Protein-linked DNA Strand Breaks Induced in Mammalian Cells by Camptothecin, an Inhibitor of Topoisomerase I

Abstract

Camptothecin was recently identified as an inhibitor of mammalian topoisomerase I. Similar to inhibitors of topoisomerase II, camptothecin produces DNA single-strand breaks (SSB) and DNA-protein cross-links (DPC) in mammalian cells. However, their one-to-one association, expected for trapped topoisomerase complexes, has not previously been demonstrated. We have studied camptothecin-induced SSB and DPC in Chinese hamster DC3F cells and their isolated nuclei, using the DNA alkaline elution technique. It was found that the SSB and DPC frequencies detected following camptothecin treatment depend upon the conditions used for lysis. When lysis was with sodium dodecyl sulfate, the observed frequencies of SSB and DPC were 2- to 3-fold greater than when sodium dodecyl sarkosinate (Sarkosyl) was used. In either case, the SSB:DPC ratio was close to 1. All of the camptothecin-induced SSB were protein linked, as indicated by the absence of DNA elution under nondeproteinizing conditions. DNA cleavage assays with purified topoisomerase I also indicated that the weaker Sarkosyl detergent fails to trap all of the enzyme-DNA complexes. In contrast, lysis conditions had little effect on levels of SSB or DPC produced by 4′-(9-acridinylamino)-methanesulfon-m-anisidide, suggesting that trapping of topoisomerase II complexes occurs equally well with either detergent. In experiments using isolated nuclei, it was found that the camptothecin-induced SSB, in contrast to trapped topoisomerase II complexes, can form and reverse within minutes at 4°C. The activity of camptothecin at low temperature was also seen with purified topoisomerase I. These results support the hypothesis that the SSB and DPC induced by camptothecin in mammalian cells are due to an action on topoisomerase I.