ABSTRACT
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.
ABSTRACT
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.
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Objective:To establish the granulocyte-differentiation model of the HL60 cells which are treated with All-trans retinoic acid(ATRA),and to use the modified two-dimensional electrophoresis conditions to analyze the differences of protein expression between treated and untreated HL60 cells.Methods:HL60 cells were induced through treatment with All-trans retinoic acid(ATRA).For selection of the appropriate drug concentration and induction time,MTT and flow cytometry are used to detect the HL60cell proliferation and the expression of differentiation antigens CD11b respectively.Cellular chemical staining was used for the verification of the differentiation of the treated HL60 cells.The protein of HL60 cell lines could be separated by modified two-dimensional electrophoresis(2-DE).PDQuest software was used to analyze the different protein expression between treated and untreated HL60 cells.The protein was analyzed by matrix-assisted laser desorption -time of flight mass spectrometry(MALDI-TOF).Results:ATRA could inhibit HL60 cell proliferation,and with the increase in drug concentration,the effect of inhibiting was more significant.Treated with 2? M ATRA for five days,there were more than 90% of HL60 cells expressing antigenCD11b.Cellular chemical staining also showed that ATRA could induce HL60 cells to granulocyte cells.By the analysis of modified 2-DE and PDQuest software,25 protein spots was detected in untreated cells,while 15 protein spots was promoted Some of them were oncogene protein and suppressor gene protein,while some of others are involved in apoptosis.Conclusion:ATRA could induce HL60 cells to granulocyte cells in selected drug concentration and induction time.Using the modified two-dimensional electrophoresis conditions,different protein expression can be found from the traditional two-dimensional electrophoresis.
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The transfection efficiency of oligonucleotide and plasmid to the HL-60 cell line with lipofectaminePLUS was compared through observing the transfection rate and the expression duration of exogenous gene in the target cells. The results showed that the transfection rate of oligonucleotide to the HL-60 was about 90 %-95 % and it had no obvious attenuation within 84 h. However,the plasmid transfection rate was only 5 %-25 % and it was decreased significantly within 60 h. It was suggested that the transfection of oligonucleotide with liposomes was better than that of plasmid.
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The HL-60 cells were transfected with chk1 antisense and sense chain, and 24 h later subjected to irradiation. Twenty-four h after irradiation, the changes in the chk1 protein expression was assayed by Western blot, and the cell cycles and apoptosis rate detected by FCM. The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line with the apoptosis rate being 26.31%, significantly higher than that by the sense blocking (10.34 %,0.025＜P＜0.05). In HL-60 cells transfected with chk1 antisense chain, the G2/M phase arrest was attenuated and the cells in G2/M phase were accounted for 38.42 %, significantly lower than those of the cells transfected with chk1 sense chain (54.64 %, 0. 005＜P＜0.01). It was concluded that antisense blocking of chk1 gene could increase the apoptosis sensitivity to irradiation.
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The effect of rhGM-CSF/IL-3 on apoptosis of Ara-C-induced myeloid leukemic cell line HL- 60 was investigated. The results indicated that treatment with rhGM-CSF/IL-3 in combination with Ara-C significantly inhibited the colony growth of HL-60 and enhanced the oligonucleosomal DNA fragmentation as compared with Ara-C alone. The Ara-C mediated apoptosis rates of HL-60 cells treated with rhGM-CSF/IL-3 fusion protein alone were markablely improved compared with treatment with rhIL-3 plus rhGM-CSF, it was noted that the Ara-C mediated of apoptosis normal peripheral white blood cells was less affected by rhGM-CSF/IL-3. It suggested that rhGM-CSF/IL-3 could be used as a possible curing drug during the phase of induced remission of leukemia.
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Objective To investigate the effect of tadpole extract(T871 3) on tumor cells and its mechanism. Methods We studied the effects of T871 3 on proliferation, differentiation and apoptosis of HL 60 cells by cytomorphological observation, cytochemistry and TUNEL method. We also examined gene expression during the induction of apoptosis and differentiation in tadpole extract treated HL 60 cells by in situ hybridization and intact cell mRNA dot blot techniques. Results 1 T871 3 was able to inhibite HL 60 cells proliferation. 2 T871 3 was able to induce HL 60 cells to differentiate along monocyte macrophage lineage at low concentration, and apoptosis at higher concentration. 3 The differentiation of HL 60 cells was accompanied by downregulations of c myc,c myb gene expression, The apoptosis of HL 60 cells was accompanied by downregulations of c\|myc bcl 2 gene expression, suggesting that these genes may be involved in the apoptosis and differentiation process. Conclusion Tadpole extract may have effects on HL 60 cells through changing the oncogene expression. [