The polyphenolic ellagitannin vescalagin acts as a preferential catalytic inhibitor of the α isoform of human DNA topoisomerase II.

Polyphenolic ellagitannins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models by a mechanism that remains unclear. Potential targets have been proposed, including DNA topoisomerases II (Top2). Top2α and Top2ß, the two isoforms of the human Top2, play a crucial role in the regulation of replication, transcription, and chromosome segregation. They are the target of anticancer agents used in the clinic such as anthracyclines (e.g., doxorubicin) or the epipodophyllotoxin etoposide. It was recently shown that the antitumor activity of etoposide was due primarily to the inhibition of Top2α, whereas inhibition of Top2ß was responsible for the development of secondary malignancies, pointing to the need for more selective Top2α inhibitors. Here, we show that the polyphenolic ellagitannin vescalagin preferentially inhibits the decatenation activity of Top2α in vitro, by a redox-independent mechanism. In CEM cells, we also show that transient small interfering RNA-mediated down-regulation of Top2α but not of Top2ß conferred a resistance to vescalagin, indicating that the α isoform is a preferential target. We further confirmed that Top2α inhibition was due to a catalytic inhibition of the enzyme because it did not induce DNA double-strand breaks in CEM-treated cells but prevented the formation of Top2α- rather than Top2ß-DNA covalent complexes induced by etoposide. To our knowledge, vescalagin is the first example of a catalytic inhibitor for which cytotoxicity is due, at least in part, to the preferential inhibition of Top2α.

Recombinant differential anchorage probes that tower over the spatial dimension of intracellular signals for high content screening and analysis.

Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.