Abstract 365: DNA double-strand break imaging with anti-γH2AX antibodies does not alter DNA repair kinetics

Abstract Introduction: DNA double-strand breaks (dsb) are caused by exposure to radiotherapy and various anticancer drugs and are very deleterious. Upon dsb formation, histone H2AX is phosphorylated to form γH2AX foci at the break site. We have reported the development of a non-invasive in vivo DNA dsb imaging agent, based on anti-γH2AX IgG, modified by addition of the cell penetrating and nuclear localizing peptide, TAT. It was demonstrated that fluorescent and radiolabelled anti-γH2AX-TAT was internalized into cancer cells, and retained specifically in cells expressing γH2AX foci, through binding to γH2AX. Using in vivo fluorescence and SPECT imaging, we showed tumour uptake correlates with the extent of DNA dsb damage after radiation therapy (Cornelissen et al. Cancer Res 2011 71:4539). Ideally, imaging γH2AX in this way should not alter the physiology/ biology of the imaged organism. Therefore, we investigated the influence of anti-γH2AX-Tat on DNA dsb repair kinetics using comet assays and γH2AX and 53BP1 foci kinetics. These data were applied using a published mathematical model of non-homologous end joining (NHEJ) by Cucinotta et al. (Rad Res 2008 169, 214-222). Methods: A panel of four carcinoma cells (MCF7, MDA-MB-231/H2N, MDA-MB-468, MCF7) was exposed to a range of concentrations of anti-γH2AX-Tat (0 - 0.5 μg/mL) and irradiated 1 h later (IR; 137Cs, 0.95 Gy/min, 0-4 Gy). At selected time points, cells were analysed using γH2AX and 53BP1 foci staining and counting and comet assays. Foci counting was performed automatically using an InCell analyser. Comet assay results were analysed using software developed in house and expressed as Olive Tail Moment. γH2AX foci kinetics data were modelled in silico using a concatenary model or an extended version of the NHEJ model, allowing for anti-γH2AX-Tat binding to γH2AX. The kinetics of dsb formation and resolution were calculated from the model (dsbcal), and validated using comet assay data. Results: The number of γH2AX foci/cell was maximal at 0.5-1 h post IR. The affinity (KD) of anti-γH2AX-Tat for γH2AX was 25 nM. There was a trend towards a dose-dependent increase of the maximum number of foci. The concatenary model revealed no significant differences between treated and non-treated cells. However, using the NHEJ model, a dose-dependent decrease in the rate of phosphorylation of γH2AX was demonstrated (in MCF7 cells exposed to 0-0.5 μg/mL anti-γH2AX-Tat, κPγ = 2507-1462 copy−1 h−1; p<0.01), as well as a lower affinity of DNA-PKcs for H2AX (κM = 0.03 - 0.10 in MCF7 cells; p<0.01). Conversely, there was no significant effect on the kinetics of the calculated number of dsbs (p > 0.05). This was confirmed by comet assays. Conclusion: Anti-γH2AX-TAT binding to γH2AX reduces the rate of formation of new γH2AX foci, but has no significant influence on DNA dsb repair. Therefore, the use of γH2AX-Tat for imaging DNA dsbs does not alter DNA dsb status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 365. doi:1538-7445.AM2012-365