Analysis of topoisomerase II-mediated DNA cleavage of the c-myc gene during HL60 differentiation.

We have investigated the effect of mAMSA, a potent topoisomerase II inhibitor, on the c-myc proto-oncogene of the acute promyelocytic leukemia HL60 cell line during its differentiation. When HL60 cells were induced by dimethylsulfoxide (DMSO) to terminally differentiate, a rapid drop in the level of c-myc mRNA was observed, followed by an arrest of cell proliferation. In contrast, the level of topoisomerase II mRNA was transiently increased with a maximum at 6 h after DMSO addition and was then completely abolished after 48 h, indicating that topoisomerase II is activated during the onset of HL60 differentiation. In exponentially growing cells, treatment by mAMSA results in the formation of topoisomerase II-mediated double strand DNA breaks in the c-myc gene at positions where topoisomerase II would normally nick and reseal the two strands. In HL60 cells treated with both mAMSA and DMSO, the sites in the c-myc gene at which mAMSA had induced cleavage were not altered. However, a DNA cleavage site located at the end of the first c-myc exon (position +3100), was strongly stimulated by mAMSA and DMSO treatment. This site fell within a DNase I hypersensitive region encompassing the MYC intron factor 1 (MIF1) binding site, where transcription elongation is normally blocked during differentiation. These data indicate that a change of topoisomerase II binding to critical regulatory region of the c-myc gene is associated with the downregulation of this gene during differentiation.