DNA damage and cell killing by camptothecin and its derivative in human leukemia HL-60 cells.

Abstract

Camptothecin (CPT) has heen recognized as a topoisomerase I (Topo I) inhibitor. However, the mechanism of cytotoxicity of this agent remains unknown. In the present study, we analyzed the kinetics of Topo I‐mediated DNA single‐strand breaks and internucleosomal DNA cleavage produced by CPT and its derivative, 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), in HL‐60 cells. DNA single‐ strand breaks were detected using alkaline sucrose gradient centrifugation when HL‐60 cells were incubated with 10 μM CPT or 10 μM SN‐38 for 30 min. These DNA single‐strand breaks were rapidly repaired after drug removal, while the cytotoxic action of these drugs was sustained. Treatment of HL‐60 cells with CPT or SN‐38 for 3 h produced extensive degradation of DNA. Agarose gel electrophoresis showed a ladder of DNA fragments consisted of multimers of approximately 200 base pairs, characteristic of apoptosis. Interestingly, this type of DNA fragmentation was also induced within 4 h after repair of DNA single‐strand breaks, and subsequently loss of cell viability was observed. When zinc ion, a potent inhibitor of endonuclease, was added to drug‐free medium after treatment with CPT or SN‐38, internucleosomal DNA cleavage was abolished. Furthermore, addition of zinc ion reduced the loss of cell viability. These data suggest that Topo I‐mediated DNA single‐strand breaks may be necessary but are not sufficient for cell death, and the endonuclease involved in induction of internucleosomal DNA cleavage may play an important role in HL‐60 cell death induced by Topo I inhibitor.