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ATM and the Mre11-Rad50-Nbs1 Complex Respond to Nucleoside Analogue–Induced Stalled Replication Forks and Contribute to Drug Resistance

Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center and The University of Texas Health Science Center at Houston 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.

Key Words: stalled replication forks • DNA damage response • gemcitabine • ara-C • troxacitabine

The Mre11-Rad50-Nbs1 complex and autophosphorylated Ser¹⁹⁸¹-ATM are involved in recognizing and repairing DNA damage, such as double-strand breaks (DSB). However, the role of these factors in response to stalled replication forks is not clear. Nucleoside analogues are agents that are incorporated into DNA during replication, which cause stalling of replication forks. The molecular mechanisms that sense these events may signal for DNA repair and contribute to survival but are poorly understood. Cellular responses to both DSBs and stalled replication forks are marked by H2AX phosphorylation on Ser¹³⁹ (-H2AX), which forms nuclear foci at sites of DNA damage. Here, concentrations of the nucleoside analogues 1-β-D-arabinofuranosylcytosine (cytarabine; ara-C), gemcitabine, and troxacitabine, which inhibited DNA synthesis by 90% within 2 hours, were determined for each agent. Using -H2AX as a marker for changes in chromatin structure, we show that Mre11, Rad50, Nbs1, and phosphorylated ATM respond to nucleoside analogue–induced stalled replication forks by forming nuclear foci that colocalize with -H2AX within 2 hours. Because neither DSBs nor single-strand breaks were detectable after nucleoside analogue exposure, we conclude that this molecular response is not due to the presence of DNA breaks. Deficiencies in ATM, Mre11, or Rad50 led to a 2- to 5-fold increase in clonogenic sensitization to gemcitabine, whereas Nbs1 and H2AX deficiency did not affect reproductive growth. Taken together, these results suggest that ATM, Mre11, and Rad50 are required for survival after replication fork stalling, whereas Nbs1 and H2AX are inconsequential. [Cancer Res 2008;68(19):7947–55]