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KMID : 0857020040190010286
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Kosin Medical Journal
2004 Volume.19 No. 1 p.286 ~ p.293
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Effects of doxorubicin on the activity and expression of topoisomerase in HL-60 human leukemia cells
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Cho Moo-Youn
Jeong In-Cheol
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Abstract
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Background DNA topoisomerases are essential enzymes present in all organisms. They modify DNA topology in connection with a number of nuclear processes, such as replication, transcription, chromatin remodeling, chromatin condensation/decondensation, recombination and repair. In the strand-breakage reaction by a DNA topoisomerase, a tyrosyl oxygen of the enzyme attacks a DNA phosphorus, forming a covalent phosphotyrosine link and breaking a DNA phosphodiester bond at the same time. DNA topoisomerase have been shown to be the molecular targets of many antimicrobial and anticancer agents. Among topoisomerase-targeting drugs in clinical use at present, most act by trapping the covalent DNA-enzyme intermediates to convert a normal cellular enzyme to a DNA damaging agent. This studies were designed to elucidate whether pretreatment of HL-60 human leukemia cells with the topoisomerase I - directed drug doxorubicin would increase the expression of topoisomerase and to investigate the activity of topoisomerase mediated by doxorubicin in nuclear extract from HL-60 human leukemia cells.
Methods We have conducted experiments on topoisomerase assay using gel electrophoresis, pUC-X I cloning, DNA sequencing, cell cytotoxicity in drug-treated cells, topoisomerase purification, quantitative RT-PCR analysis, northern blotting techniques, respectively.
Results Doxorubicin inhibited the relaxation activity of topoisomerase in pUC19 DNA at warious concentrations (0.4-50 μM), while it enhanced the cleavage of topoisomerase in the pUC-X I by forming a cleavable complex at 0.4-2μM. The levels of the topoisomerase IIα mRNA from RT-PCR analysis and northern blot were increased in HL-60 cells treated with doxorubicin, whereas the expression of topoisomerase I and IIβ mRNA from RT-PCR analysis remained no significant change.
Conclusion Our results suggest that overexpression of topoisomerase IIα mRNA by topoisomerase II-directed drug treatment is due to stabilizing drug-enzyme-DNA "cleavable complex".
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KEYWORD
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Doxorubicin, Topoisomerase activity, Expression, HL-60 cells
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