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Research Articles: Therapeutics, Targets, and Development

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

Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas and The University of Texas Graduate School of Biomedical Sciences, Houston, Texas

Requests for reprints: William Plunkett, Department of Experimental Therapeutics, Unit 71, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3335; Fax: 713-794-4316. E-mail: wplunket{at}mdanderson.org

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 (-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 -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 -H2AX–positive cells increased with concentrations of gemcitabine up to 0.1 µmol/L, and -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]

Grant support: Grants CA32839 and CA55164 and Cancer Center support grant CA16672 from the National Cancer Institute Department of Health and Human Services.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supplementary material for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

² B. Ewald, D. Sampath, and W. Plunkett. Colocalization of the Mre11-Rad50-Nbs1 complex, phosphorylated ATM, and -H2AX may identify sites of nucleoside analogue-induced stalled replication forks. AACR Meeting Abstracts 2007, Abstract #4037, unpublished.

Received 10/12/06; revised 1/26/07; accepted 2/21/07.

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