Targeted si-RNA mediated down-regulation of topoisomerase II α or β isozymes defines selectivity to inhibitors of the enzyme

2402 The type I and type II topoisomerases are essential enzymes involved in altering DNA topology for the processing of genetic material. In humans the type II enzymes consist of two highly homologous isozymes, topoisomerase (topo) IIα and topo IIβ of molecular weight 170 and 180 kDa, respectively. The interaction of these enzymes with DNA, make them important targets for cancer therapeutic agents. In vitro the topo IIα and β isoforms catalyze similar enzymatic reactions and do not differ in their ability to form a stable DNA cleavable complex with topo II targeting drugs. However, tumor models expressing different levels of topo IIα or topo IIβ following selection for resistance to specific topo II inhibitors are reported to differ in their response to clinically active topo II targeting drugs. In this study we used an isogenic model system, in which the topo IIα or the topo IIβ proteins were selectively down-regulated by stable transfection of the respective siRNAs in human leukemia HL-60 cells, to determine whether different topo II inhibitors target distinct topo II isoforms. The siRNA targeting topo IIα (sitopo IIα ) and topo IIβ (sitopo IIβ ) cloned into a pBabe vector led to a >20-fold and >30-fold stable down-regulation of the respective protein in HL-60 cells. A control transfection with green fluorescent protein (GFP) siRNA (siGFP) was also carried out. Comparison of drug-stabilized topo II-DNA cleavable complex (DNA-CC) formation in the siRNA transfected cells revealed that etoposide and amsacrine induced significantly (p=0.05) lower DNA-CC in cells transfected with sitopo IIα and sitopo IIβ , respectively as compared to those transfected with siGFP. In contrast, DNA-CC with camptothecin, a topo I-targeting drug, did not differ in these cells (p=0.30). Determination of drug induced cytotoxicity, measured by the soft-agar colony assay, revealed that sensitivity to etoposide was significantly (p=0.002) lower only in sitopo IIα transfected cells as compared to sitopo IIβ and siGFP transfected cells. In contrast, sensitivity to amsacrine and mitoxantrone, but not doxorubicin, was significantly (p=0.001) lower in sitopo IIβ transfected cells as compared to sitopo IIα and siGFP transfected cells. Induction of apoptosis by mitoxantrone was also attenuated only in sitopo IIβ transfected cells. These results suggest that the DNA damaging and down-stream cytotoxic effects of etoposide are dependent on adequate expression of topo IIα protein levels. In contrast, the anti-tumor effects of amsacrine and mitoxantrone are dependent on adequate expression of topo IIβ . Model systems with targeted down-regulation of topo II isoforms could be useful for testing novel topo II inhibitors. Further, profiling of patient tumors for differential expression of the topo II isozymes, α and β , could be a useful strategy to improve anti-tumor activity of clinically active topo II targeting drugs.