J Korean Cancer Assoc.  1999 Dec;31(6):1279-1287.

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

• Lim K
• Son MY
• Choi BI
• Yun KA
• Zheng M
• Kang TW
• Lee YC
• Park JI
• Yoon WH
• Hwang BD

Abstract

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.

Keyword

DNA topoisomerase II a; Nocodazole; Taxol; ATF; HL-60

MeSH Terms

Anti-Bacterial Agents
Binding Sites
Blotting, Northern
Cell Division*
DNA Topoisomerases, Type I*
DNA Topoisomerases, Type II*
DNA*
Edetic Acid
Electrophoretic Mobility Shift Assay
Genes, vif
Glycerol
HEPES
HL-60 Cells
Humans
Hydrogen-Ion Concentration
Magnesium Chloride
Nocodazole
Paclitaxel
Repression, Psychology
RNA
RNA, Messenger
Anti-Bacterial Agents
DNA
DNA Topoisomerases, Type I
DNA Topoisomerases, Type II
Edetic Acid
Glycerol
HEPES
Magnesium Chloride
Nocodazole
Paclitaxel
RNA
RNA, Messenger