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KMID : 0857020040190010279
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Kosin Medical Journal
2004 Volume.19 No. 1 p.279 ~ p.285
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Idenfitication of type IB topoisomerase from HL-60 human leukemia cells
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Jeong In-Cheol
Kwak Choong-Keun
Cho Moo-Youn
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Abstract
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Background DNA topoisomerases fall into two categories - type Ⅰand typeⅡ. For the type I enzymes. the DNA strands are transiently broken one at a time; for the type II enzymes. by contrast, a pair of stands in a DNA double helix are transiently broken in concert by a dimeric enzyme molecule. The two types can be further divided into four subfamilies : IA, IB, IIA and IIB. DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. Camptothecin is an antitumor alkaloid that has been isolated from the Chinese tree. Camptotheca acuminata.. We have developed a procedure for the simultaneous purification of type I and II topoisomerase from HL-60 human leukemia cells, and identified type IB topoisomerase.
Methods DNA topoisomerase was purified from HL-60 human leukemia cells by a simple and fast four-step procedure : selective ammonium sulfate precipitation, chromatography on Ultrogel A6 and DNA-cellulose. followed by ultracentrifugation on a glycerol gradient. Enzyme activity, and molecular weight were assayed by measuring the relaxation of supercoiled pUC19 DNA using agarose gel electrophoresis and sodium dodesyl sulfate denaturing gel electrophoresis, respectively,
Results Type I and II topoisomerases were isolated from extracted nucleoprotein complexes by glycerol gradient centrifugation, respectively. Human type IB topoisomerasewas purified 8.6-fold as compared to the whole cell homogenate, with 12% yield. The purified type I topoisomerase has a molecular weight of 100 kDa ad determined by SDS polyacrylamide gel electrophoresis. The enzyme relaxes supercoiled DNA in the absence of ATP of Mg2+. Camptothecin inhibited the relaxation activity of type I topoisomerase in pUC19DNA by forming a cleavable complex at various concentratrions (0.4-50 μM).
Conclusion The results showed that a type IB topoisomerase has been purified to near homogeneity from HL-60 human leukemia cells, and partial purification of type IIA from small samples was achieved by isolation of cell nuclei. Thus, the potential antitumor activity of a type I and II topoisomerase-targeted drug might be easily screened by testing the drug’s ability to cause DNA cleavage in the presence of enzymes.
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KEYWORD
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Identification, Type IB topoisomerase, HL-60 cells
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