DNA signals for G2 checkpoint response in diploid human fibroblasts.
DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included gamma-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4-12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.
Department of Pathology and Laboratory Medicine, Curriculum in Toxicology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599-7295, USA.
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Kaufmann
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Kies
D002138:Calibration; D002166:Camptothecin / Q000494:pharmacology; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods; D019457:Chromosome Breakage; D004247:DNA / Q000502:physiology*; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004171:Diploidy; D005347:Fibroblasts / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D016195:G2 Phase / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D006801:Humans; D008938:Mitosis / Q000187:drug effects
D004247:DNA; D002166:Camptothecin