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Poisoning of Topoisomerase I by an Antitumor Indolocarbazole Drug

Stabilization of Topoisomerase I-DNA Covalent Complexes and Specific Inhibition of the Protein Kinase Activity¹

Institut de Génétique Moléculaire, Université de Montpellier II, 34293 Montpellier cedex [E. L., J. T.]; Rhône-Poulenc Rorer, Centre de Recherche de Vitry-Alfortville, 94403 Vitry sur Seine [J-F. R.]; Synthèse Electrosynthèse et Etude de Systèmes à Intènêt Biologique, Université Blaise Pascal, 63177 Aubière cedex [M. P.]; and Laboratoire de Pharmacologie Moléculaire Antitumorale du Centre Oscar Lambret et U-124 INSERM, 59045 Lille cedex [C. C., C. B.], France

We have investigated the mechanism of topoisomerase I inhibition by an indolocarbazole derivative, R-3. The compound is cytotoxic to P388 leukemia cells, but not to P388CPT5 camptothecin-resistant cells having a deficient topoisomerase I. R-3 can behave both as a specific topoisomerase I inhibitor trapping the cleavable complexes and as a nonspecific inhibitor of a DNA-processing enzyme acting via DNA binding. In addition, the drug is a potent inhibitor of the kinase activity of topoisomerase I. Unlike camptothecin, R-3 completely inhibits the phosphorylation of SF2/ASF, a member of the SR protein family, in the absence of DNA. The inhibitory effect is also observed using mutant enzyme Y723F that lacks DNA cleavage/religation activity but does not affect phosphotransferase activity, indicating, therefore, that R-3 acts independently at both DNA cleavage and protein kinase sites. R-3 is the only compound known thus far that interferes specifically with the kinase activity of topoisomerase I and not with other kinases, such as protein kinase C and the cdc2 kinase. The study reinforces the view that topoisomerase I is a dual enzyme with a DNA cleavage site juxtaposed to a functionally independent kinase site and shows for the first time that indolocarbazole drugs can inhibit both the DNA cleavage/religation and kinase activities of the enzyme.

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