ABSTRACT
AgNORs are nucleolar proteins that interact specifically with silver salts. The size of silver precipitates measured by image analysis (ICM) in cycling cells proved to be inversely proportional to the cell cycle time and provided a significant correlation with prognosis for a large spectrum of cancers. Because ICM is time-consuming and poorly reproducible among laboratories using different imaging settings, this article presents a new approach to AgNOR quantitation based on flow cytometry (FCM). We report that silver precipitates caused a great decrease in the forward scattered light and that this effect was correlated with the AgNOR's relative area as measured by ICM. These results were confirmed by measuring cell lines having different cell cycle durations. Moreover, double staining using APase-Fast red fluorescence to reveal the Ki-67/MIB 1 antigen of cycling cells and silver nitrate to stain the AgNORs was successfully analyzed by FCM. The procedure makes it possible, for the first time, to validly and rapidly compare the growth fraction and cycling speed of partially proliferating cell populations, such as tumors.
Subject(s)
Nuclear Proteins/metabolism , Nucleolus Organizer Region , Alkaline Phosphatase , Antigens, Nuclear , Azo Compounds , Cell Cycle , Cell Line , Coloring Agents , Flow Cytometry/methods , Humans , Image Processing, Computer-Assisted , Ki-67 Antigen/metabolism , Light , Scattering, Radiation , Silver Nitrate , Silver StainingABSTRACT
BACKGROUND: The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. METHODS: After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. RESULTS: Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. CONCLUSIONS: The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units.
