Role of AP-1 Transcriptional Regulation of Vimentin Gene during 12-O-Tetradecanoylphorbol-13-Acetate Differentiation of HL-60 Cells.
- Author:
Kyu LIM
1
;
Jin Hee KIM
;
Do Won KWEON
;
Sung Min KIM
;
Myung Sun LEE
;
Kyung Ah YUN
;
Mee Young SON
;
Jong Il PARK
;
Wan Hee YOON
;
Byung Doo HWANG
Author Information
1. Department of Biochemistry, Chungnam National University, Daejeon, Korea.
- Publication Type:Original Article
- Keywords:
Vimentin;
AP-1;
Staurosporin;
TPA;
HL-60
- MeSH:
Anti-Bacterial Agents;
Blotting, Northern;
DNA;
Edetic Acid;
Electrophoretic Mobility Shift Assay;
Glycerol;
HEPES;
HL-60 Cells*;
Humans;
Hydrogen-Ion Concentration;
Magnesium Chloride;
Protein Kinases;
RNA;
RNA, Messenger;
Signal Transduction;
Transcription Factor AP-1*;
Vimentin*
- From:Journal of the Korean Cancer Association
1998;30(5):997-1004
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
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.
