Role of ATF on Transcriptional Regulation of DNA Topoisomerase II a Gene in HL - 60 Arrested to G2 / M and M Phase / 대한암학회지

PURPOSE: To gain insight on transcriptional repression of Topo II a in HL-60 cells arrested to G2/M and M phase, the levels of Topo IIa mRNA and the binding activity of ATF have been investigated with Northern blot hybridization and DNA mobility shift assay, respectively. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-mactivated fetal bovine serum and antibiotics in a humidified 5% CO2 at 37C degree. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. A Xho I-Mlu I fragment of phTOP2 was used as probe for Northern blot analysis of Topo II a mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA oligomer (upper strand, 5-TCTCCGCTATGACGCCGAGTGGTG-3) for ATF binding activity was mixed with nuclear extracts in a 20 pl reaction volume containing 60 mM KC1, 12 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 12% glycerol, and 2 ug of poly [dI-dC]. RESULTS: HL-60 cells were arrested at G2/M phase and M phase after taxol or nocodazole treatment. The levels of Topo II a mRNA were reduced at 24 hours after exposure with nocodazole or taxol but the unknotting activities were not changed. DNA mobility shift assay using oligonucleotide containing the ATF binding site showed that ATF binding activity was reduced after pretreatment of nododazole or taxol. CONCLUSIONS: These results suggest that the reduction of ATF binding activity may be important to transcriptional repression of Topo II a gene by nocodazole and taxol in HL- 60 cells.

Transcriptional REpression of Vimentin Gene During All-TTrans Retinoic Acid-Induced Differentiation of HL-60 Cells / 대한내분비학회지

BACKGROUND: Vimentin is the major intermediate-size filament in the cytoplasm of cells from mesenchymal origin. The HL-60 cell is a unique human leukemic cell line capable of terminal differentiation with several chemical inducers, and then the cell line becomes a fre#quently described model system for cell differentiation in vitro. Vimentin mRNA is reduced during all-trans retinoic acid (retinoic acid) -dependent differentication but increased by 12-0-tetradecanoylphorbol-13-acetate (TPA). In this paper, we have investigated on the mechanism of transcriptional repression of vimentin gene during retinoic acid-dependent differentication of HL-60 cell. METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics in a humidified 5% CO at 37C. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. EcoRI fragment of pVIM-GEM was used as probe for vimentin mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA probe (Upper strand, 5-CGCITGATGAGTCAGCCG-3) for AP-1 binding activity was mixed with nuclear extracts in a 20 pL reaction volume containing 300 mM KCI, 60 mM HEPES, pH 7.9, 25mM MgC1, 1mM EDTA, 1mM DTT, 60% glycerol, and 2 pg of poly[dI-dC]. RESULTS: The level of vimentin mRNA was decreased at 12 hours after retinoic acid treatment, and not detected at 48 hours. The level of vimentin mRNA was reduced in proportion to concentration of retinoic acid, Retinoic acid-reduced vimentin mRNA was no change in cells treated with cycloheximide. Retinoic acid-dependent decrease of vimentin mRNA was partially recovered by staurosporin pretreatment. In DNA mobility shift assay, AP-1 binding activity was reduced at 48 hr during retinoic acid-induced differentiation. CONCLUSION: These results suggest that the transcriptional repression of vimentin gene during retinoic acid-induced differentiation in HL-60 cells is correlated with reduction of DNA binding activity of AP-1.

Role of AP-1 Transcriptional Regulation of Vimentin Gene during 12-O-Tetradecanoylphorbol-13-Acetate Differentiation of HL-60 Cells / 대한암학회지

PURPOSE: To gain insight on the role of AP-1 in transcriptional regulation of vimentin gene during differentiation of HL-60 cells by 12-0-tetradecanoylphorbol-13-acetate (TPA), the levels of vimentin mRNA and AP-1 have been investigated with Northern blot hybridization and DNA mobility shift assay. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics in a humidified 5% CO2 at 37 degree C. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. EcoRI fragment of pVIM-GEM was used as probe for vimentin mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA probe(Upper strand, 5'-CGCTTGATGAGTCAGCCG- 3') for AP-1 binding activity was mixed with nuclear extracts in a 20 microliter reaction volume containing 300 mM KC1, 60 mM HEPES, pH 7.9, 25 mM MgCl2, 1 mM EDTA, 1 mM DTT, 60% glycerol, and 2 microgram of poly[dI-dC]. RESULTS: TPA increased vimentin mRNA levels, with maxima1 stimulation reached at 24 hr. The level of vimentin mRNA was induced in proportion to the concentration of TPA. TPA-induced vimentin mRNA was almost reduced by actinomycin-D pretreatment. TPA- induced stimulation of vimentin gene was completely reduced by staurosporin pretreatment. In DNA mobility shift assay, AP-I newly appeared at 24 hr during TPA- induced differentiation and was almost not detected after the pretreatment of staurosporin. CONCLUSIONS: These results suggest that the induction of vimentin mRNA during TPA- dependent differentiation in HL-60 cells may be mediated by protein kinases C signal transduction and AP-1 is important to transcriptional regulation.