The nuclear enzyme DNA topoisomerase II, involving in DNA replication and transcription, mediates DNA scission in cells exposed to certain classes of intercalating agents, and is trapped as a covalent protein-DNA complex. In a previous study, we have found that benzo(a)pyrene(BP) and it's metabolites specifically bind to the DNA topoisomerase II fraction of mouse fibroblast C3H/10Tl/2 cell cultures. The enzyme was predicted to be a primary target of BP, causing malignancy. We therefore investigated in the present study changes in DNA cleavage by various concentrations of BP bound to DNA topoisomerase II in the human c-myc protooncogene. At the same time, the effects of m-AMSA(amsacrine), ellipticine and doxorubicin were also investigated. DNA topoisomerase II was purified by glycerol gradient centrifugation from mouse leukemia L1210 cells which easily form pro tein-DNA cross linkage. In the experiments of agarose gel electrophoresis which analyzed genomic changes in the human c-myc DNA, the pBR322 DNA was completely reversed from a supercoiled to a relaxed form in the presence of 160ng of purified DNA topoisamerase II. In BP-treated Hind III/Xba I cutting c-myc DNA, DNA cleavage was most apparently increased at 1.7 kb site, the effect being particularly pronounced with 0.1 ￥i M BP. Genomic changes by drug-induced DNA topoisomerase II in the labelled with P32 to restricted c-myc DNA were enhanced predominantly in the cleavage sites of P2 promoter region with m-AMSA, ellipticine and doxorubicin, and in the upstream of exon 1 with BP. In in vivo experiments, in which the effect of BP exposure of in human lymphoblast NC- 37 cells was tested, there was no change in c-myc RNA expression by 0.25 ￥ig/ml of BP. However, the c-myc RNA expression increased significantly with BP of 2.5 ￥ig/mL A simi lar increase in RNA expression was observed in Burkitt lymphoma cells. These resu1ts indicate that an increase of DNA cleavage or increased cleavage at specific sites induce gene expression of abnormal pattern.
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