Anti-BrdUrd labeling of newly synthesized DNA in HL-60 cells triggered to apoptosis.
Apoptosis is an active process that takes place during pre- and postnatal life. It can be viewed as the essential counterpart to cell proliferation, both phenomena 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 between 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 synthesis 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, therefore, 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 apoptosis is gradually downregulated; after 6 h of camptothecin treatment, virtually no residual DNA replication activity can be detected in micronuclei. DNA repair does not appear to be involved in bromode-oxyuridine incorporation during the apoptotic process.
Zamai
L
L
Istituto di Anatomia Umana Normale, Università di Bologna, Italy.
Falcieri
E
E
Gobbi
P
P
Santi
S
S
Falconi
M
M
Marhefka
G
G
Vitale
M
M
D000911:Antibodies, Monoclonal / Q000276:immunology; D016590:Aphidicolin / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology*; D001973:Bromodeoxyuridine / Q000276:immunology / Q000493:pharmacokinetics*; D002166:Camptothecin / Q000494:pharmacology*; D004273:DNA, Neoplasm / Q000096:biosynthesis* / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D013997:Time Factors; D059004:Topoisomerase I Inhibitors
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D059004:Topoisomerase I Inhibitors; D016590:Aphidicolin; D001973:Bromodeoxyuridine; D002166:Camptothecin