Quantitative analysis of three-dimensional distribution of AgNOR proteins during interphase in leukemic cells

Acidic proteins of the nucleolar organizer regions, selectively stained by silver (AgNOR-proteins), were investigated during interphase in leukemia cells with a confocal scanning laser microscope (CSLM). Simultaneous confocal fluorescence (for specific labeling of DNA, using propidium iodide) and transmitted light microscopy combined with digital deconvolution (for the location of the AgNOR proteins in nonconfocal mode) were used. The distribution of the AgNOR proteins measured by 3D microscopy was described by their number, the volume occupation of the nucleus by the AgNOR aggregates, the distance between each AgNOR, the distance of each AgNOR to the nucleolar border, and their anisotropy. The results of the 3D analysis were compared to those obtained by conventional 2D analysis, cytogenetical analysis of metaphase nucleolar organiser regions (NORs), and cell duplication rate. The descriptive power of these 3D parameters were assessed for nine leukemic cell lines. The measurements of the 3D spatial distribution of AgNORs was a better discriminant parameter than the morphological parameters (i.e., number and volume). The 3D expression of AgNORs is also a reliable parameter for assessing proliferative activity of leukemic cells and seems to be in relation with the differentiation stage of these leukemic cells.