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 Ser(139) (gamma-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 gamma-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 gamma-H2AX-positive cells increased with concentrations of gemcitabine up to 0.1 micromol/L, and gamma-H2AX was most evident in the S-phase fraction. Phosphorylation of ataxia-telangiectasia mutated (ATM) on Ser(1981) 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.
Highlights
Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematologic malignancies [1]
This study identifies H2AX phosphorylation as a pharmacodynamic indicator of gemcitabine-induced stalled replication forks that correlates with cellular reproductive viability
To determine if there is an association between stalled replication forks, the S-phase population, and H2AX phosphorylation, the action of gemcitabine on the rate of DNA synthesis was investigated
Summary
Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematologic malignancies [1] On cellular entry, these nucleic acid antagonists are phosphorylated to a triphosphate form and incorporated into DNA [2, 3]. Incorporation causes steric hindrance of extending replication forks, leading to fork stalling, which induces S-phase checkpoint activation, a subsequent decrease in initiation of replication origins [4], and S-phase arrest [5, 6]. Whether H2AX is phosphorylated and forms nuclear foci at sites of nucleoside analogue – induced stalled replication forks has not been studied in detail [36]. The mechanisms by which this drug class induces cell killing have been well studied, the molecules that sense nucleoside analogue – induced stalled replication forks are unknown. This study identifies H2AX phosphorylation as a pharmacodynamic indicator of gemcitabine-induced stalled replication forks that correlates with cellular reproductive viability
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