UC Riverside

Topoisomerase II (topo II) is an essential nuclear enzyme that plays a role in maintaining DNA topology during several cellular processes such as DNA replication, transcription, DNA repair, and mitosis. While topo II inhibitors are commonly used in chemotherapy, their use is limited by their causal association with therapy-related acute myeloid leukemias. Similarly, several environmental toxicants, including flavonoids, have been reported to inhibit topo II in cell free assays. Topo II poisons, such as etoposide, act to stabilize the cleavage complex and inhibit the religation step, an important step which when inhibited leads to the formation of unprotected double stranded breaks. Catalytic inhibitors affect other parts of the topo II catalytic cycle and can act either prior to the cleavage step or after religation of the DNA strand break. The focus of this dissertation research was to more thorough investigate the types of genotoxic effects caused by topo II poisons, catalytic inhibitors, as well as flavonoids fisetin and genistein.

Fisetin, a flavonoid, has previously been reported to inhibit both topo II as well as Aurora kinases. In a comparative study, between fisetin and two Aurora kinase inhibitors, VX-680 and ZM-447439, all three compounds were effective aneuploidy and polyploidy-inducing agents in vitro. While the Aurora kinase inhibitors also resulted in increases in chromosome breakage, treatment with fisetin at low-cytotoxic concentrations resulted almost exclusively in chromosome loss, highlighting a key difference between these agents. Topo II inhibitors, aclarubicin, merbarone, etoposide, mitoxantrone, ICRF-154, and ICRF-87, all induced concentration-dependent increases in micronuclei, primarily through chromosome breakage. These results indicated that stabilization of the cleavage complex might not be necessary for chromosome breakage. Because of this, we characterized and quantified stabilized topo II cleavage complexes (SCCs) in intact cells. While topo II poisons are known to cause chromosome breakage by persistent stabilization of the enzyme-DNA cleavage complex, treatment of cells with post-religation catalytic inhibitors ICRF-154 and ICRF-187 also led to increases in cells with SCCs. In contrast, treatment with either aclarubicin or merbarone led to no increases in cells with SCCs. With regards to flavonoids of possible concern, cells treated with genistein, but not fisetin, led to increases in cells with SCCs.