[Effect of mPGES-1 inhibitor MK886 on cell cycle of leukemia HL-60 cells].
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To investigate the effect of a microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor MK886 on cell cycle of the human acute myeloid leukemia HL-60 cells. HL-60 cells were treated with different concentration of MK886 (10, 25, 50 µmol/L) for 24 h. Flow cytometry, Western blot and ELISA were used to measure cell cycle, cyclin D1, mPGES-1, PGE(2), Akt, P-Akt and C-MYC. The results indicated that after treated with MK886, the percentage of HL-60 cells decreased in G(0)/G(1) phase and increased in S phase, and expressions of mPGES-1, cyclin D1, P-Akt and C-MYC and synthesis of PGE(2) decreased significantly. It is concluded that MK886 can arrest HL-60 cells in G(0)/G(1) phase, the mechanism of which is possibly associated to inhibition of mPGES-1 expression, reduction of PGE(2) synthesis, suppression of Akt phosphorylation and C-MYC expression, down-regulation of cyclin D1 expression.Cite
To explore the possible mechanism(s) of taurine-inhibiting the proliferation of hepatic stellate cells (HSC), this study investigated the effect of taurine on the HSC cell cycle and its regulatory protein expression.Cell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. Cell cycle regulatory protein Cyclin D1 and P21waf1 expression were determined by immunocytochemistry and image-analysis system, and real-time quantitative PCR.HSC proliferation was markedly inhibited when HSC were treated with taurine at concentrations of 5, 10, 20, 30, 40 and 50 mmol/L for 48 hours, and the inhibition rates were 6.7%, 14.4%, 23.3%, 32.2%, 36.7% and 45.6% respectively (P < 0.05-0.01). In the flow cytometry analysis, it was found that taurine could block HSC in the G0/G1 phase from entering the S phase, resulting in more cells in the G0/G1 phase and fewer in the S phase. The percentage of the cells in the G0/G1 phase and the S phase at the dosage of 40 mmol/L were 68.2%+/-1.4% and 26.2+/-1.3% respectively, which was significantly different in comparison to the controls (56.2%+/-1.7% and 38.5%+/-0.8% respectively, P < 0.01). HSC expressed cyclin D1 and P21waf1. Taurine inhibited cyclin D1 expression and induced P21waf1 expression. The cyclin D1 protein and mRNA in the HSC treated with 40 mmol/L taurine were significantly reduced compared with the controls [protein (optical density value): 0.13+/-0.02 versus 0.18+/-0.02, P < 0.01; mRNA: 5776.7+/-3345.0 versus 18,400.6+/-1374.8 copies/10(6) GAPDH, P < 0.01]; and the P21waf1 protein and mRNA were markedly increased compared with the controls [protein (optical density value): 0.19+/-0.02 versus 0.14+/-0.01, P < 0.01; mRNA: 44,866.7+/-3910.7 versus 16,933.3+/-960.9 copies/10(6) GAPDH, P less than 0.05].Cyclin D1 and P21waf1 were cell cycle regulatory proteins in HSC, and taurine can inhibit the HSC cyclin D1 expression and stimulate P21waf1 expression, facilitate arresting cells in G0/G1 phase, and suppress cell proliferation.
Hepatic stellate cell
Cyclin E
Cyclin B1
MTT assay
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目的：建立稳定表达Δ42PD1的293T细胞系，并探索其对AKT，NF-κB和Erk1/2磷酸化的影响。方法：分别将含有人PD1和Δ42PD1基因的真核表达质粒稳定转染293T细胞，用流式细胞术筛选稳定细胞系，用RT-PCR和Western blot进一步鉴定目基因的表达。将稳定细胞系分别于PBMCs进行孵育，以激活Δ42PD1和PD1，用流式细胞术检测细胞内AKT，NF-κB和Erk1/2的磷酸化水平。结果：通过流式细胞术筛选出稳定表达PD1和Δ42PD1的293T细胞系，RT-PCR和Western blot确认了目的基因的表达。与PBMC混合孵育后，293T细胞内AKT的磷酸化水平被Δ42PD1或PD1显著抑制，而NF-κB和Erk1/2的磷酸化水平没有显著变化。结论：本研究发现Δ42PD1与PD1的功能类似，可能是一种重要的抑制性免疫调节受体。 Objective: To establish Δ42PD1-expressing stable 293T cell line and explore the effect of Δ42PD1 on phosphorylation of AKT, NF-κB and Erk1/2. Methods: The human PD1- and Δ42PD1-carrying eukaryotic expression plasmids were stably transfected into 293T cells respectively. Stable cell lines were screened using flow cytometry, and confirmed by RT-PCR and Western blot. The cell lines were co-cultured with PBMC to activate PD1 and Δ42PD1, and then analyzed the phosphorylation of AKT, NF-κB and Erk1/2 via flow cytometry. Results: We obtained PD1- and Δ42PD1-stably expressing 293T cell lines by flow cytometry. The expression of genes of interest was confirmed by RT-PCR and Western blot. After co-culture with PBMC, phosphorylation of AKT but not NF-κB or Erk1/2 in 293T cells was significantly inhibited by Δ42PD1 or PD1. Conclusion: The present study found that functionally similar to PD1, Δ42PD1 could be an important inhibitory immune-regula- tory receptor.
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Objective To explore the putative effect of deoxynivalenol on cell cycle and apoptosis of human gastric carcinoma cell line HGC-27 in vitro.Methods HGC-27 cells were treated with DON at different concentrations(50、100、1 000、2 000μg/L) for 12,24 and 48 hours,and then cells were harvested for analysis of apoptosis,cell cycle distribution and expression of Bax,Bcl-2 and Caspase-3 with flow cytometric(FCM) and Western Blot methods.Results At relatively higher concentrations(1 000 and 2 000 g/L),DON could affect cell cycle distribution in a time dependent manner.DON treatment for 12 h,the percentages of cells in G0/G1 phases was significantly decreased and that in S phase increased.As the treatment time lasted for 24 h and 48 h,the percentages of cells in G0/G1 phases were significantly increased while that in S phase decreased(P0.05).No significant effects on G2/M phase could be found.The apoptosis rates in all DON treated groups for 12 h,24 h and 48 h were all higher than that in control.Though statistical significance in the difference of apoptosis rate between control and the DON treated groups was not found 12 h after DON treatment but it could be seen 24 and 48 h after DON treatment(P0.05).Significant dose-effect relationships were found between DON concentrations and the apoptosis rates.Western Blot analysis showed bax and caspase-3 expression was up-regulated and bcl-2 expression was down-regulated in DON treated HGC-27 cells in vitro.Conclusion DON could significantly affect the cell cycle distribution of HGC-27 cells in vitro and the effects of DON on cell cycle varied as the concentratrion and treatment time of DON changed.DON treatment could induce apoptosis of HGC-27 cell possibly by up-regulating bax and caspase-3 expression and down-regulating bcl-2 expression.
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Background A protocol to measure a wide range of Bcl-2 protein expression using quantitative fluorescence cytometry (QFCM) in different cell types was developed for use with flow cytometry. Bcl-2 measurements obtained by flow cytometry were correlated with Western blot Bcl-2 measurements to confirm specificity of the Bcl-2-FITC staining. This protocol was applied to measure absolute levels of Bcl-2 protein in different tumor cell lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymphocytes (PBL). Methods HL-60, K562, DOHH2, Jurkat, MDA435/LCC6, MCF7 cell lines, and PBL derived from normal donors were fixed, permeabilized, stained with anti-Bcl-2-FITC antibody and evaluated by QFCM. In parallel, the same cells were evaluated for Bcl-2 protein expression by Western blot analysis. Mitochondrial localization of anti-Bcl-2-FITC antibody inside cells was confirmed using fluorescence imaging microscopy. Results Bcl-2 expression in different cell types could be accurately quantified based on antibody-binding capacity (ABC) ranging from 12.6 × 103 antibody-binding sites in HL-60 cells to 1.64 × 106 antibody-binding sites in a Bcl-2-transfected MDA435/LCC6 clone. The data from flow cytometry analysis correlated well with Western analysis (R2 = 0.78). Bcl-2-FITC staining colocalized with dyes specific for mitochondria. Conclusions The Bcl-2 staining protocol described here was shown to be specific, sensitive, and it was able to provide higher resolution as well as more reproducible quantitation of Bcl-2 protein content in cells when compared with Western blot methods. Quantitation of Bcl-2 content in cells by QFCM may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo. Cytometry 40:346–352, 2000 © 2000 Wiley-Liss, Inc.
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clone (Java method)
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AIM:To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS:We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria.The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad-p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry.Cell cycle was determined by flow cytometry.DNA fragment analysis identified the occurrence of apoptosis. RESULTS:The recombinant adenovirus which already contained p27mt target gene was successfully constructed.When multiplicity of infection was ≥ 50, the infection efficiency was 100%.After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay.PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G0/G1, while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively.DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells.CONCLUSION: p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G0/G1.Therefore, p27mt can induce apoptosis in colorectal cells.
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Aim: To investigate the effects of tetramethypyrazine (TMP) on proliferation and apoptosis of the human gastric carcinoma cell line 7901 and its possible mechanism of action. Methods: The viability of TMP-treated 7901 cells was measured with a 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and cell apoptosis was analyzed by flow cytometry. The distribution of cells in different phases of cell cycle after exposure of TMPs was analyzed with flow cytometry. To investigate the molecular mechanisms of TMP-mediated apoptosis, the expression of NF-κBp65, cyclinD1 and p16 in SGC-7901 cells was analyzed by reverse transcriptionpolymerase chain reaction (RT-PCR) and western blotting. Results: TMP inhibited the proliferation of human gastric carcinoma cell line 7901 in dose and time dependent manners. Cell growth was suppressed by TMP at different concentrations (0.25, 0.5, 1.0, 2.0 mg/ml), the inhibition rate is 0.46%, 4.36%, 14.8%, 76.1% (48h) and 15.5%, 18.5%, 41.2%, 89.8% (72h) respectively. When the concentration of TMPs was 2.0mg/ml, G1-phase arrest in the SGC-7901 cells was significant based on the data for cell cycle distribution. RT-PCR demonstrated that NF-κBp65 and cyclin D1 mRNA expression was significantly down-regulated in 7901 cells treated with 2.0 mg/ml TMP for 72h (p<0.05), while the p16 mRNA level was up-regulated (p<0.05). The protein expression of NF-κBp65 and cyclin D1 decreased gradually with the increase in TMP concentration, compared with control cells (p<0.05), while expression of protein p16 was up-regulated (p<0.01). Conclusion: TMP exhibits significant anti-proliferative and pro-apoptotic effects on the human gastric carcinoma cell line SGC-7901. NF-κBp65, cyclinD1 and p16 may also play important roles in the regulation mechanisms.
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Objective To investigate the impact of PI3K-AKT pathway inhibitor (AKTi Ⅳ) on the acute crisis of chronic myeloid leukemia (CML) and its regulatory effect on PI3K-AKT pathway in Wnt/β-catenin signal pathway. Methods K562 cells were cultured to logarithmic growth phase, and they were treated with 0, 2.5, 5, 10μmol/L AKTi Ⅳ respectiv ely. Cell growth was determined with MTT test. The ability of cell colonization was assessed by colony-forming assay. The protein expression of pAKT (Thr308) and pGSK-3β(Ser9) was determined by Western blotting. The protein expression and mRNA levels of β-catenin and its down-stream targets c-myc and cyclin D1 were analyzed by Western blotting and realtime fluorescence quantitative polymerse chain reaction (Q-PCR), respectively. Results Cell growth and colony-forming ability of K562 cells were inhibited significantly after treatment with AKTi Ⅳ for 6, 10, 16h, and the best exposure time for K562 cells was 10h. PI3K-AKT pathway was inhibited obviously and the protein level of pAKT (Thr308) was lowered apparently after 2.5, 5, 10μmol/L AKTi Ⅳ treatment. When K562 cells were treated by 5μmol/L AKTi Ⅳ for 10h, pGSK-3β (Ser9) and β-catenin protein levels were lowered significantly, while the mRNA of β-catenin was not affected. The mRNA and protein levels of c-myc and cyclin D1 in the downstream of β-catenin were also decreased obviously after treatment with 5μmol/L AKTi Ⅳ for 10h. Conclusion AKTi Ⅳ can inhibit the proliferation and colony-forming ability of K562 cells in critical stage of CML. The mechanism may be related to down-regulation of expression of Wnt/β-catenin pathway by blocking the signal transduction of cell growth.
DOI: 10.11855/j.issn.0577-7402.2015.09.05
K562 cells
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Objective:To prepare the recombinant neuron protective protein TAT-Bcl-XL in E.coli expression system and to determine its anti-apoptosis activity.Methods:DNA fragment of TAT-Bcl-XL was obtained by RT-PCR using primers that were specific for Bcl-XL gene. Prokaryotic expression vector(pTBTOPO) was constructed by insertion of TAT-Bcl-XL DNA fragment into pCRT7/CT-TOPO vectors, and the recombinant protein was detected by SDS-PAGE and Western blot analysis using anti-V5 tag epitope antibody as the primary antibody. The recombinant fusion protein was purified by affinity chromatography, and the anti-apoptosis function of the purified protein was determined by flow cytometry.Results:A molecular weight of 30 000 protein was detectable in both SDS-PAGE and Western blot analysis. Immunofluorescent staining showed the fusion protein was distributed in cell plasma after incubation with 200 nmol/L fusion protein. Flow cytometry data indicated that the recombinant protein could enhance cell survival by 40% when 293T cells were treated with 250 μmol/L zinc chloride as a supplementation in cell culture media.Conclusion:High level expression of the reported neuron protective protein was obtained in E.coli expression system, and the purified recombinant proteins remained its anti-apoptosis activity in our preliminary characterization.
Myc-tag
Protein A/G
Protein G
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Objective:To investigate the influence of combined application of histone deacetylase inhibitor(HDACi)MS-275 and 5-FU on the apoptosis and cycle arrest on human hepatoma cell line HepG2 and unclose the related mechanism.Methods:Divided all the cells into 4 groups:control group,MS-275 group,5-FU group and drug combination group.Flow cytometry(FCM)was used to examine the effects of 5-FU with MS-275 on the apoptosis and cell cycle of HepG2 cells.Bcl-2,Bax,CyclinD1 and P21 protein were determined by Western blot assay.Results:5-FU and MS-275 combination could inhibit HepG2 cell growth through G0-G1 arrest,and induce apoptosis,both time and dose dependently.The combination of two agents increased P21 protein levels and decreased Bcl-2,CyclinD1 protein levels.The levels of Bax protein were not changed.Conclusion:The combination of 5-FU and MS-275 can induce apoptosis and cell cycle arrest,the effect might be associated with down-regulating expression of Cyclin D1 and Bcl-2 protein and upregulating the expression of P21 protein.
Histone deacetylase inhibitor
Cyclin B1
Cyclin E
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Objective To explore the possible mechanism(s) of taurine-inhibiting the proliferation of hepatic stellate cells (HSC), this study investigated the effect of taurine on the HSC cell cycle and its regulatory protein expression. Methods Cell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. Cell cycle regulatory protein Cyclin D1 and P21waf1 expression were determined by immunocytochemistry and image-analysis system, and real-time quantitative PCR. Results HSC proliferation was markedly inhibited when HSC were treated with taurine at concentrations of 5, 10, 20, 30, 40 and 50 mmol/L for 48 hours, and the inhibition rates were 6.7%, 14.4%, 23.3%, 32.2%, 36.7% and 45.6% respectively (P 0.05-0.01). In the flow cytometry analysis, it was found that taurine could block HSC in the G0/G1 phase from entering the S phase, resulting in more cells in the G0/G1 phase and fewer in the S phase. The percentage of the cells in the G0/G1 phase and the S phase at the dosage of 40 mmol/L were 68.2% ± 1.4% and 26.2 ± 1.3% respectively, which was significantly different in comparison to the controls (56.2% ± 1.7% and 38.5% ± 0.8% respectively, P 0.01). HSC expressed cyclin D1 and P21waf1. Taurine inhibited cyclin D1 expression and induced P21waf1 expression. The cyclin D1 protein and mRNA in the HSC treated with 40 mmol/L taurine were signifi- cantly reduced compared with the controls [protein (optical density value): 0.13 ± 0.02 versus 0.18 ± 0.02, P 0.01; mRNA: 5776.7 ± 3345.0 versus 18 400.6 ± 1374.8 copies /106 GAPDH, P 0.01]; and the P21waf1 protein and mRNA were markedly increased compared with the controls [protein (optical density value): 0.19 ± 0.02 versus 0.14 ± 0.01, P 0.01; mRNA: 44 866.7 ± 3910.7 versus 16 933.3 ± 960.9 copies/106 GAPDH, P 0.05]. Conclusions Cyclin D1 and P21waf1 were cell cycle regulatory proteins in HSC, and taurine can inhibit the HSC cyclin D1 expression and stimulate P21waf1 expression, facilitate arresting cells in G0/G1 phase, and suppress cell proliferation.
Hepatic stellate cell
Cyclin E
Cyclin B1
MTT assay
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