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Here we describe methods to prepare a mammalian expression plasmid encoding EGFP fused to the amino terminus of human DNA topoisomerase IIα (TopoIIα). This plasmid is transfected into LLC-Pk cells, a porcine epithelial cell line that remains relatively flat during mitosis. After selection for stable integration, cells are cloned by serial dilution in microwells and used to grow a stable cell line expressing EGFP-TopoIIα. This line is termed LPk-GT2. Using photobleaching methods with conventional and patterned photobleaching LPk-GT2 cells are used demonstrate the rapid dynamics of TopoIIα exchange in both interphase nuclei and mitotic chromosomes. These rapid dynamics are dependent on enzyme activity since ICRF159 a catalytic inhibitor of TopoIIα, slows dynamics significantly.
DNA topoisomerase IIα (TopoIIα) is expressed primarily in actively dividing cells and shows dynamic changes at various stages of the cell cycle. In proliferating cells, both protein and message levels are low during the G1 phase and begin to rise during S phase, with a peak of protein expression during G2 and M phases 1–3. After mitosis TopoIIα reconcentrates in the nuclei, and subsequently during G1 message levels decrease and the protein is degraded by the proteasome 1–5. In interphase cells, antibody labeling and expression of tagged forms of the protein show that the enzyme is nuclear with a high concentration in the nucleolus 5–7. As the chromosomes condense during early mitosis, TopoIIα concentrates at centromeres and along the axes of the chromosome arms 7–10.
This pattern of association with mitotic chromosomes and the persistence of TopoIIα in salt- and DNAse-treated chromosome fractions led to the idea that TopoIIα might form a stable mitotic chromosome scaffold 11. To test this notion we and others created cell lines that express TopoIIα fused to fluorescent proteins that were then used for photobleaching analyses 5, 6, 12. Surprisingly, TopoIIα showed rapid turnover in photobleaching experiments in both interphase nuclei and in mitotic chromosomes. When inhibitors of topoisomerase II catalytic activity were applied to cells TopoIIα became stably anchored on chromatin in both interphase and mitotic cells 10. These observations led to the conclusion that TopoIIα exhibits rapid exchange on chromatin that is dependent on enzyme activity. Further studies have implicated TopoIIα in a host of important roles for chromatin regulation in interphase and mitosis 13–17. The dynamic exchange of TopoIIα on chromatin is likely to be a key factor in these functions. Finally several chemotherapeutic agents function through the activity of TopoIIα 18, 19.
Overexpression of TopoIIα in mammalian cells is toxic and induces apoptosis 20, 21. For this reason, establishment of cell lines stably expressing TopoIIα tagged with fluorescent proteins is dependent on spontaneous down regulation of the endogenous TopoIIα or by purposefully targeting endogenous gene expression 5, 12. Keeping in mind this caveat, the techniques described here will facilitate analysis of TopoIIα dynamics in living cells under various experimental conditions.
Support was provided by the National Institutes of General Medical Sciences, from the Oklahoma Medical Research Foundation and from the McCasland Foundation. We thank Dr. Penny Tavormina for helpful suggestions.