The promyelocytic leukemia (PML) nuclear body (NB) is a dynamic subnuclear compartment that is implicated in tumor suppression, as well as in the transcription, replication, and repair of DNA. PML NB number can change during the cell cycle, increasing in S phase and in response to cellular stress, including DNA damage. Although topological changes in chromatin after DNA damage may affect the integrity of PML NBs, the molecular or structural basis for an increase in PML NB number has not been elucidated. We demonstrate that after DNA double-strand break induction, the increase in PML NB number is based on a biophysical process, as well as ongoing cell cycle progression and DNA repair. PML NBs increase in number by a supramolecular fission mechanism similar to that observed in S-phase cells, and which is delayed or inhibited by the loss of function of NBS1, ATM, Chk2, and ATR kinase. Therefore, an increase in PML NB number is an intrinsic element of the cellular response to DNA damage.
Hypoxia exists in all solid tumors and leads to clinical radioresistance and adverse prognosis. We hypothesized that hypoxia and cellular localization of gold nanoparticles (AuNPs) could be modifiers of AuNP-mediated radiosensitization. The possible mechanistic effect of AuNPs on cell cycle distribution and DNA double-strand break (DSB) repair postirradiation were also studied. Clonogenic survival data revealed that internalized and extracellular AuNPs at 0.5 mg/mL resulted in dose enhancement factors of 1.39 ± 0.07 and 1.09 ± 0.01, respectively. Radiosensitization by AuNPs was greatest in cells under oxia, followed by chronic and then acute hypoxia. The presence of AuNPs inhibited postirradiation DNA DSB repair, but did not lead to cell cycle synchronization. The relative radiosensitivity of chronic hypoxic cells is attributed to defective DSB repair (homologous recombination) due to decreased (RAD51)-associated protein expression. Our results support the need for further study of AuNPs for clinical development in cancer therapy since their efficacy is not limited in chronic hypoxic cells.
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
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