Abstract 3916: PML nuclear bodies are juxtaposed to DNA-DSBs following IR-induced DNA damage

Abstract

Abstract The promyelocytic leukemia (PML) tumor suppressor protein aggregates in discrete PML nuclear bodies (PML-NBs) that are detectable by immunofluorescent microscopy. PML-NB number can change in response to cell cycle and various cell stresses, including DNA damage. Intra-nuclear γH2AX foci form following ionizing radiation (IR) as a function of dose and time and are used as an indicator of DNA double strand breaks (DNA-DSBs). However, whether PML-NBs are associated with exogenous or endogenous (e.g. DNA replication associated) damage was unclear. Using G0-G1 synchronized human fibroblast (GM05757) to exclude endogenous DNA damage, we examined the intra-nuclear locales of γH2AX and other DNA repair biomarkers with respect to PML-NBs following IR. PML +/+ and -/- MEFs did not vary in DNA-DSB repair based on comet assay. Consistent with this, PML-NBs did not respond to immediate DNA damage. However, 3D-confocal microscopy studies showed that PML-NBs associated with the majority of residual γH2AX sites at later times (24h post-IR). Using quantitative microscopy, these associations were distinguishable as juxtaposition and not true co-localization (i.e., between γH2AX and DSB marker 53BP1). Continued presence of damage sensing and signaling proteins (MRE11, NBS1, MDC1 and 53BP1) and persistent kinase activity (DNA-PK, ATM, CHK2) was observed at these residual γH2AX sites. Absence of RAD51 and BRCA1 indicated that these were not replication-associated breaks and the exclusion of TRF2 suggested that these were not telomeric regions; rather PML-NBs associated specifically with unrepaired exogenous DSBs. Residual γH2AX sites were not enriched in eu- or heterochromatin. Interestingly, scanning electron imaging revealed a lower chromatin density near residual γH2AX foci, suggesting chromatin remodeling in the vicinity of unrepaired breaks. Live cell experiment using GFP-PML IV and mCherry-53BP1tudor domain suggested that PML-NB mobility facilitated the association with residual 53BP1 foci. Our data suggests that maintenance of genomic stability may depend, in part, on the association between PML-NBs and residual γH2AX foci. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3916. doi:10.1158/1538-7445.AM2011-3916