THE ANTILEUKEMIC ALKALOID FAGARONINE IS AN INHIBITOR OF DNA TOPOISOMERASE-I AND TOPOISOMERASE-II

The antileukemic alkaloid, fagaronine, is a potent differentiation ind ucer of various hematopoietic cell lines. We show here that fagaronine is a DNA base-pair intercalator with a K(app) of 2.1 x 10(5) M-1 for calf thymus DNA. Fagaronine inhibits the catalytic activity of purifie d calf thymus topoisomerase I as shown by relaxation of supercoiled pl asmid DNA followed by electrophoresis in neutral as well as in chloroq uine-containing gels. The catalytic activity of topoisomerase I is inh ibited at concentrations above 30 muM. Fagaronine also inhibits the ca talytic activity of purified calf thymus topoisomerase II at concentra tions above 25 muM as shown by decatenation of kinetoplast DNA. Fagaro nine stabilizes the covalent DNA-enzyme reaction intermediate (the cle avable complex) between topoisomerase I and linear pBR322 DNA at conce ntrations up to 1 muM. Further increase of the fagaronine concentratio n leads to a progressive decrease in the cleavable complex formation, which is totally inhibited at 100 muM. In contrast, up to 1 muM fagaro nine has no effect on cleavable complex formation between purified cal f thymus topoisomerase II and linear pBR322 DNA, whereas cleavable com plex formation is inhibited at higher concentrations. Exposure to faga ronine results in an increase in DNA-protein complex formation in inta ct P388 murine leukemia cells. P388CPT5 cells, which have an altered t opoisomerase I activity, are 4-fold resistant to the growth inhibitory effects of fagaronine compared to the parental cell line. Similarly, DC-3F/9-OH-E Chinese hamster fibrosarcoma cells, which have an altered topoisomerase II activity, are about 5-fold resistant to the growth i nhibitory effects of fagaronine. We conclude that fagaronine is an inh ibitor of both DNA topoisomerase I and II and propose that this might play a role in the cytotoxic activity.