PO-093 A hep-2 cell model as tool for analysing DNA double strand breaks in cancer patients

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IntroductionDNA double strand breaks (DSB) are one of the most severe DNA damages and as thus can lead to genomic instability favouring cancer. The number of phosphorylated histone variant H2AX (γH2AX) correlates with the number of DSB and can be quantitatively detected by immunofluorescence imaging. In particular, we are interested on the individual response and adverse effects of DSB–inducing chemotherapeutic drugs such as etoposide in the context of the chemoresistance–inducing and tumour–associated antigen dense fine speckled protein of 70 kDa (DFS–70).Material and methodsHEp-2 wild type and CRISRP/Cas9-generated DFS-70 knock-out (DFS-70 KO) cells were treated with the topoisomerase inhibitor etoposide (0–10 µM) to induce DSB. The AKLIDES NUK system (Medipan GmbH, Dahlewitz, Germany), a fully automated and standardised immunofluorescence imaging and data processing platform, was used to measure the number of γH2AX foci and nucleus size. Additionally, immunoblotting was performed to analyse yH2AX.Results and discussionsBoth the protein analysis by Western blotting and Immunofluorescence analysis by AKLIDES NUK system showed an elevated formation of γH2AX with increased etoposide concentration. HEp-2 wild type cells indicated a significant induction (p<0.01) of DSB up to 13 foci per cell after 16 hours of etoposide treatment. Adverse effects could be observed on nucleus size that increased significantly upon treatment in all cell lines. No difference in the basal level of γH2AX foci were detectable comparing wild type and DSF-70 KO cells. In contrast, two out of five DFS-70 KO clones exhibited an increased foci number in comparison to the wild type control after 16 hours of etoposide treatment. This finding might indicate that DSF-70 KO sublines are more sensitive to topoisomerase inhibition than HEp–2 wild type cells.ConclusionThe automated multiparameter imaging platform AKLIDES allows an individual and differential detection of γH2AX foci formation and nuclei size in human laryngeal carcinoma cells, HEp–2 and sublines. Therefore, this system can be useful to identify cancer progression e.g. analysing peripheral blood mononuclear cells of patients with malignant lymphoma. Moreover, additional biomarkers defining cancer progression can be integrated into the analysis.