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Objective To evaluate the role of recombinant human soluble Tim-3 (hTim-3-Fc) in regulating immune response.Methods Soluble hTim-3 was incubated with human macrophage cell line U 937, human T cell line Jurkat and normal human PBMC before cytokines secreted by or expressed in different immune cells were analyzed using ELISA , RT-PCR and Western-blotting, respectively.Results Soluble hTim-3 significantly promoted the activation of different immune cells.Our data showed that IL-8 secretion by U937 cells, IL-2 secretion by Jurkat cells , IL-2 and IFN-γsecretion by human PBMCs were all significantly increased .In addition , soluble hTim-3 significantly increased the IFN-α2 and IFN-β1 mRNA expression in U937, Jurkat and PBMCs and increased the phosphorylation of stat-1 in Jurkat and U937 cells.Conclusion Recombinant soluble hTim-3 can significantly promote the activation of immune cells in vitro, which shows its therapeutic potential .
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To construct soluble TNF related apoptosis inducing ligand (TRAIL) expression system and investigate the effect of the expression product on tumor cell. It may provide valuable information for research into the immune system of the finless porpoise. The full-length cDNA of TRAIL (designated fTRAIL) was cloned from the total RNA of the finless porpoises blood using RT-PCR techniques and then the extracellular soluble fragments of fTRAIL (designated fsTRAIL) was ligated into pET43.1a. Recombinant soluble fTRAIL (pET43.1a-fsTRAIL) fused with Nus-his tag was efficiently expressed in Escherichia coli BL21 (DE3) and the Nus-His-fsTRAIL protein was purified. The expression of Nus-His-fsTRAIL was verified by Western blotting. In vitro, the effects of the purified Nus-His-fsTRAIL protein on Jurkat and HeLa cells were etected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) assay, TrypanBlue and Flow Cytometry analysis. The expression system pET43.1a-fsTRAIL was constructed and Nus-His-fsTRAIL protein was expressed successfully. In vitro, the Nus-His-fsTRAIL protein was able to inhibit the proliferation and induce apoptosis of Jurkat and HeLa cells in a dose-dependent manner. The Nus-His-fsTRAIL protein has anti-tumor activity against Jurkat and HeLa cells in vitro.
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Animals , Humans , Apoptosis , Blotting, Western , Cloning, Molecular , DNA, Complementary , Escherichia coli , HeLa Cells , Jurkat Cells , Porpoises , TNF-Related Apoptosis-Inducing LigandABSTRACT
Objective: To investigate the specific cytotoxicity of MUC1-specific chimeric antigen receptor (CAR)-engineered Jurkat T cells against hepatocellular carcinoma (HCC) cells. Methods: The expression cassettes of both the first and the third generation MUC1-specific CAR gene (i.e. MUC1-CAR and G3MUC1-CAR) were constructed and cloned into lentivirus transfer plasmids, respectively. Then the obtained recombinant lentiviruses carrying the MUC1-specific CAR gene and hrGFP reporter genes were used to infect Jurkat T cells in vitro to establish MUC1-sepcific CAR-engineered Jurkat cells (i.e. CAR-T cells). Subsequently, the assays of CCK-8, ELISA, and LDH were used to detect the cell proliferation, IL-2 secretion and killing effect of the CAR-T cells, respectively. Results: We successfully constructed the expression cassettes of MUC1-sepcific CAR gene and the corresponding recombinant lentivirus, established the MUC1-specific CAR-engineered Jurkat T cells. Both types of MUC1-specific CARs-engineered Jurkat T cells could recognize MUC1 molecule and specifically kill MUC1 over-expressed HCC cells while left normal hepatic cells almost undamaged. In addition, the cell proliferation, the secretion of IL-2 and killing effect of the G3MUC1-CAR-engineered Jurkat T cells was significantly superior to the MUC1-CAR-engineered counterpart (P<0.05 or 0.01). Conclusion: The MUC1-sepcific CAR-engineered Jurkat T cells can specifically kill MUC1 over-expressed HCC cells.
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In order to assess the anticancer action of extracts obtained by latex from Calotropis procera and Pedilanthus tithymaloides, samples were collected from adult plants. Soluble proteins were extracted with 16 uL of 50 mM sodium acetate pH 5/ug integral latex and centrifugation at 16,000 x g for 15 min, the supernatant was named "latex crude extract" (LCE). The "latex methanolic extract" (LME) was obtained on dried latex. Both extracts were tested in vitro by cytotoxic and cytostatic activity in Jurkat cell cultures. Cellular viability, proliferation, necrosis and apoptosis were evaluated. LCE and LME of C. procera were found with cytotoxic and cytostatic activity after 24 incubation hours (p < 0,05) with doses from 1ug/mL. The LCE and LME of P. tithymaloides presented cytotoxic effect (p < 0,05) from 50 ug/mL and from 1ug/mL, respectively.
Con el objetivo de evaluar el potencial anticanceroso de extractos de látex de Calotropis procera y Pedilanthus tithymaloides se colectaron muestras de plantas adultas. Las proteínas solubles fueron extraídas con 16 uL de acetato de sodio 50 mM pH 5/ug de látex integral y centrifugación a 16.000 x g durante 15 min, denominándose al sobrenadante extracto crudo de látex (ECL). El extracto metanólico de látex (EML) se obtuvo sobre látex deshidratado. Ambos extractos fueron probados en su actividad citotóxica y citostática in vitro sobre cultivos de células Jurkat. Se realizaron estudios de viabilidad, proliferación, necrosis y apoptosis celular. El ECL y el EML de C. procera presentaron actividad citotóxica y citostática después de 24 y 48 horas de incubación (p < 0,05) con dosis desde 1 ug/mL. Los ECL y EML de P. tithymaloides presentaron efectos citotóxicos (p < 0,05) a partir de 50 ug/mL y desde 1 ug/mL respectivamente.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Calotropis/chemistry , Euphorbia/chemistry , Plant Extracts/pharmacology , Apoptosis , Cell Culture Techniques , Cell Survival , Jurkat Cells , Latex , Methanol , Cell ProliferationABSTRACT
Objective To investigate the effects of escin sodium on proliferation of human leukemia Jurkat cells and its possible mechanism. Methods The reduction of cellular viability was determined by MTT assay. Hoechst 33258 staining, DNA fragmentation assay, FITC-Annexin V-FITC/PI staining assay, and cytometric analysis were used to confirm the features of apoptosis and cell cycle. Western blotting assays were performed to explore the apoptotic pathway. Results Escin sodium inhibited the proliferation of Jurkat cells in both dose- and time-dependent manners. The morphological apoptosis, DNA fragmentation pattern, and the percentage of Annexin V+/PI- (early apoptosis) cells were markedly increased in escin sodium-treated Jurkat cells. Escin sodium activated Caspase-8, Caspase-9, and Caspase-3, degraded poly (ADP-ribose) polymerase (PARP), and attenuated Bcl-2 expression. Conclusion Escin sodium could inhibit the proliferation of Jurkat cells via the induction of apoptosis effectively.
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Objective To construct Gadd45a expression plasmid and induce its expression in human T cells.Methods Gadd45a was amplified by reverse transcription PCR from human embryonic stem cells,and cloned into the pcDNA3.1 vector.The recombinant plasmid or blank plasmid was transfected into Jurkat cells or normal human CD4+T cells using electroporation,and the expression of Gadd45a was detected by quantitative RT-PCR and Western blot.Results Human Gadd45a expression plasmid was constructed successfully.Gadd45a was overexpressed both in Jurkat cells and normal human CD4+T cells after these cells were transfected with pcDNA3.1-Gadd45a.Conclusion The construction of Gadd45a expression plasmid and induction of Gadd45a overexpression in human T cells lay the foundation for further research on the role of Gadd45a in the epigenetic mechanism.
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Objective To investigate the inhibition of proliferation and the regulation of histone acetylation modification in Jurkat cells treated by sodium valproate(VPA). Methods Jurkat cells were treated with VPA.Cell proliferation was determined by CCK-8 assay, and cell cycle were analyzed by flow cytometry (FCM). mRNA of HDAC1 was detected by semi-quantitative RT-PCR, and protein expression of HDAC1 and acetylation of histone H3, H4 was examined by Western blotting. Results VPA inhibited the proliferation of Jurkat cells in concentration-and time-dependent manners. After exposure to VPA in different concentrations for 48h,cell cycle was arrested obviously at G0/G1 phase (P <0.05), and with increasing concentration, the percentage of G0/G1 phase cells was increased and that of S phase were decreased. HDAC1 mRNA expression were inhibited with the increasing concentration of VPA. The protein level of HDAC1 was down-regulated, while acetylation of histone H3、H4 was up-regulated in Jurkat cells by VPA. Conclusion VPA can inhibit proliferation of Jurkat cells and induce G0/G1 phase arrest. The mechanism may be that VPA increase acetylation of histone H3/H4 by inhibiting expressions of HDAC1 gene.
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Objective To explore the effects of UVB on the expression of Gadd45a gene and DNA methylation levels in Jurkat cells. Methods Jurkat cells were irradiated with UVB of 1.0 J/cm2 and 1.5 J/cm2 respectively, and collected at 6, 12, 24 and 48 hours after the irradiation. Real-time RT-PCR was used to detect the mRNA expression of Gadd45a gene and methylation-sensitive genes CD11a and CD70. Global methylation level was also measured by MethylAmp global DNA methylation quantification kit. Results After irradiation with UVB at 1.0 J/cm2, the mRNA level of Gadd45a increased but global methylation level decreased at 6, 12, 24 and 48 hours, and significant changes were observed at 6 and 12 hours for the level of both Gadd45a mRNA expression and global methylation (P ＜ 0.01 or 0.05). Elevated mRNA expressions of CD11 a and CD70 were also noted in Jurkat cells after irradiation with UVB of 1.0 J/cm2, and significant elevation was observed at 12 hours (both P ＜ 0.05 ). After irradiation with UVB of 1.5 J/cm2, there was a statistical increase in the mRNA expressions of Gadd45a, CD11 a, CD70, together with a statistical decrease in global methylation level in Jurkat cells, at 6, 12, 24 and 48 hours (P ＜ 0.01 or 0.05). The mRNA expression of Gadd45a negatively correlated with the global level of DNA methylation in Jurkat cells (r = -0.395, P ＜ 0.05). Conclusion UVB irradiation can upregulate the expression of Gadd45a, but downregulate the global DNA methylation level in Jurkat cells.
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Objective To observe the effects of triptolide(TPL) on the anti-oncogene-APC gene of acute lymphoblastic leukemia cell line Jurkat in vitro. Methods The effects of TPL on proliferation Jurkat cells were assayed by using cell culture, MTT. The effects of TPL on APC gene of Jurkat cells were analyzed by nested methylation specific PCR and RT-PCR. The effects of TPL on the proteinum expression of APC gene were detected by Western blotting analysis. Results Following the treatment of TPL, the cell proliferation rate was degraded as the treatment concentration increased and the culture time extended. The effects were dose and time-dependent. The 48 hour IC50 was 19.7 ng/ml. TPL can reverse hypermethylation of APC gene,and induce the expression of the mRNA and the proteinum. Conclusion Low dose TPL could depress the proliferation rate of Jurkat. The possible mechanism might be its reversing the hypermethylation of APC gene and activiting the expression of APC gene.
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Objective To investigate the chemotherapeaties sensitivity and expression of bcl-2, bcl-xL and bax of Jurkat cells co-cultured with bone marrow stromal cells (BMSC) isolated and cultured from leukemia patients. Methods BMSC were isolated and cultured from leukemia patients routinely. To construct the co-cultured model, Jurkat cells were co-cultured with of the irradiated layer BMSC by 60Co and observed the model with scanning electron microscope. The Jurkat cells suspension-cultured were used as control. The apoptosis and IC50 were detected by the FACS can machine and MTT, respectively. The expression of bcl-2,bcl-xL and bax in Jurkat cells was detected by Western blotting. Results We found that the Jurkat cells in the model showed a decreased sensitivity to DNR, IC50 values for leukemic BMSC and nonadhered contol were of 2.30 μmol/L and 0.45 μmol/L, respectively. Moreover, Jurkat cells adhered to BMSC have a survival advantage over suspended cells following DNR exposure for 24 h, apoptosis percentages for leukemic BMSC group and nonadhered controls were of (6.05±0.54)% and (25.74±6.15)%, respectively. As compared with controls, leukemic BMSC group had significant difference in apoptosis percentages (P <0.01). The expression of bcl-2 in Jurkat cells was up-regnlated when adhered to BMSC for 4 h and the higher expression emerged after adhering for 24 h and 48 h. No marked change of bcl-xL and bax expressions were observed in the adhered Jurkat cells. Conclusion The adhered-culture with bone marrow stromal cells isolated from leukemia patients could make the leukemia cells acquire drug resistance, which was associated with the up-regulated expression of bcl-2 in the leukemia cells.
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The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 μmol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G0/G1 phase and decrease in S phase. After treatment with 0, 20, 60, 100 μmol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00±1.25)% to (58.84±0.32)% in G0/G1 phase, whereas it was decreased from (61.45±1.04)% to (35.82±1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3,bel-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is coneluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle,arrest cells at G0/G1 phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.
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Objective To investigate the effect of resveratrol on proliferation inhibition,cell cycle arrest and apoptosis of Jurkat cell line in acute T lymphoblast leukemia.Methods MTT assay was used to determine the cell vitality.Wright-Giemsa,Hoechest 33258/PI staining and transmission electron microscope technique were used to detect the apoptosis status of Jurkat cells.The cell cycle arrest was analyzed by flow cytometry.(Results Resveratrol) had 64.01% inhibitory rate on the growth of Jurkat cells at 0.2 mmol?L~(-1) and inhibited the growth of Jurkat cells in dose-and time-dependent manner.24 h after treated with resveratrol,the typical features of apoptosis were observed under light and electron microscope in all treatment groups.Some nuclei showed bright blue under fluorescence microscope in the resveratrol-treated Jurkat cells,and the number of cells with bright blue fluorescence increased with time.Nuclei condensation and fragmentation were observed.Cell shrinkage,chromatin condensation,and marginalization were found by Wrigh-Giemsa staining and transmission electron microscope technique.By flow cytometry,62.57% of the cells were arrested at the S phase after exposured to(0.05 mmol?L~(-1)) resveratrol for 48 h,the rate of apoptotic cells to total cells was 12.01% in 0.05 mmol?L~(-1) treatment groups,and that in the control groups was 2.05%. Conclusion Resveratrol can inhibit the proliferation,cause S-phage arrest and induce the apoptosis of Jurkat cells.
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CD43 (sialophorin, leukosialin) is a heavily sialylated surface protein expressed on most leukocytes and platelets including T cells. Although CD43 antigen is known to have multiple and complex structure, exact function of CD43 in each cell type is not completely understood. Here we evaluated the role of CD43 in Fas (CD95)-induced cell death in human T lymphoblastoid cell line, Jurkat. Crosslinking CD43 antigen by K06 mAb increased the Fas-mediated Jurkat cell apoptosis and the augmentation was inhibited by treatment with caspase inhibitors. Further, CD43 signaling of Jurkat cells induced Fas oligomerization on the cell surfaces implying that CD43 ligation have effects on early stage of Fas-induced T cell death. These also suggest that CD43 might play an important role in contraction of the immune response by promotion of Fas-induced apoptosis in human T cells.
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Humans , Receptor Aggregation/immunology , Jurkat Cells , Caspases/metabolism , Apoptosis/immunology , Antigens, Surface/metabolism , fas Receptor/metabolism , Leukosialin/metabolism , Antibodies, Monoclonal/metabolismABSTRACT
AIM:To study the changes of mitochondria during apoptosis in Jurkat cells induced by arsenic oxide(As2O3).METHODS:By treated with 4?10-6 mol/L As2O3,apoptosis and necrosis of Jurkat cells were assessed by annexin V-FITC/PI double staining flowcytometry.Mitochondrial mass and its membrane potential(△?m)was measured by NAO/PI and DiOC6(3)/PI staining,respectively.Free radical formation was detected by DCFDA staining.RESULTS:After 48 h of As2O3 treatment,the rates of early apoptotic Jurkat cells in As2O3 and control groups were(18.98?1.40)% and(5.17?0.80)%,respectively(P
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AIM: To explore the effect of hTERT antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis induced by chemotherapeutic drugs in Jurkat cell lines. METHODS: Cell viability was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation, DNA gel electrophoresis and flow cytometry analysis. RESULTS: The survival rates of Jurkat cells cultured with daunorubicin, vincristin, and etoposide, respectively were similar with that cultured with those chemotherapic drugs plus hTERT ASODN. The survival rates of Jurkat cells cultured with cis-diamminedichicloroplatinum(DDP) added 24 hours later were higher than that cultured with hTERT ASODN and DDP added 24 hours later. The survival rates of Jurkat cells cultured with DDP were similar with that cultured with hTERT SODN and DDP. In morphological observation of apoptotic cells using Giemsa staining, cells displayed classic apoptotic changes treated with DDP or DDP combined with hTERT ASODN or SODN at 48 hours. Agarose gel electrophoresis of genomic DNA from Jurkat cells treated with ASODN and DDP combination for 48 hours showed typical DNA “ladder”. Neither the DNA from Jurkat cells treated with SODN plus DDP nor the DNA from the cells treated with DDP alone showed ladder pattern. Apoptosis rates of Jurkat cells treated with DDP for 48 hours after 24 hours of exposure to ASODN significantly increased. There was significant difference in the percentage of apoptotic Jurkat cells between hTERT ASODN plus DDP and SODN plus DDP or DDP alone, respectively. CONCLUSION: The hTERT ASODN complementary to the translation initiation region of hTERT mRNA enhanced DDP-induced apoptosis in Jurkat cells.
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AIM: To study the changes of mitochondrial membrane potential(△?m) and mitochondrial mass in apoptosis of Jurkat cells induced by camptothecin(CPT).METHODS: Jurkat cells were treated with CPT.Annexin V-FITC/propidium iodine(PI) double stainig was used to detected early stage of apoptosis and PI staining for analyzing the cell cycle.Jurkat cells were stained by annexin V-PE/DiOC_6(3) to detect changes of △?m.The mitochondrial mass was measured by cytometry with NAO staining.RESULTS: 6 h after treated with 10 ?mol/L CPT,the rate of early apoptotic cells(22.59?1.04)% had significantly difference compared with control group(3.93?0.73)%(P0.05).Apoptotic peak appeared obviously after treated with CPT,the percentage of late apoptotic cells(13.58?0.97)% had distinctly difference compared with control group(3.18?0.51)%(P
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Objective To investigate whether difluromethylornithine (DFMO) can be used in the treatment of human leukemia. Methods The cell proliferation was detected by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay after treatment of human lymphocyte Jurkat cells by DFMO (0 to 10 mmol/L) for 24 to 72 h. Enzyme activity of spermine oxidase (SMO) and acetylpolyamine oxidase (PAO) was determined by chemiluminesence assay. DNA fragmentation assay was used to evaluate cell apoptosis. Fluorescent dye assay was performed to determine the changes in mitochondrial membrane potential. Western blotting was used to determine Bax content. Casepase-3 enzyme activity was measured by spectrophotometric method. Results DFMO treatment inhibited the proliferation of Jurkat cells significantly in a dosage- and time-dependent manner (P
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AIM: To investigate the expression of Fas/FasL system in human lung carcinoma cell lines (A549, EBC-1, LCSC) and T-lymphocytes (Jurkat), and to search the possibilities of immune escape and counterattack mediated by Fas/FasL pathway in human lung carcinoma cells. METHODS: The protein and mRNA expression of Fas and FasL were detected by FACScan, RT-PCR, respectively. Cell apoptosis was assessed by fluorescent staining. Cell growth was determined by a trypan blue exclusion assay. RESULTS: Fas and FasL were expressed in 3 human lung carcinoma cell lines and T-cells, Jurkat. The lung carcinoma cells inhibited the growth (P
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AIM: To observe the effect of antisense oligodeoxynucleotide of human telomerase reverse transcriptase(hTERT) on the telomerase activity in Jurkat cells and its mechanism. METHODS: The telomerase activity was detected by TRAP. hTERT protein and mRNA expressions were detected by flowcytometry and RT-PCR. RESULTS: The absorbency of the cells ( A values) was used to represent the telomerase activity. At 48 h and 72 h, the A values of the Jurkat cells treated with antisense oligodeoxynucleotide of hTERT decreased to 0.351?0.051 and 0.238?0.024, respectively, compared to the A value of 0.492?0.051 in the control cells without treatment ( P