To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml ...To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.展开更多
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 targe...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.展开更多
Aim To study the effect of 8 chloroadenosine (8 CA)on undifferentiatied HL 60 cell line. Methods The IC 50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 54...Aim To study the effect of 8 chloroadenosine (8 CA)on undifferentiatied HL 60 cell line. Methods The IC 50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB). Morphology of HL 60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL 60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL 60 cells was analyzed by flow cytometry. Results 8 CA inhibited proliferation of eight human cancer cell lines. The IC 50 ranked in the following order: KB (0 05 μmol·L -1 ) < HL 60 (0 25 μmol·L -1 ) < Bel 7402 (0 56 μmol·L -1 ) < MCF 7 (0 65 μmol·L -1 ) < HCT (0 79 μmol·L -1 ) < HeLa (0 89 μmol·L -1 ) < BGC 823 (1 149 μmol·L -1 ) < PG (2 50 μmol·L -1 ). The scanning and transmission electron microscopy showed that the microvilli of HL 60 cell surface shortened, and the shape of HL 60 cells nuclei changed to kidney shaped, horse shoe shaped and bilob ated after treatment with 8 CA. Meanwhile, 8 CA promoted NBT reduction and increased activity of acid phosphatase in HL 60 cells in a time and concentration dependent manner. Flow cytometry analysis indicated that 8 CA induced an appreciable increase of the cell population in G 1 phase with a marked reduction in S phase. Conclusion 8 CA can induce differentiation of HL 60 cells and block the cells at G 1 phase, thus inhibiting proliferation of HL 60 cells.展开更多
Summary： The HL-60 cells were transfected with chkl 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 Weste...Summary： The HL-60 cells were transfected with chkl 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 chkl antisense chain, the G2/M phase arrest was attenua：ted and the cells in G2/M phase were accounted for 38.42 %, significantly lower than those of the cells transfected with chkl 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.展开更多
Background p27 is an essential mediator of cell cycle control,which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects...Background p27 is an essential mediator of cell cycle control,which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects of p27 on the proliferation and apoptosis of HL-60 and Raji cell lines.Methods HL-60 and Raji cells were transfected with p27 via an adenovirus-mediated approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR, and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by means of trypan blue staining, MTT assay, Annexin V/PI, and DNA ladder electrophoresis. Results The infection efficiencies in HL-60 and Raji cells were 40.3% and 32.0%, respectively. RT-PCR and flow cytometry showed that there was significant expression of p27 mRNA and protein in HL-60 and Raji cells infected with Adp27; on the other hand, uninfected HL-60 cells showed faint traces of p27 mRNA and protein and Raji cells showed nearly no signs of p27 mRNA and protein. As demonstrated by a cell growth curve and by an MTT assay, strong time-dependent proliferation inhibition was apparent in HL-60 and Raji cells infected by Adp27. After 72 hours of infection, the Annexin V+/PI- apoptotic cell rates in HL-60 and Raji cell lines were 46.9% and 35.7%, respectively, significantly higher than in the control groups (4.7% and 5.6%, respectively). Typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 hours of Adp27 infection. Conclusions Adenoviral vector-mediated p27 gene transfection of HL-60 and Raji cells leads to the inhibition of cellular proliferation and the promotion of cell apoptosis. This technique may provide an approach to gene therapy for leukemia or lymphoma.展开更多
Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective...Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it's mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.展开更多
文摘To explore the effect of NF κB on bcl x gene transcription in extended drug resistance leukemia cell line HL 60/E6, drug resistant subline HL 60/E6 was derived by intermittently exposing HL 60 cells to 6 ng/ml epirubicin. Indirect immunofluorescence was used to demonstrate the location of NF κB RelA in HL 60/E6 cells. FCM analysis and RT PCR were used to detect the efficiency of liposome mediated ODN transfection and the change of bcl x L mRNA levels after 5 μmol/L phosphorothioate (PS) derivatized antisense (AS) oligodeoxynucleotide (ODN) directed to RelA was transferred into HL 60/E6 cells. The results showed that RelA remained persistently active and located at the nuclei of HL 60/E6 cells,but in the cytoplasm of HL 60 cells, the efficiency of liposome mediated ODN transfection was significantly higher than that of null ODN ( P <0.01 in 4 h, 6 h, 12 h, 24 h). Exposure of HL 60/E6 cells to 5 μmol/L AS PS ODN directed to RelA led to a maximal 40 % decline of bcl x L mRNA levels within 8 h. The inhibition rate of bcl x L mRNA was (15±1.79) %, (28±2.34) %, (40±3.47) %, (20±1.54) % in 4 h, 6 h, 8 h, 15 h, respectively, but it was less than 15 % in control group. It was concluded that NF κB was involved in regulating bcl x transcription. It was suggested that NF κB was an important factor for drug resistance in leukemia cells.
基金This project was supported by a grant from National Natu ral Sciences Foundation of China (No. 39800149).
文摘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.
文摘Aim To study the effect of 8 chloroadenosine (8 CA)on undifferentiatied HL 60 cell line. Methods The IC 50 of cancer cell proliferation was determined using a microculture plate reader at 570 nm (MTT) and 540 nm (SRB). Morphology of HL 60 cells was observed under a scanning electron microscope and a transmission electron microscope. The differentiation of HL 60 cells was examined by nitro blue tetrazolium reduction (NBT) and acid phosphatase assay. The cycle of HL 60 cells was analyzed by flow cytometry. Results 8 CA inhibited proliferation of eight human cancer cell lines. The IC 50 ranked in the following order: KB (0 05 μmol·L -1 ) < HL 60 (0 25 μmol·L -1 ) < Bel 7402 (0 56 μmol·L -1 ) < MCF 7 (0 65 μmol·L -1 ) < HCT (0 79 μmol·L -1 ) < HeLa (0 89 μmol·L -1 ) < BGC 823 (1 149 μmol·L -1 ) < PG (2 50 μmol·L -1 ). The scanning and transmission electron microscopy showed that the microvilli of HL 60 cell surface shortened, and the shape of HL 60 cells nuclei changed to kidney shaped, horse shoe shaped and bilob ated after treatment with 8 CA. Meanwhile, 8 CA promoted NBT reduction and increased activity of acid phosphatase in HL 60 cells in a time and concentration dependent manner. Flow cytometry analysis indicated that 8 CA induced an appreciable increase of the cell population in G 1 phase with a marked reduction in S phase. Conclusion 8 CA can induce differentiation of HL 60 cells and block the cells at G 1 phase, thus inhibiting proliferation of HL 60 cells.
文摘Summary： The HL-60 cells were transfected with chkl 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 chkl antisense chain, the G2/M phase arrest was attenua：ted and the cells in G2/M phase were accounted for 38.42 %, significantly lower than those of the cells transfected with chkl 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.
文摘Background p27 is an essential mediator of cell cycle control,which plays a key negative role in the proliferation and tumorigenesis of certain cell types. Here, we designed this study to explore the possible effects of p27 on the proliferation and apoptosis of HL-60 and Raji cell lines.Methods HL-60 and Raji cells were transfected with p27 via an adenovirus-mediated approach. The efficiency of Adp27 infection and the expression of p27 mRNA and protein were evaluated by X-gal staining, RT-PCR, and flow cytometry. The proliferation and apoptosis of HL-60 and Raji cells were estimated by means of trypan blue staining, MTT assay, Annexin V/PI, and DNA ladder electrophoresis. Results The infection efficiencies in HL-60 and Raji cells were 40.3% and 32.0%, respectively. RT-PCR and flow cytometry showed that there was significant expression of p27 mRNA and protein in HL-60 and Raji cells infected with Adp27; on the other hand, uninfected HL-60 cells showed faint traces of p27 mRNA and protein and Raji cells showed nearly no signs of p27 mRNA and protein. As demonstrated by a cell growth curve and by an MTT assay, strong time-dependent proliferation inhibition was apparent in HL-60 and Raji cells infected by Adp27. After 72 hours of infection, the Annexin V+/PI- apoptotic cell rates in HL-60 and Raji cell lines were 46.9% and 35.7%, respectively, significantly higher than in the control groups (4.7% and 5.6%, respectively). Typical DNA ladder bands were detectable in HL-60 and Raji cells after 48 hours of Adp27 infection. Conclusions Adenoviral vector-mediated p27 gene transfection of HL-60 and Raji cells leads to the inhibition of cellular proliferation and the promotion of cell apoptosis. This technique may provide an approach to gene therapy for leukemia or lymphoma.
文摘Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it's mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.
