ABSTRACTS â ORAL PRESENTATIONS - AMCA, spol. s r.o.

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two sophisticated systems: Metafer (Metasystems, Germany) and ImageStream (Amnis, USA). Metafer is automatized microscopic system that has recently been introduced for analyzing DNA repair foci. ImageStream is a combination of flow cytometer with fluorescent microscope. Thus, we compare ImageStream with Metafer in analyzing IRIF. We found that sensitivity of both systems to low-dose-induced γH2AX foci was very similar: ImageStream show statistical significant induction at the dose of 5cGy and Metafer at the dose of 10cGy while prior analysis using classical flow cytometry showed statistical significance at doses ≥ 50cGy. Analysis of 53BP1 foci by ImageStream was less sensitive then by Metafer. In conclusion, we show for the first time that analysis of low-dose-induced γH2AX foci by ImageStream system is very appropriate method, that reach sensitivity of automatized Metafer microscopic system and is more sensitive than measuring γH2AX fluorescence by classical flow cytometry. Thus, ImageStream is the system that has great potential for analyzing DNA repair foci. Acknowledgements This work was supported by the Slovak Research and Development Agency (APVV 0669- 10) and the VEGA Grant Agency (2/0150/11) of the Slovak Republic. References Bekker-Jensen, S., Lukas, C., Kitagawa, R., Melander, F., Kastan, M. B., Bartek, J. and Lukas, J.: Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks. - Journal of Cell Biology 173, 195-206, 2006. Belyaev, I. Y.: Radiation-induced DNA repair foci: Spatio-temporal aspects of formation, application for assessment of radiosensitivity and biological dosimetry. - Mutation Research-Reviews in Mutation Research 704, 132-141, 2010. Goodarzi, A. A. and Jeggo, P. A.: Irradiation induced foci (IRIF) as a biomarker for radiosensitivity. - Mutat Res 736, 39-47, 2012. Wakeford, R.: The risk of childhood leukaemia following exposure to ionising radiation--a review. - J Radiol Prot 33, 1-25, 2013. Legend to figure Typical data obtained by ImageStream after irradiation with the dose of 50cGy (transformed in black and white). There are 6 channels shown, from the left: brightfield, γH2AX stained with monoclonal mouse antibody and FITC., granularity, DNA stained with DAPI, 53BP1 stained with polyclonal rabbit antibody and AF647 and colocalization of γH2AX and 53BP1. Analytical Cytometry VII 49
25. PROFILING OF CHILDHOOD ACUTE LEUKAEMIA CELLS USING A NOVEL FLOW CYTOMETRY-BASED METHOD OF AFFINITY PROTEOMICS Daniela Černá 1 , Veronika Kanderová 1 , Jan Stuchlý 1 , Karel Fišer 1 , WeiWei Wu 2 , Anders Holm 2 , Ondřej Hrušák 1 , Fridtjof Lund-Johansen 2 , Tomáš Kalina 1 1 Pediatric Hematology and Oncology, Charles University Prague, 2 nd Medical Faculty, Praha 5, Czech Republic; cerna.daniela@lfmotol.cuni.cz 2 Dpt. of Haematology, Rikshospitalet, Oslo, 02770, Norway Acute leukaemia (AL) is the most common childhood malignancy driven by a number of aberrations at the DNA and mRNA level. Current research is mainly focused on the detection of mutations at the DNA level while functional consequences of these alterations on cellular level are not fully understood. Proteins are entities that form connection between gene expression and cell physiology, therefore more effective and sensitive approaches that could search for new prognostic markers on protein level are in development. Using SEC-MAP technology (Size-exclusion Chromatography - Microsphere-based Affinity Proteomics), we are searching for the expression and activation (e.g. phosphorylation) of differentially expressed proteins in AL cells regarding to cell type (B-cell, T-cell, myeloid), genotype (fusion genes, aneuploidy) and early response to treatment detected as minimal residual disease. SEC-MAP is a set of 1728 populations of fluorescently-labeled latex microbeads, each carrying an antibody against a human protein. We isolate the cellular proteins from membranes, cytoplasm and nuclei using detergents, label them with biotin and separate them using gel chromatography into 24 fractions. These fractions are incubated with SEC-MAP microbeads and the antibody-protein binding is detected using fluorescentlylabeled streptavidin by flow cytometry. We have examined the expression of cytoplasmic (n=980) and membrane (plus DNAbinding) (n=769) proteins in 69 diagnostic samples of AL. The analysis was performed using automatic software created in R-project. For the normalization of protein expression we have used Loess normalization commonly used in mRNA profiling studies. Due to ability of SEC-MAP to separate proteins according their molecular weight we have identified not only the expression of particular proteins but also the size that could serve as a control of proteolysis. We have detected proteolysis in 12 samples, which have been therefore excluded from the analysis. We have revealed the sensitivity to proteolysis of four standard house-keeping proteins (Akt, Abl, β-actin and β2-microglobulin). Abl and Akt have been cleaved while β-actin and β2-microglobulin have not been detected in their cleaved forms in the diagnostic AL samples. Therefore we have decided to use Abl and Akt as house-keeping proteins to reveal the proteolysis. So far we have identified 44 proteins (e.g. SH2D1A, FAS, LAT, KIT, CD72) differentially expressed in different subtypes of AL. We have discovered e.g. higher expression of cAMP-dependent protein kinase PRKACA in TEL-AML1 positive AL. Recently we are verifying SEC-MAP data using other proteomic approaches. SEC-MAP is a novel method of functional proteomics combining the capacity of DNA 50 Analytical Cytometry VII
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Acknowledgements This work was supp
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