In the post-genomic era, the structure of the cell nucleus has become the key to understanding the most important cellular processes such as cell
differentiation, apoptosis or transformation. In order to investigate highly organized nuclear structure, methods of high-resolution cytometry developed in our laboratory in combination with techniques that enable visualization of nuclear structures in living cells will be used. DNA constructs coding fusion proteins will be employed to study DNA-protein interaction in vivo. As a model system proteins related to acute
promyelocytic leukemia will be used. Nuclear localization of PML, RARa, PML/RARa tagged to GFP, RFP and YFP will be investigated in PR-9 cells in which the PML/RARa protein can be induced by increased concentration of Zn ions. Interaction with DNA will be studied using subsequent 3D FISH in cells previously investigated in vivo. For both in vivo studies and FISH the technology of high-resolution cytometry will be used. Automated image acquisition, on-line analysis, storage and evaluation of the arrays of parameters in computer memory will be used. The main objective of the project will be the introduction of the methodology of GFP techniques in our laboratory and its combination with high-resolution cytometry. Substantially deeper understanding of the functional topography of proteins related to APL is expected. The long-term practical goal of the project will be the use of experimental results and technology developed in our laboratories in diagnostics and therapy of cancer.

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