Optimization of comet experiment method and analysis of influencing factors / 安徽医科大学学报

Objective To detect cell DNA damage more effectively by optimizing the influencing factors of Comet assay and establishing a positive control.Methods Based on the traditional alkaline comet assay,the gel prepara-tion method,gel concentration,agarose solvent,lysis time,cell density and electrophoresis voltage were opti-mized.HL-60 cells were induced by hydrogen peroxide(H2O2)as a DNA damage inducer as a positive control.Results The optimized experimental results showed that the gel preparation method improved from three-step meth-od to two-step method.PBS was used as agarose solvent,0.8％normal melting point agarose was used to prepare the bottom layer,and 0.7％low melting point agarose was mixed with cell suspension as the second layer.The gel preparation effect was better,the operation was simpler,the time was saved,and the degumming problem was bet-ter solved.After lysis for 1 h and electrophoresis at 1 V/cm voltage,representative comet images were obtained.The results were easy to interpret and could effectively avoid false positive results.The positive control of comet as-say was successfully established by comparing the DNA damage of HL-60 cells induced by different concentrations of H2 O2.Conclusion Compared with the traditional method,the optimized comet assay method is simpler,faster,more accurate,lower cost and has better repeatability,which can quickly detect DNA damage in cells.

Study on the mechanism of diterpenoid DP from Euphorbia fischeriana against leukemia / 中国药房

OBJECTIVE To explore whether diterpenoid 12-deoxyphorbol-13-palmitate （DP） from Euphorbia fischeriana can exert anti-leukemia effects through the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signal pathway， and to provide experimental evidence for developing it into a new anti-leukemia drug. METHODS Using LY294002 （PI3K specific inhibitor） as tool drug， the effects of 24 h DP treatment on the proliferation and apoptosis of human myeloid leukemia HL60 cells were detected by MTT method， Annexin Ⅴ-FITC/PI staining and AO-EB staining. ELISA method was used to detect lactic dehydrogenase （LDH） release and the activities of cysteinyl aspartate specific proteinase 3 （caspase-3） and caspase-9. The transcriptional level of caspase-3， caspase-9， forkhead box O3a （FoxO3a） and B cell lymphoma 2 interacting mediator of cell death （Bim） mRNA were detected by real-time quantitative polymerase chain reaction （qRT-PCR）. The protein expression of phosphorylated FoxO3a （p- FoxO3a） and phosphorylated Akt （p-Akt） were detected by Western blot method. The nuclear translocation of FoxO3a protein was detected by immunostaining combined with laser confocal microscopy. RESULTS 10 μmol/L DP and 10 μmol/L DP+LY294002 could inhibit the proliferation and induce the apoptosis of HL60 cells （P＜0.01）. After treatment of 5， 10， 20 μmol/L DP， HL60 cells showed typical morphological characteristics of apoptosis； DP could significantly increase the levels of LDH release and the activities of caspase-3 and caspase-9 （P＜0.05 or P＜0.01）， in dose-dependent manner. After treatment of 10 μmol/L DP and 10 μmol/L DP+LY294002， the transcriptional levels of caspase-3， caspase-9 and Bim mRNA were increased significantly （P＜0.05 or P＜0.01）， and transcriptional level of FoxO3a mRNA and protein expressions of p-FoxO3a and p-Akt were decreased significantly （P＜0.05 or P＜0.01）. Nuclear translocation changes were observed in FoxO3a protein in 10 μmol/L DP+LY294002 group， and the change was more significant than that of LY294002 group. CONCLUSIONS DP can inhibit the proliferation and induce the apoptosis of HL60 cells via inhibiting PI3K/Akt signaling pathway.

Preliminary Study on the Effect of Silencing Nucleostemin Com- bined with Rapamycin on Autophagy and Apoptosis of HL-60 Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of knocking down nucleostemin ( NS) combined with rapamycin (RAPA) on autophagy and apoptosis in HL-60 cells , and to explore its role in HL-60 cells .@*METHODS@#The expression of NS protein was detected using Western blot , after transfection of HL-60 cells was achieved by the recombinant lentviral vector NS -RNAi-GV248 . Flow cytometry was used to detect changes in cells apoptosis after NS silencing/ rapamycin for 24 , 48 hours , and the expressions of NS , LC3 , p62 , BCL-2 and Bax proteins in cells were detected by Western blot.@*RESULTS@#The expression of NS in HL-60 cells was successfully down-regulated by recombinant lentiviral vector. After treatment with rapamycin for 24 and 48 h , the apoptosis rate of cells in each group increased (P < 0.05) , and the apoptosis was more obvious at 48 hours . Compared with the NS silencing group or rapamycin group , after treated with NS down-regulation combined with rapamycin for 48 hours , the apoptosis of HL-60 cells was significantly increased ( P < 0.05 ) , LC3 -II/LC3 -I ratio was significantly increased ( P < 0.05 ) , p62 protein expression was significantly decreased (P < 0.05) , and BCL-2/Bax ratio was significantly decreased ( P < 0.05) .@*CONCLUSION@#NS down-regulation combined with rapamycin can enhance the apoptosis and autophagy of HL-60 cells , and the induction of apoptosis of HL-60 cells may be related to the expression of BCL-2 and Bax proteins .

The method transfer and validation of freeze-dried rabies vaccine pyrogen detection in HL-60 monocyte activation assay / 中国药理学通报

Aim To study the application of HL-60 monocyte activation test in the pyrogen detection of freeze-dried rabies vaccine for human use.Methods The established HL-60 mononuclear cell activation test (MAT) was transferred between laboratories and the method was verified; referring to the interference test in the photometric method of the bacterial endotoxin test method of the Chinese Pharmacopoeia, HL-60- IL- 6 MAT was used to detect the recovery and pyrogen content of 13 batches of freeze-dried rabies vaccine for human use.Results The linearity of the amount of 1L-6 secreted by HL-60 cells, which stimulated by dif¬ferent concentrations of endotoxin standards was above 0.95; the calculated minimum detection limit was not more than 0.125 EU • mL"1 ; the recovery experiment with a solution containing 0.5 and 1.0 EU • mL"1 of endotoxin was performed to cheek the accuracy of the method.HL-60-IL-6 was used to detect 13 hatches of Freeze-dried rahies vaccine for human use, and the re¬covery of endotoxin was between 50 % to 200%.It was consistent that HL-60-IL-6 with pyrogens and en¬dotoxin test for 4 batches of freeze-dried rabies vaccine for human use which pyrogens and endotoxin test failed and the 3 batches of water for injection.Conclusion The HL-60 MAT using IL-6 as a detection indicator is suitable for the detection of pyrogenic substances in freeze-dried rabies vaccine for human use.

Hop-derived Xanthohumol Induces HL-60 Leukemia Cells Death

Background : Acute promyelocytic leukemia (APL) affects both kids and adults, however it is more prevalent in younger population. Although APL has a favorable prognostic, patients that relapse often do not respond positively to additional chemotherapy. Therefore, there is a need to further identify ways to overcome these challenges. Hypothesis: In this study, we examined antileukemic effects of xanthohumol (XN), a prenylated flav onoid derived from hops ( Humulus lupulus L ), on human promyelocytic HL - 60 cells. Materials and Methods : HL - 60 cells were exposed to different concentrations of XN (?M) for 24 h. Cell viability, cell morphology, chromatin condensation, cPARP - 1 level, and caspase - 3 activation, and the expression of p21 WAF1/Cip1 were analyzed. Results : XN reduced HL - 60 cell viability in a dose - dependent manner. XN induced a dose - dependent morphological changes including cell shrinkage and b lebbing , and significantly increased the number of cells with condensed chromatin. XN significantly increased the level of cPARP - 1, active caspase - 3, and the expression of p21WAF/CIP mRNA. Conclusion : These data indicate that XN induces HL - 60 cell death by regula ting cell cycle progression and apoptosis. This study suggests that XN may have antileukemic preventive effects.

Polyphenolic extracts of walnut (Juglans regia) green husk containing juglone inhibit the growth of HL-60 cells and induce apoptosis

BACKGROUND: Juglone is a naphthoquinone currently obtained by chemical synthesis with biological activities including antitumor activity. Additionally, juglone is present in the green husk of walnut, which suggests evaluating the effect of GH extracts on carcinogenic cell lines. RESULTS: Walnut green husk ethanolic extract was obtained as 169.1 mg juglone/100 g Green Husk and antioxidant activity (ORAC) of 44,920 µmol Trolox Equivalent/100 g DW Green Husk. At 1 µM juglone in HL-60 cell culture, green husk extract showed an antiproliferative effect, but pure juglone did not; under these conditions, normal fibroblast cells were not affected. A dose-dependent effect on mitochondrial membrane potential loss was observed. Apoptosis of HL-60 was detected at 10 µM juglone. Despite high ORAC values, neither purified juglone nor the extract showed protective effects on HL-60 cells under oxidative conditions. CONCLUSIONS: Green husk extract generates an antiproliferative effect in HL-60 cells, which is related to an induction of the early stages of apoptosis and a loss of mitochondrial membrane potential. The normal cells were not affected when juglone is present at concentrations of 1 µM, while at higher concentrations, there is loss of viability of both cancerous and healthy cells.

Comparison of Four Statistical Tools for Running Four-Parameter Logistic Model in the Establishment of Endotoxin Standard Substance / 中国药学杂志

OBJECTIVE: To study the algorithm of four-parameter logistic regression and apply the model in the potency calibration of bacterial endotoxin standard. METHODS: The potency of endotoxin reference standard was estimated by a novel monocyte activation test,HL60-IL6 assay.The endotoxin national standard was set as standard, while the endotoxin working standard was set as test. The four-parameter logistic model was fitted by the method of nonlinear least squares when observations of standard or sample solutions were plotted against endotoxin concentration. The algorithm was performed by C Language, the RESULTS: of which were compared by those of softmax, R Language as well as Microsoft Office Excel. RESULTS The estimated parameters as well as estimated potency were parallel between the four statistical tools. The goodness-of-fit were all over 0.99.The variance was within 0.1%. CONCLUSION: The four-parameter logistic model can be established by the four statistical tools. Researchers can select a suitable tool according to demands.

Effect of SALL4 on radiosensitivity of leukemia cell line HL-60 / 中华放射肿瘤学杂志

Objective@#To evaluate the effect of down-regulating SALL4 on the radiosensitivity of leukemia cells, aiming to provide new ideas for improving radiosensitivity of leukemia patients.@*Methods@#Human acute myeloid leukemia cell line HL-60 infected with shRNA SALL4 and shRNA control lentivirus was classified into the Lv-shSALL4 group and Lv-shNC group. The levels of SALL4 mRNA and protein in cells were detected by RT-PCR and Western blot. The infected cells treated with 8 Gy dose irradiation were assigned into the Lv-shSALL4+ radiation and Lv-shNC+ radiation groups. The cell apoptosis was detected by flow cytometry. The levels of cleaved Caspase-3, cleaved Caspase-9 and Bax proteins in cells were determined by Western blot. The cells in the Lv-shSALL4 and Lv-shNC groups were exposed to 0, 2, 4, 6 and 8 Gy irradiation. The radiosensitivity ratio was determined by cell clone test.@*Results@#The level of SALL4 in the Lv-shSALL4 group was significantly lower than that in the Lv-shNC group (P<0.05). The cell apoptosis rate was significantly increased, the levels of cleaved Caspase-3, cleaved Caspase-9 and Bax proteins were remarkably up-regulated in cells compared with those in the Lv-shNC group (all P<0.05). The cell proliferation ability in the Lv-shSALL4+ radiation was significantly reduced, the cell apoptosis rate was considerably increased, the levels of cleaved Caspase-3, cleaved Caspase-9 and Bax proteins were significantly up-regulated compared with those in the Lv-shSALL4 and Lv-shNC+ radiation groups (all P<0.05). The cell radiosensitization ratio in the Lv-shSALL4 group was 1.323.@*Conclusion@#Down-regulating SALL4 can increase the radiosensitivity of leukemic cells, inhibit the cell proliferation and induce the apoptosis of leukemic cells.

Effect of SALL4 on radiosensitivity of leukemia cell line HL-60 / 中华放射肿瘤学杂志

Objective To evaluate the effect of down-regulating SALL4 on the radiosensitivity of leukemia cells,aiming to provide new ideas for improving radiosensitivity of leukemia patients.Methods Human acute myeloid leukemia cell line HL-60 infected with shRNA SALL4 and shRNA control lentivirus was classified into the Lv-shSALL4 group and Lv-shNC group.The levels of SALL4 mRNA and protein in cells were detected by RT-PCR and Western blot.The infected cells treated with 8 Gy dose irradiation were assigned into the Lv-shSALL4 + radiation and Lv-shNC+radiation groups.The cell apoptosis was detected by flow cytometry.The levels of cleaved Caspase-3,cleaved Caspase-9 and Bax proteins in cells were determined by Western blot.The cells in the Lv-shSALL4 and Lv-shNC groups were exposed to 0,2,4,6 and 8 Gy irradiation.The radiosensitivity ratio was determined by cell clone test.Results The level of SALL4 in the Lv-shSALL4 group was significantly lower than that in the Lv-shNC group (P＜0.05).The cell apoptosis rate was significantly increased,the levels of cleaved Caspase-3,cleaved Caspase-9 and Bax proteins were remarkably up-regulated in cells compared with those in the Lv-shNC group (all P＜ 0.05).The cell proliferation ability in the Lv-shSALL4 + radiation was significantly reduced,the cell apoptosis rate was considerably increased,the levels of cleaved Caspase-3,cleaved Caspase-9 and Bax proteins were significantly up-regulated compared with those in the Lv-shSALL4 and Lv-shNC+ radiation groups (all P＜0.05).The cell radiosensitization ratio in the Lv-shSALL4 group was 1.323.Conclusion Down-regulating SALL4 can increase the radiosensitivity of leukemic cells,inhibit the cell proliferation and induce the apoptosis of leukemic cells.

Inhibitory effect of PARP inhibitor olaparib on acute myeloid leukemia HL-60 cells / 实用肿瘤学杂志

Objective The inhibitory effect of the PARP inhibitor olaparib on human acute myeloid leukemia HL-60 cells was studied. Methods The HL-60 cells in logarithmic growth phase were treated with different concentrations(1. 25,2. 5,5 and 10 μmol/L) of olaparib for different time. The CCK-8 assay was used to detect the inhibitory effect of olaparib on HL-60 cells. The apoptotic level of HL-60 cells was detected by Annexin-V/PI double staining method,and the expression of related signal proteins ( PARP-1 and caspase-3)in HL-60 cells was detected by Western blot. Results HL-60 cells were inhibited by olaparib at dif-ferent concentrations(1. 25,2. 5,5 and 10 μmol/L) for 48 h,and the inhibition rate gradually increased with the prolongation of the action time;at the same time,the apoptotic rate was increased in HL-60 cells after olaparib treatment for 48 h,showing a dose-de-pendent manner;the PARP activity was inhibited and caspase-3 was activated in HL-60 cells treated with olaparib. Conclusion The PARP inhibitor olaparib not only inhibits proliferation of HL-60 cells,but it also promotes apoptosis of HL-60 cells by inhibi-ting PARP activity and activating caspase-3.

Induction effects of rabdosin B on differentiation of human promyelocytic leukemia HL-60 cells / 中草药

Objective To investigate the characteristics and mechanism of spironolactone ent-kaurane diterpene type diterpene rabdosin B (RB) induced HL-60 differentiation into mature granulocytes. Methods The effects of RB on proliferation, cell cycle, NBT reducing power, phagocytosis, and cell surface antigen CD11b expression of HL-60 were detected by trypan blue staining, Giemsa staining, NBT reducing power assay, and flow cytometry, respectively. Results RB inhibited the proliferation of HL-60 cells at the concentration of 1.0, 3.0, and 5.0 μmol/L, which resulted in the arrest of G0/G1 phase, increased the ratio of renal nucleated and lobate nucleus, and markedly enhanced the NBT reducing power and phagocytic capacity and the significant increase of CD11b positive cell rate. Further tests showed that the active oxygen scavenger NAC (300 μmol/L), NADPH oxidase inhibitor APO (100 μmol/L), and DPI (0.1 μmol/L) not only significantly inhibited the intracellular ROS upregulation induced by RB, but also inhibited various features of RB induced HL-60 cell differentiation. Conclusion RB can increase the activity of NADPH oxidase in HL-60 cells, increase the intracellular ROS concentration, and induce the differentiation of HL-60 cells to mature granulocytes.

Effect of 12-Deoxyphorbol-13-Palmitate on Proliferation Inhibition and Apoptosis Induction of HL60 Cells / 中国药学杂志

OBJECTIVE: To investigate the effects of 12-deoxyphorbol-13-palmitate (DP) on the proliferative inhibition and induction apoptosis of human acute myelocytic leukemia cell line HL60. METHODS: HL60 cells were treated with different concentrations of DP. The morphological changes were observed with optical microscope. The effect of DP in cell proliferation was tested using cell counting kit-8(CCK-8) assay, to analyze the cell cloning capacity by methyl cellulose colone-forming experiment. DNA agarose electrophoresis method was used to detect apoptosis. The expression of pro-and anti-apoptotic genes Bcl-2 and Bax were examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: CCK-8 METHODS and methyl cellulose colone-forming experiment indicated DP had an certainly inhibitory effect on HL60 cells(P<0.05, P<0.01). The characteristics of apoptosis was presented under optical microscope. DNA agarose electrophoresis showed the obvious DNA 1adder. In accordance with RT-PCR experiments, compared with the control group(0 mg·L-1), DP could down-regulate the gene expression of Bcl-2 and up-regulate the gene expression of Bax in HL60 cells(P<0.01). CONCLUSION: DP can inhibit the proliferation and induce the apoptosis of cultured HL60 cells in a dose-effect and time-effect relationship in vitro, and its mechanism may be related to down-regulating gene expression of Bcl-2, up-regulating gene expression of Bax, rising Bax/Bcl-2 value.

Effects of Treponema pallidum on the expression of chemokine ligand 6, 8, 10 in human brain microvascular endothelial cells / 中华皮肤科杂志

Objective To evaluate the effects of Treponema pallidum (T.pallidum) on the expression of chemokine ligands (CXCL) in human brain microvascular endothelial cells (HBMECs).Methods HBMECs were randomly divided into 4 groups,which were treated with viable T.pallidum suspension (T.pallidum group),heat-inactivated T.pallidum suspension (inactivated T.pallidum group),200 μg/L lipopolysaccharide (LPS group) and cell culture medium (blank control group),respectively,for 6,12 and 24 hours.Fluorescence-based quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were performed to determine the mRNA and protein expression of CXCL6,CXCL8 and CXCL10 in HBMECs in the above groups respectively.Transwell migration assay was conducted to evaluate the effects of T.pallidum-stimulated HBMECs on the chemotaxis of human promyelocytic HL-60 leukemia cells (HL-60 cells).Results At 6,12 and 24 hours,the T.pallidum group showed significantly higher mRNA expression of CXCL6,CXCL8 and CXCL10 in HBMECs compared with the blank control group and inactivated T.pallidum group (all P ＜ 0.05),while there were no significant differences between the blank control group and inactivated T.pallidum group (all P ＞ 0.05).Compared with the LPS group,the T.pallidum group showed significantly decreased mRNA expression of CXCL6 and CXCL8 (P ＜ 0.05),but similar mRNA expression of CXCL10(P ＞ 0.05)at 6,12 and 24 hours.At these time points,the levels of CXCL6 and CXCL8 in the culture supernatant of HBMECs were significantly higher in the T.pallidum group than in the blank control group and the inactivated T.pallidum group (all P ＜ 0.05),but no significant differences were observed between the blank control group and the inactivated T.pallidum group (both P ＞ 0.05).Moreover,there were no significant differences in the level of CXCL10 in the culture supernatant of HBMECs among the T.pallidum group,the inactivated T.pallidum group and the blank control group (all P ＞ 0.05).The number of migratory HL-60 cells in the lower Transwell chambers was significantly higher in the T.pallidum group than in the inactivated T.pallidum group and the blank control group (both P ＜ 0.05).Conclusion Viable T.pallidum can up-regulate the gene expression of CXCL6,CXCL8 and CXCL10 in HBMECs,promote the secretion of CXCL6 and CXCL8,and enhance the chemotactic effect of HBMECs on HL-60 cells,which may play a certain role in the occurrence of neurosyphilis.

Mechanism of coke oven emissions-induced acute toxicity in HL-60 cells / 中国职业医学

OBJECTIVE: To establish the cell model using human leukemia cell line HL-60 for exposure of coke oven emissions( COE) in vitro and to explore the mechanism of COE-induced acute toxicity in HL-60 cells. METHODS: HL-60 cells were collected in their logarithmic growth phase and cultured in medium that had final concentrations of COE in 2. 5,5. 0,10. 0 and 20. 0 mg / L for 24 hours. Cell survival rate was examined by CCK-8 assay. The cytotoxicity was evaluated using lactate dehydrogenase release assay. Reactive oxygen species( ROS) production was determined by the 2',7'-dichlorofluorescein diacetate and nitroblue tetrazolium method. The activation of nuclear factor-κB( NF-κB) pathway was evaluated by western blot. RESULTS: With the increasing exposure concentrations of COE,the cytotoxicity of HL-60 cells increased( P < 0. 01),the cell survival rate decreased( P < 0. 01),intracellular ROS decreased( P < 0. 01),whereas extracellular ROS increased( P < 0. 01). These changes had a dose-effect relationship. The levels of phospho-nuclear factor-kappa B p65 and phospho-inhibitor of kappa Bα were higher in all the COE-treated cells compared with untreated cells( P < 0. 05),with no dose-effect relationship. CONCLUSION: COE could cause acute toxicity in HL-60 cells in a doseeffect relationship. The mechanism may be related to the COE-induced in-balanced ROS release and removal,leading to the activation of NF-κB pathway. HL-60 cells can be used as a common cell line for COE hematotoxicity analysis.

Effects of ICAT silencing in Wnt signaling pathway and NSC67657 induced cell differentiation of HL-60 cells / 中华血液学杂志

Objectives@#To investigate the effect of β-catenin interacting protein 1 (ICAT) silence in Wnt signaling pathway and sterol drug NSC67657 induced cell differentiation of HL60 cell.@*Methods@#HL-60 cells were treated with NSC67657, the cell surface antigen CD14 expression was detected by flow cytometry. Lentivirus LV-ICAT-RNAi vector was constructed and infected HL-60 cells. Then the ICAT gene and protein expression were analyzed using real-time qPCR and Western blot technique. Furthermore, Co-immunoprecipitation assay was used to confirm the interaction of β-catenin/ICAT proteins, and Western blot was employed to compare the expressions of Wnt signaling pathway downstream targets Cyclin D1, TCF-1 and c-Jun between Lentivirus LV-ICAT-RNAi vector infected HL-60 (HL-60i) cells and un-infected HL-60 (HL-60v) cells. The cellular differentiation of HL-60i and HL-60v cells treated with NSC67657 for 24 h was evaluated by Wright’s staining, transmission electron microscopy and flow cytometry analysis.@*Results@#HL-60 cells could be induced to differentiate into monocytes by 10 μmol/L NSC67657. The CD14 positive cells could reach to (92.30±5.14) % after NSC67657 treatment for 5d. The co- immunoprecipitation assay demonstrated that ICAT protein did interact with β-catenin protein, and the absorbance of protein electrophoresis bands increased in differentiated cells. The expressions of Wnt signaling pathway downstream target proteins in HL-60i cells were higher than that in HL-60v cells when they were treated by 10 μmol/L NSC67657, but lower than NSC67657 untreated cells. CD14 positive HL-60i cells were significantly lower than that of HL-60v cells[ (8.33±3.14) % vs (19.08±4.73) %]when treated with NSC67657, but still higher than that of uninfected and untreated HL60 cells[ (0.60±0.03) %] (F=119.24, P=0.010) . The results of cellular morphology and ultrastructure observation were also in accord with that of cell surface antigen analysis.@*Conclusions@#ICAT does participate in HL-60 cells monocytic differentiation induced by NSC67657, and Wnt/β-catenin signaling pathway might play a bridge role.

Effect of LSD1 knock-down by small hairpin RNA on the apoptosis and cell cycle of human acute myelogenous leukemia cells / 实用医学杂志

Objective To investigate the effect of down-regulation of lysine specific demethylase 1 (LSD1) by shRNA on the apoptosis and cell cycle of human acute myelogenous leukemia cells.Methods The lentiviral vector-mediated LSD1-shRNA was transfected into human acute promyelocytic leukemia HL-60 cells and acute monocytic leukemia SHI-1 cells.The expressions of LSD1 mRNA and protein were examined by real time quantitative PCR and Western blot,respectively.The flow cytometry was applied to detect the apoptosis and cell cycle distribution after AnnexinV-PE/7-AAD and PI dying,respectively.Results The expressions of LSD1 mRNA and protein in HL-60 and SHI-1 LSD1-shRNA group were significantly decreased compared with the blank control group and the negative shRNA group (P ＜ 0.01,respectively).The apoptosis levels of HL-60 and SHI-1 cells were significantly increased after knockdown of LSD1 (P ＜ 0.01).Moreover,the cell cycle distribution in the G0/G1 phases was also significantly increased(P ＜ 0.01).Conclusion LSDI-shRNA promotes cell apoptosis and increases the percentage of cells in the G0/G1 phases of human acute myelogenous leukemia cells.

Arsenic Trioxide Induces Apoptotic Cell Death through Mitochondrial Pathway in Human Leukemia HL-60 Cells / 现代生物医学进展

Objective:To investigate the potential pro-apoptotic activity of arsenic trioxide (ATO) in human leukemia HL-60 cells,as well as the potential mechanism with focus on mitochondrial pathway.Methods:After treatment with different concentrations of ATO (1 μg/mL,5 μg/mL or 10 μg/mL) for 24 h,apoptotic cell death was detected by flow cytometry,oxidative stress was determined by measuring MDA and GSH levels,the expression of apoptotic factors was detected by western blot,and mitochondrial membrane potential (MMP) was determined by immunofluorescence staining.Results:ATO at the concentrations of 5 μg/mL or 10 μg/mL induces apoptotic cell death and increases oxidative stress in human leukemia HL-60 cells.ATO significantly increases the expression of pro-apoptotic factors (Bax and Caspase-3),whereas decreases the expression of anti-apoptotic factor Bcl-2.Compared with the control group,ATO treatment significantly decreases the MMP level in HL-60 ceils.Conclusions:Arsenic trioxide induces apoptotic cell death through mitochondrial pathway in human leukemia HL-60 cells.

Suppression of mel18 enhances differentiation of acute myeloid leukemia cell line HL60 induced by cinnamaldehyde / 中国病理生理杂志

AIM:To investigate the effect of suppression of mel18 gene on the differentiation of human acute myeloid leukemia cell line HL 60 induced by cinnamaldehyde ( CA) .METHODS:HL60 cells were treated with low con-centration of CA or all-trans retinoic acid (ATRA).shRNAmel18 vector and shRNALuc control vector were employed to package lentiviruses which were then used to infect HL 60 cells.The virus-infected HL60 cells were treated with low con-centration of CA , and ATRA was used as a positive control of differentiation-inducing agent .The differentiation markers on the cell surface and cell cycle of virus-infected HL60 cells were analyzed by flow cytometry .Western blot was used to deter-mine the expression of MEL18, cyclin D1 and p27, as well as the phosphorylation level of Akt .RESULTS:Low concen-tration of CA and ATRA increased the expression of granulocytic differentiation marker CD 11b on the HL60 cells, with the decreased expression of MEL 18 in the HL60 cells.The expression of MEL18 decreased in shmel18 virus-infected HL60 cells (shmel18-HL60 cells), but did not change in shLuc-HL60 cells.The expression of CD11b on shmel18-HL60 cells increased with G 1-phase arrest , which went even higher after treatment with CA .The phosphorylation level of Akt and the expression of cyclin D1 decreased in shmel18-HL60 cells with the increase in the expression of p27.CONCLUSION:In-hibition of mel18 gene leads HL60 cell granulocytic differentiation .mel18 gene may affect the differentiation of HL 60 cells by regulating cyclin D1 and p27 via PI3K/Akt signaling pathway.PI3K/Akt signaling pathway is also involved in CA-in-duced differentiation of HL60 cells by suppressing mel18 gene expression.

Suppression of mel18 enhances differentiation of acute myeloid leukemia cell line HL60 induced by cinnamaldehyde / 中国病理生理杂志

AIM:To investigate the effect of suppression of mel18 gene on the differentiation of human acute myeloid leukemia cell line HL 60 induced by cinnamaldehyde ( CA) .METHODS:HL60 cells were treated with low con-centration of CA or all-trans retinoic acid (ATRA).shRNAmel18 vector and shRNALuc control vector were employed to package lentiviruses which were then used to infect HL 60 cells.The virus-infected HL60 cells were treated with low con-centration of CA , and ATRA was used as a positive control of differentiation-inducing agent .The differentiation markers on the cell surface and cell cycle of virus-infected HL60 cells were analyzed by flow cytometry .Western blot was used to deter-mine the expression of MEL18, cyclin D1 and p27, as well as the phosphorylation level of Akt .RESULTS:Low concen-tration of CA and ATRA increased the expression of granulocytic differentiation marker CD 11b on the HL60 cells, with the decreased expression of MEL 18 in the HL60 cells.The expression of MEL18 decreased in shmel18 virus-infected HL60 cells (shmel18-HL60 cells), but did not change in shLuc-HL60 cells.The expression of CD11b on shmel18-HL60 cells increased with G 1-phase arrest , which went even higher after treatment with CA .The phosphorylation level of Akt and the expression of cyclin D1 decreased in shmel18-HL60 cells with the increase in the expression of p27.CONCLUSION:In-hibition of mel18 gene leads HL60 cell granulocytic differentiation .mel18 gene may affect the differentiation of HL 60 cells by regulating cyclin D1 and p27 via PI3K/Akt signaling pathway.PI3K/Akt signaling pathway is also involved in CA-in-duced differentiation of HL60 cells by suppressing mel18 gene expression.

Construction of ICAT interference lentivirus expression vector and establishment of stable HL 60 cell lin / 国际检验医学杂志

Objective To construct the ICAT gene interference lentivirus expression vector targeting and to establish stable transfected cell line HL60 .Methods The interference sequence targeted at human ICAT gene was designed and synthesized ,after annealing ,which was connected to PGLV3 interference vector and with PG‐p1‐VSVG ,PG‐p1‐REV ,PG‐p1‐RRE were co‐transfected into 293T cells The lentivirus particles were packaged and generated .The virus titer was detected .HL60 cells were transfected for establishing the stable cell line ;RT‐PCR and Western Blot techniques were used to detect ICAT gene and protein expression in sta‐ble HL60 cells ,then the results were compared with those in the control group .Results The lentivirus expression vector targeted at ICAT was successfully constructed and the virus titer was 2 × 108 U/mL .Stable transfected HL60 cell line was established .The effective interference verification revealed that shICAT could significantly reduce the mRNA and protein level of ICAT ( P<0 .001) .Conclusion The shRNA lentiviral expression vector of ICAT gene is successfully constructed and the HL 60 cell line stably interfering ICAT expression is established .