4037

Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematological malignancies. They are metabolized to their active triphosphate form, which is incorporated into DNA during replication or excision repair synthesis. Since DNA polymerases have a greatly decreased affinity for primers terminated by nucleoside analogues, drug incorporation likely leads to stalling of replication forks. Excision of these analogues may lead to drug resistance, resulting in clinical relapse. The Mre11-Rad50-Nbs1 (MRN) complex has both exonuclease and endonuclease DNA repair capacities, but has not been studied for its capabilities in repairing nucleoside analogue-induced DNA damage. Cellular responses to stalled replication forks and double-strand breaks are marked by H2AX phosphorylation on serine 139 (γ-H2AX), which forms nuclear foci at DNA damage sites. Ataxia-telangiectasia mutated (ATM) is autophosphorylated on serine 1981 and has been identified as a sensor molecule that is a key signaling kinase for initiating cell cycle arrest, DNA repair, and apoptosis in response to such damage. Here, we demonstrate that the MRN complex and phosphorylated ATM co-localize with γ-H2AX when replication forks are stalled by exposure to nucleoside analogues. DNA synthesis was inhibited in human acute myelogenous leukemia cells (OCI-AML3) by 90% within 2 h and was sustained for 4 h in response to 0.1 μM gemcitabine, 0.5 μM ara-C, and 2 μM troxacitabine. This was associated with an accumulation of cells in S-phase within 24 h. Immunofluorescent imaging by confocal microscopy was used to determine if ATM and the MRN complex localized to sites of nucleoside analogue-induced stalled replication forks. Mre11, Rad50, Nbs1, and phosphorylated ATM each formed distinct nuclear foci that co-localized with γ-H2AX foci within 2 h after exposure to the same concentrations of gemcitabine, ara-C, and troxacitabine as described above. Similarly, the same molecules co-localized with γ-H2AX after exposure to 10 Gy ionizing radiation (IR). Double-strand breaks were not evident after gemcitabine, ara-C, or troxacitabine nucleoside treatment, but were present after IR exposure, as measured by pulsed-field gel electrophoresis and neutral comet assays. Thus, the MRN complex and phosphorylated ATM co-localize with nuclear γ-H2AX foci in the absence of double-strand breaks after exposure to nucleoside analogues. Such foci may identify the sites of stalled replication forks.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA