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© 1994 Oxford University Press

research-article

The effects of 5-fluoropyrimidines on nascent DNA synthesis in Chinese hamster ovary cells monitored by pH-step alkaline and neutral elution

Suzanne Jones, Elaine Willmore and Barbara W. Durkacz

Cancer Research Unit Medical School, University of Newcastle upon Tyne Newcastle upon Tyne NE2 4HH, UK

Nascent DNA (nDNA) replication Intennediates can be isolated and quantified by pH-step alkaline elution (L.C. Erikson et al., Chromosoma, 74, 125–139, 1979). The effects of 5-fluorouradil (FU) and 5-fluoro-2'-deoxyuridine (FdU) on the formation and persistence of nascent DNA structures were studied in Chinese hamster ovary K1 cells. Exogenous thymidine (dT) rescued FdU-lnduced, but had little effect on FU-induced, cytotoxicity, indicating DNA- and RNA directed cytotoxic mechanisms respectively. Drug-treated cells were pulse labelled with [3H]dT and the percentage of total counts eluting from and retained by filters estimated at successive pH Increments (11.0, 11.3, 11.5 and 12.1). A 1 min pulse of control cells resulted in >75% of the DNA eluting at pH 12.1 or being retained by the filter. By 1 h, >90% of the DNA was retained by the filter. FdU treatment resulted in the persistence of DNA in all four pH bands. More than 95% of total DNA synthesized during a 5 mm pulse eluted as discrete peaks at each pH. This DNA was processed to higher Mr species during a 1 h chase, but following a 24 h chase, 68% of the DNA still eluted at pH 12.1. In contrast, EU treatment caused only a transient accumulation (≤10 min) of the DNA (e.g. -26% at 5 mm) in the pH 11.0 and 11.3 bands only, indicating a selective effect of EU on the maturation of very short size classes of DNA. Neutral elution was used to assess the effect of FdU on double-strand break (dsb) formation in nDNA. Whereas no dsbs were evident in untreated cells, dsbs appeared In FdU-treated cells even following the shortest [3H]dT pulse times (1–10 min). Although these were processed into higher Mr species with longer pulse times, dsbs were still evident at 2 h.


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