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.
Subject(s)
Humans , Anti-Bacterial Agents , Blotting, Northern , Cell Differentiation , Cell Line , Cycloheximide , Cytoplasm , DNA , Edetic Acid , Electrophoretic Mobility Shift Assay , Glycerol , HEPES , HL-60 Cells , Hydrogen-Ion Concentration , Repression, Psychology , RNA , RNA, Messenger , Transcription Factor AP-1 , Tretinoin , VimentinABSTRACT
Sequestration of disc herniation is important cause of failure of chemonucleolysis. It is important to recongnize this particular variety of disc herniation before considering enzymatic discetomy. Disc herniation were classified into three-anatomical categories. type I: Subligamentous Herniation type II: Extra-ligamentous Herniation type III: Free-extraligamentous Herniation, Sequestratid disc. one-hundred and eight-six cases of herniatid intervertebral disc were evaluated in Department of Orthopedic Surgery, Wonju College of Medicine, Yonsei University and in Department of Orthopedic Surgery, Inchon Christian Hospital from March 1982 to March 1988. The results of study are as follows; l. Among 186 patients, type III sequestrated disc patients were 32 cases(17.2%). 2. The ratio between male and female was about 2:l. 3. The duration of symptoms in type II,III were longer than type I. 4. On examination, the physical changes were more common in type III than in other types. Positive well leg rasing test was prominant finding especially in type III. 5. The myelographic findings, in type III, were irregularity of dural sac at the level of vertebra body both on obligue and lateral view. 6. The C-T findings, in type III, were irregular protruded disc, or caudal or cephalsd migrated disc materials.