Anti-BrdUrd labeling of newly synthesized DNA in HL-60 cells triggered to apoptosis.

Apoptosis is an active process that takes place dur- ing pre- and postnatal life. It can be viewed as the essential counterpart to cell proliferation, both phe- nomena being aimed at the maintenance of tissue and organ homeostasis. Because apoptosis often takes place during the S phase of the cell cycle, we describe the spatial and temporal correlation be- tween DNA synthesis and DNA cleavage taking place in the same nucleus at the same time as a result of the action of camptothecin on proliferating HL-60 cells in vitro. The relationship between DNA syn- thesis and DNA fragmentation was studied at the single-cell level by bromodeoxyuridine (BrdUrd) incorporation revealed by flow cytometry, electron microscopy, and confocal microscopy. Most HL-60 cells are triggered to apoptosis during the first hour of treatment with camptothecin, and only cells in early-middle S phase are sensitive to the drug effect, whereas late S phase cells appear insensitive to camptothecin-induced apoptosis. Our data, there- fore, reinforce the hypothesis of a DNA strand break threshold that may exist in the cell, beyond which the apoptotic program is activated. Moreover, DNA synthesis activity in the nucleus committed to apop- tosis is gradually downregulated; after 6 h of camp- tothecin treatment, virtually no residual DNA repli- cation activity can be detected in micronuclei. DNA repair does not appear to be involved in bromode- oxyuridjne incorporation during the apoptotic pro- CeSS.