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

Camptothecin (CPT) has been 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 microM CPT or 10 microM 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.

Camptothecin

DNA topoisomerase I

phosphate‐buffered saline

7‐ethyl‐10‐ hydroxycamptothecin

7‐ethyl‐10–[4–(l‐piperidino)‐l‐ plperidino carbonyloxycamptothecin

50 mM Tris‐HC1 (pH 7.5)‐25 mM KC1‐5 mM MgCl2

50 mM Tris‐HCl (pH 7.5)‐25 mM KC1‐1 mM EDTA

dithiothreitol

phenylmethylsulfonyl fluoride

sodium dodecyl sulfate