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H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation

2007; American Association for Cancer Research; Volume: 6; Issue: 4 Linguagem: Inglês

10.1158/1535-7163.mct-06-0633

1538-8514

Brett Ewald, Deepa Sampath, William Plunkett,

Carcinogens and Genotoxicity Assessment

Abstract Gemcitabine is a nucleoside analogue that is incorporated into replicating DNA, resulting in partial chain termination and stalling of replication forks. The histone variant H2AX is phosphorylated on Ser139 (γ-H2AX) and forms nuclear foci at sites of DNA damage. Here, we characterize the concentration- and time-dependent phosphorylation of H2AX in response to gemcitabine-induced stalled replication forks. The number of γ-H2AX foci increased with time up to 2 to 6 h after exposure to gemcitabine, whereas longer exposures did not cause greater phosphorylation or increase cell death. The percentage of γ-H2AX–positive cells increased with concentrations of gemcitabine up to 0.1 μmol/L, and γ-H2AX was most evident in the S-phase fraction. Phosphorylation of ataxia-telangiectasia mutated (ATM) on Ser1981 was also associated with S-phase cells and colocalized in the nucleus with phosphorylated H2AX foci after gemcitabine exposure. Chemical inhibition of ATM, ATM- and Rad3-related, and DNA-dependent protein kinase blocked H2AX phosphorylation. H2AX and ATM phosphorylation were associated with inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1/Cdc25A/cyclin-dependent kinase 2). Exposure of previously gemcitabine-treated cultures to the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01) caused a 10-fold increase in H2AX phosphorylation, which was displayed as an even pan-nuclear staining. This increased phosphorylation was not due to apoptosis-induced DNA fragmentation and was associated with the S-phase fraction and decreased reproductive viability. Thus, H2AX becomes phosphorylated and forms nuclear foci in response to gemcitabine-induced stalled replication forks, and this is greatly increased upon checkpoint abrogation. [Mol Cancer Ther 2007;6(4):1239–48]