J Korean Soc Endocrinol.  1998 Dec;13(4):601-611.

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

• Lim K
• Kwon DW
• Kim SM
• Yoon KA
• Son MY
• Lee MS
• Park JI
• Yoon WH
• Hwang BD

Abstract

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.

MeSH Terms

Anti-Bacterial Agents
Blotting, Northern
Cell Differentiation
Cell Line
Cycloheximide
Cytoplasm
DNA
Edetic Acid
Electrophoretic Mobility Shift Assay
Glycerol
HEPES
HL-60 Cells*
Humans
Hydrogen-Ion Concentration
Repression, Psychology*
RNA
RNA, Messenger
Transcription Factor AP-1
Tretinoin
Vimentin*
Anti-Bacterial Agents
Cycloheximide
DNA
Edetic Acid
Glycerol
HEPES
RNA
RNA, Messenger
Transcription Factor AP-1
Tretinoin
Vimentin