin (DDP) and IL-12. The levels of IFN-γ and TNF-α in culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA); The content of IFN-γ and TNF-α, TRAIL (TNF-related apoptosis inducing ligand) and transcription factors including T-bet and p-STAT-4 in NK cells were analyzed by Flow cytometry. The cytotoxicity of purified NK cells (pretreated with/without chemotherapeutics and IL-12 for 48 h) to Jurkat cells was measured by Flow cytometry. Results: Chemotherapeutics significantly inhibited the production of IFN-γ, TNF-α and the expression of TRAILin NK cells, which were significantly reversed by IL-12 (P<0.05 or P<0.01). Further study revealed that chemotherapeutics down-regulated while IL-12 reversed the expression of p-STAT4 to restore cytokine production. In addition, DDP also inhibited but IL-12 recovered the cytotoxicity of NK cells against tumor cells by inducing the expression of TRAIL (P<0.05 or P<0.01). Conclusion: Chemotherapeutics inhibited the cytotoxicity of NK cells and its secretion of cytokines (IFNγ and TNF-α), which were reversed by IL-12 via up-regulating TRAIL and p-STAT-4; this might provide experimental evidence for the clinical application of IL-12 for rebuild the immune function of tumor patients receiving chemotherapy.
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Objective To explore the effect of proliferation and apoptosis of Solanine on acute T lymphocyte leukemia (T-ALL) Jurkat cells and its mechanism.Methods After treated with different concentrations of Solanine,the proliferation of Jurkat cells was detected by CCK-8 assay,and the effect of Solanine on apoptosis of Jurkat cells were detected by flow cytometry.The expression of Bcl-2 and Bax in Jurkat cells were detected by Western blot,and the expression levels of Bcl-2 mRNA and Bax mRNA were detected by real-time fluorescence quantitative polymerase chain reaction.Results CCK-8 assay showed that Solanine significantly inhibited the proliferation of Jurkat cells in a dose-and time-dependent manner.The results of flow cytometry showed that the apoptosis rates of Jurkat cells treated with Solanine for 24 h were (2.40-± 0.98) ％,(28.43-± 4.86) ％,(41.56-± 1.87) ％,respectively,in a dose-dependent manner.Western blot showed that Solanine could increase the expression of Bax and decrease the expression of Bcl-2 in Jurkat cells,and they all were dose-dependent.Conclusion Solanine can significantly inhibit the proliferation and induce apoptosis of Jurkat cells.The mechanism is related to the up-regulation of Bax expression and down-regulation of Bcl-2 expression.
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Objective:To establish T lineage leukemia Jurkat cell mice model of over expression of C-terminal Src kinase binding protein( Cbp ) and Cbp palmitoylation and to research the effect of Cbp and Cbp palmitoylation to proliferation of Jurkat cell.Methods:Virus transfected cell of neg-EGFP,Cbp-EGFP and Cbp-m-EGFP were used in mice model.24 female BALB/c-nu mice were randomly divided into blank control group,empty virus control group,over expression of CBP group and Cbp palmitoylation group, 6 mice in each group.The nude mice were weighed in 0,1,2,3,4,5 weeks.The amount of white blood cell in peripheral blood were counted in 0, 1, 2, 3, 4, 5 weeks.The proliferation of Jurkat cell in peripheral blood of mice were observed by laser confocal microscope.The pathological changes of liver were observed using HE staining.The proliferation of Jurkat cell in the bone marrow and peripheral blood of mice were detected with flow cytometry.Results:The weight of mice in over expression of Cbp group was less than that in blank control group,but higher than that in empty virus control group and Cbp palmitoylation group.The weight of mice in Cbp palmitoylation group was less than that in blank control group,empty virus control group and over expression of Cbp group.The amount of white blood cell in peripheral blood and proliferation of Jurkat cell in liver, bone marrow and peripheral blood of mice in over expression of Cbp group was higher than that in blank control group, but less than that in empty virus control group and Cbp palmitoylation group.The amount of white blood cell in peripheral blood and proliferation of Jurkat cell in liver, bone marrow and peripheral blood of mice in Cbp palmitoylation group was higher than that in blank control group,empty virus control group and over ex-pression of Cbp group.Conclusion:Over expression of Cbp and Cbp palmitoylation in T lineage leukemia Jurkat cell mice model was established.Over expression of Cbp has inhibitory effect on the proliferation of Jurkat cell.Cbp palmitoylation has promotable effect on the proliferation of Jurkat cell.
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@#To determine the effects of celastrol on the proliferation and apoptosis of cell lines HL-60 and Jurkat in human leukemia. Human leukemia cell lines were treated with celastrol at different concentrations. The inhibitory rate of cell proliferation was detected by MTT assay; the apoptosis rate was detected by AnnexinV-FITC/PI double staining; cell cycle was observed by PI staining; cell morphology was observed by transmission electron microscope(TEM). Cell proliferation was inhibited by celastrol, with IC50 of(0. 46±1. 05)μmol/L and(0. 88±1. 13)μmol/L at 24 h. Celastrol induced cell apoptosis in a dose-dependent manner. The cell cycle distribution of G1 phase rate increased, S phase rate decreased(P< 0. 05)with typical cell apoptosis-induced morphological changes. Results showed that celastrol could significantly inhibit cell proliferation and induce apoptosis in human leukemia cell lines of HL-60 and Jurkat.
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Objective Ouabain is a cardiotonic steroid that can induce the apoptosis of many tumorous cells .This study was to investigate the anti-tumor mechanisms of ouabain by observing its effects on the apoptosis of T lymphoblastic leukemia Jurkat cells and the expressions of hTERT and c-myc mRNA and protein . Methods Jurkat cells were treated with ouabain at the concentrations of 50 and 100 nmol/L for 24 and 48 hours, and those treated with 1 ×PBS served as the control .Then the apoptosis rate of the cells was detected by flow cytometry after Annexin V/PI staining, the expressions of hTERT and c-myc mRNA determined by RT-PCR, and those of hTERT and c-myc protein by Western blot . Results The apoptosis rates of the Jurkat cells in the 50 and 100 nmol/L oua-bain groups were (5.67 ±3.71)%and (9.63 ±4.83)%respectively at 24 hours, and (19.67 ±4.55)%and (37.60 ±11.89)%at 48 hours, significantly higher than (4.23 ±1.01)%in the PBS control group at 48 hours (P<0.05).Compared with the control, the expressions of hTERT and c-myc mRNA were decreased by 200%and those of hTERT and c-myc protein by 224%and 400%, re-spectively, at 48 hours (P<0.05).There was a positive correlation between the reduction of the mRNA levels and that of the protein levels of hTERT and c-myc (P<0.05). Conclusion Ouabain can down-regulate the mRNA and protein expressions of hTERT and c-myc, which may be one of the mechanisms of its induction of the apoptosis of Jurkat T lymphocyte leukemia cells .
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Objective To identify and analyze a novel transcript 630 in acute T-cell lymphoblastic leukemia cell (T-ALL) Jurkat cell lines.Methods RNA-sequencing was applied to transcriptome profiling.The novel transcript 630 was identified by reverse transcription polymerase chain reaction (RT-PCR) combined with sequencing.The expression level of the novel transcript was detected in some tumor cell lines and normal cells.The sequences for small interfering RNA (siRNA) against 630 were designed and synthesized.Then,the effects of siRNA were determined by real-time PCR.Apoptotic cells and cell cycle analysis were investigated with the flow cytometry after siRNA-mediated knockdown of 630.Results The novel transcript 630 was identified.High expression level of 630 was detected in Jurkat E6-1,HL60 and human umbilical vein endothelial(HUVE) cells.The knockdown of transcript 630 induced a decrease of cellular proliferation.Conclusions The novel transcript 630 is one of the novel transcripts in Jurkat cells.The decreased expression level of transcript 630 can suppress the proliferation of leukemia cells.The novel transcript 630 might contribute to the treatment of T-ALL.
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Objective To investigate the effects of ionizing radiation on the expression of P21 protein in Jurkat cell line and p21 gene in thymocytes and splenocytes of mice.Methods Flow cytometry (FCM)was used to analyze the expression of P21 protein in Jurkat cells at 12 and 24 h after irradiation to 0,0.5,1.0,2.0,4.0,and 6.0 Gy.Real-time PCR was used to detect the expression of p21 gene in thymocytes and splenocytes of mice at4 and 24 h after irradiation to 0,0.5,1.0,2.0,4.0,and 6.0 Gy.Multi-staining was used to analyze the micronucleus rates of Rct in bone marrow.Results The expressions of P21 protein were increased in a dose-dependent manner during 0.5-4.0 Gy(t=-24.23--3.96,P<0.05),but decreased at 6.0 Gy at 12 and 24 h post-irradiation(t=-11.19,-14.50,P<0.05).The expressions of p2 1 gene in both thymocytes and splenocytes of mice were increased in dose-dependent manner in the range of 0-6.0 Gy(including 6.0 Gy)(t=-29.96-8.80,P<0.05),and reached to the peak at 6.0 Gy at 4 and 24 h post-irradiation(t=-11.84--3.42,P<0.05),except thymocytes at 4 h and 1.0 Gy post-irradiation(t=-3.42,P>0.05).Conclusions The expressions of P21 protein and p21 gene could be increased by X-ray irradiation.which shows good dosedependent manners in certain range of dose.
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Objective:To investigate mitochondrial-dependent molecular mechanisms of a novel agonistic anti-human death receptor 5 (DR5) monoclonal antibody(mDRA-6) inducing apoptosis in Jurkat cell.Methods:The dose-dependent and time-dependent cell growth suppression of mDRA-6 in Jurkat cells was determined by MTT assay.The measurement of the mitochondrial transmembrane potential(ΔΨm) of Jurkat cells was detected by flow cytometry with JC-1 single staining.Caspase-8,9 as well as Bid,Bax,Bcl-2 and Cyto c of apoptotic Jurkat cells were analyzed by Western blot after mDRA-6 treatment.Results:The mDRA-6 induced cell growth suppression and cytotoxicity in dose-dependent manner and time-dependent manner.After mDRA-6 treatment at 2.0 μg/ml for15 min,30 min,60 min and 120 min,the change in ΔΨm were 20.14 %,19.34 %,21.11% and 30.90% respectively by JC-1 single staining.Western blot revealed that the level of active fragments of Caspase-8,9 and Bid,Bax,Bcl-2 and Cyto c respectively,and the amount of Cyto c was increased in cytosol concomitant with the related attenuation of Cyto c in mitochondria.Conclusion: Apoptotic pathway of Jurkat cells induced by mDRA-6 is initiated upon DR5 ligatian to mDRA-6 and exogenic Caspase-dependent cell apoptotic cascades is activated,and endogenic mitochondrial-dependent cell apoptosis pathway is activated.mDRA-6 may be a useful agent in investigating human leukemia therapy by using TRAIL/DR5.
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PURPOSE: This study demonstrates that pharmacologic induction of heme oygenase-1 (HO-1) along with catalytic activation significantly modulated apoptosis of Jurkat cells induced by mycophenolic acid (MPA). METHODS: Cells were cultured with the presence or absence of MPA. Flow cytometric analysis was performed after propidium iodide staining. Western blotting of HO-1, Bcl, and Bax was also performed. Cells were stained 4'-6-Diamidino-2-phenylindole (DAPI) and measured by flow cytometry in the absence or presence of CoPPIX. RESULTS: Treatment of MPA decreased cell viability in a dose- and time-dependent manner. MPA-induced cell death was confirmed as apoptosis characterized by sub G0/G1 phase arrest. Expression of HO-1 assumes a pattern of decline after rising at the initial phase. CoPPIX, HO-1 inducer, significantly inhibited the cisplatin-induced apoptosis. Treatment of MPA resulted in reactive oxygen species (ROS) generation in Jurkat cells. CoPPIX attenuated ROS production in MPA-treated cells. CONCLUSION: This result suggests that the protective mechanism of HO-1 on MPA-induced cytotoxicity is associated with direct inhibition of ROS generation and mitochondrial permeability transition.
Subject(s)
Humans , Apoptosis , Blotting, Western , Cell Death , Cell Survival , Flow Cytometry , Heme , Heme Oxygenase-1 , Jurkat Cells , Mycophenolic Acid , Permeability , Propidium , Reactive Oxygen SpeciesABSTRACT
PURPOSE: FK506 (tacrolimus) is a widely used immunosuppressive agent in the treatment of various medical conditions, including autoimmune disease, bone marrow and organ transplantations. Previously FK506 was known to cause apoptotic death of human Jurkat T cells. METHODS: The current study was designed to analyze the gene expression pattern of Jurkat T cells after FK506 application by using cDNA microarray. Treatment of Jurkat T cells with FK506 resulted in a decrease of cell viability in a time- and dose-dependent manner. Next, total RNA of Jurkat T cells was extracted by using TRIzol reagent and used to carry out a confirmation test for the purity and integrity of total RNA. RESULTS: Gene expression levels related to apoptosis and cell cycle process were mainly focused to analyze in FK506-treated Jurkat T cells. According to the inhibition of calcineurin activity, MARCKS in PKC substrates and Sp3 transcription factor was markedly increased in FK506-treated cells. Also, cell cycle control gene Id1 and Id3 were induced in expression from FK506-treated Jurkat T cells. However, FK506 decreased the expression of Src homology 2, G protein, and MEK 2 genes in bioactive peptide induced signaling pathway. It also reduced the expression level of the insulin receptor, DRPLA and Bai1-associated protein 2 genes, which are involved in the regulation of cell motility and morphology control. CONCLUSION: The author will continue to pursue the exact functional roles of genes that are markedly changed in expression by FK506 in human Jurkat T cells in vitro and in vivo experimental models.
Subject(s)
Humans , Apoptosis , Autoimmune Diseases , Bone Marrow , Calcineurin , Cell Cycle , Cell Cycle Checkpoints , Cell Movement , Cell Survival , Gene Expression , Gene Expression Profiling , GTP-Binding Proteins , Guanidines , Jurkat Cells , Models, Theoretical , Oligonucleotide Array Sequence Analysis , Organ Transplantation , Phenols , Receptor, Insulin , RNA , Sp3 Transcription Factor , T-Lymphocytes , Tacrolimus , TransplantsABSTRACT
PURPOSE: FK506 (Tacrolimus) has been widely used as an immunosuppressant. We examined the effects of FK506 on the activation, proliferation and expression of cytotoxic effector molecules of Jurkat human T-lymphocytes. METHODS: We investigated the effects of this compound on cell viability, the production of reactive oxygen species and mitochondrial dysfunction. The cells were cultured in the presence or absence of FK506. Flow cytometric analysis was performed after staining with PI. The viability of the Jurkat cells was decreased by the addition of FK506 in a dose-and time-dependent manners. RESULTS: FK506-induced cytotoxicity was characterized by G0/G1 phase cell-cycle arrest. FK506 induced cell death was confirmed by the caspase-3 protease activation. In addition, the pharmacologic scavenging study of reactive oxygen species (ROS), including H2O2, revealed that cytotoxicity was achieved by the generation of ROS, which might modulate the mitochondrial dysfunction. CONCLUSION: These results suggest that FK506 functions in CDK4-cyclin D1 mediated cell-cycle arrest of Jurkat cells via generation of ROS and mitochondrial dysfunction.
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Humans , Caspase 3 , Cell Death , Cell Survival , Jurkat Cells , Reactive Oxygen Species , T-Lymphocytes , TacrolimusABSTRACT
PURPOSE: This study examined the effects of Tacrolimus (FK506) on the expression of the apoptotic signal transduction proteins of Jurkat human T-lymphocytes. METHODS: The cell viability was examined by a MTT assay, DAPI stain, enzyme activity of caspase family proteins, and western blotting for Bcl-2, Bak, Fas, and Fas-L. The cells were cultured in the presence or absence of FK506. FK506 induced cell death was confirmed to be apoptosis by the observation of nuclear fragmentation. RESULTS: The viability of Jurkat cells was decreased by the addition of FK506 in a dose- and time- dependent manner. The FK506 induced activation of caspase-3 protease was observed. FK506 didn't increase the catalytic activity of caspase -6, -8, and -9 proteases of Jurkat cells in a time-dependent manner. The viability was improved when a caspase-3 inhibitor was added. However, the caspase-9 inhibitor did not affect the viability. Bak protein expression was increased, and the Bcl-2 protein was decreased for some time. The expression of Fas and Fas-L were unaffected by FK506. CONCLUSION: FK506 induces dose- and time-dependent apoptotic cell death, and enhances the apoptosis of Jurkat cell by increasing the expression of Bak and caspase-3.
Subject(s)
Humans , Apoptosis , bcl-2 Homologous Antagonist-Killer Protein , Blotting, Western , Caspase 3 , Caspase 9 , Cell Death , Cell Survival , Jurkat Cells , Peptide Hydrolases , Signal Transduction , T-Lymphocytes , TacrolimusABSTRACT
In this study, we examined the effects of aspirin on the growth rates, subcellar distribution of β-catenin protein, the expression of β-catenin/TCF signaling pathway target gene cyclinD1 mRNA,and cell cycle of Jurkat cell line (Human T-acute lymphoblastic leukemia). Our results showed that the treatment with aspirin inhibited the growth of Jurkat cell line. Jurkat cells treated with 3 mmol/L of aspirin could significantly decrease nuclear localization of β-catenin, and at 5 mmol/L of aspirin,the nuclear localization of β-catenin was undetectable. QRT-PCR showed that the target gene cyclinD1 mRNA expression was gradually decreased with the dosage of aspirin. Aspirin induced G0/G1cell cycle arrest in Jurkat cells. We are led to conclude that aspirin acts through β-catenin-independent mechanisms. The effects of aspirin include down-regulation of β-catenin nuclear localization and G0/G1 cell cycle arrest, which might serve as a means of growth inhibition in aspirin-treated human Jurkat cell line.
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Objective:To study the effects of ICA on HepG2.2.15 cell proliferation, their sensitivity to the lysis by CD3AK effector cell, to investigate the reversal action of ICA on hepatocarcinoma cells from immune escape through Fas/FasL pathway.To provide the theoretical and experimental bases for ICA development.Methods:MTT assay was used to detect cell proliferation and CD3AK cells cytotoxicity activity;flow cytometry assay was used to examine expression of surface molecules and apoptosis rate of HepG2.2.15 cells.Results:When HepG2.2.15 cells line was treated with 50 ?g/ml ICA,a significant reduction of the rate of cell proliferation was observed. Inhibition rate at 48h was 22.04%,and 29.68% at 72h.Kinetic study showed that inhibition of cell proliferation was time dependent (P0.05).ICA could inhibit apoptosis of Jurkat cells induced by HepG2.2.15 cells. In the co-culture system of HepG2.2.15 cell and Jurkat T cell, apoptosis ratio of Jurkat cell was reduced from 46.66% to 18.20% by ICA (P
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Expression of PRL receptor mRNA in Jurkat D1.1 cell line was confirmed by RT PCR. CD154 expression of Jurkat cells was significantly increased when these cells were incubated with PHA and recombinant human PRL, suggesting that binding of PRL to its receptor promotes CD154 expression which plays a role in immune regulation.