Functions of human DNA topoisomerases in cel proliferation and effects of anticancer drungs and natural compounds on the type II enzymes

Human DNA topoisomerases are ubiquitous enzymes that remove topological constraints like superhelical tension, knots or tangles from the cellular DNA, and are thus essentially required for DNA metabolism. They are divided into type I and type II enzymes, which transiently cleave one or both strands of DNA, respectively. Although the strand breaks generated by these enzymes are transient in nature, they could be converted into permanent breaks when the topoisomerase catalytical cycle is inhibited. Several identified topoisomerase-inhibitory drugs are in wide clinical use as anticancer therapeutics since they represent some of the most successful drugs used for the treatment of human malignancies. On the other hand, the DNA-damaging and recombinogenic potential of topoisomerases is detrimental, since it eventually leads to the development of secondary leukemias by inducing translocations of the MLL locus in patients treated with regimens including topoisomerasedirected drugs. Yet, a source of environmental topoisomerase toxication could be the diverse group of bioflavonoids, which are not only an integral component of the human diet but are also probably the most abundant source of natural antioxidants. However, these polyphenols are also known to be potent topoisomerase inhibitors. Therefore, they are suspected to have profound genotoxic potential. It is now well established that DNA topoisomerases play essential roles in all DNA metabolic processes like replication, transcription, chromosome segregation and DNA repair. A number of questions, however, are still open such as to what extent these functions are partitioned between the major players topoisomerase I, IIα and IIβ. In addition, it is unclear, in which cell cycle phase topoisomerase-targeted anti-cancer drugs most efficiently exert their effect, and whether certain food components such as polyphenols, that were shown to inhibit topoisomerases in vitro, have indeed the same effect in a living cell, where e.g. limited permeation and cellular metabolism could have counteracting consequences. Thus, this work aimed at characterizing the activity and mechanism of action of bioflavonoids against topoisomerases in comparison to standard topoisomerase toxins, which have been widely applied in cancer therapy. Firstly, biomarkers were established to allow distinction between mitosis and interphase, as well as between sub-stages of interphase in a given cell. For this purpose, proliferating cell nuclear antigen (PCNA) and the replicationinitiating factor Cdc6 were chosen as biomarkers and tagged with a fluorescent protein CFP or YFP, respectively. These proteins were stably expressed in two different human cell lines.Co-expression of these markers in a cell yielded a number of unexpected, so far unknown features of Cdc6 in mitosis, and, thus provided new insights into the still discussed regulation of Cdc6 during the initiation of replication. Co-expression of PCNA or Cdc6 with topoisomerases I, IIα and IIβ, respectively, as differently coloured bio-fluorescent proteins allowed the detailed microscopic analysis of the cell cycle-specific behaviour of the topoisomerases and their response to drug-inhibition of their catalytic cycle. Observed effects could then be correlated to in vitro-effects by means of conventional relaxation and cleavage assays.