Morphometry of nucleoli and expression of nucleolin analyzed by laser scanning cytometry in mitogenically stimulated lymphocytes

Background. Various attributes of nucleoli, including abundance of the nucl eolar product (rRNA), correlate with cell-proliferative status and are usef ul markers for tumor diagnosis and prognosis. However, there is a paucity o f methods that can quantitatively probe nucleolus. The aim of the present s tudy was to utilize the morphometric capacity of the laser scanning cytomet er (LSC) to analyze nucleoli and measure expression of the nucleolar protei n nucleolin (NCL) in individual cells and correlate it with their state of proliferation. Materials and Methods. Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the l evel of expression of NCL, separately in the nuclear and nucleolar compartm ents, were estimated in relation to the G(0) to G(1) transition and the cel l cycle progression. Results. During the first 24 h of stimulation, when the cells underwent G(0 ) to G(1) transition, their RNA content was increased nearly 8-fold, the le vel of NCL per nucleus also increased 8-fold, the NCL per nucleolus, increa sed 12-fold, nucleolear area increased 3-fold, and NCL/nucleolar area incre ased nearly 4-fold. During the subsequent 24 - 48 h of stimulation, when ce lls were progressing through S, G(2), and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocatio n of NCL from nucleoli to nucleoplasm was observed; its overall level per n ucleus, however, still remained high, at 6-fold above of that of G(o) cells . Conclusions. While high expression of NCL in the nucleolar compartment corr elates with the rate of rRNA accumulation in the cell and is a sensitive ma rker of the G(o) to G(1) transition, the cells progressing through the rema inder of the cycle are better distinguished from G(o) cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kin etic status of the tumor-cell population and tumor prognosis. The capabilit y of LSC to measure the protein translocation between nucleolus and nucleop lasm can be used to study the function and regulatory mechanisms of other p roteins that reside in these compartments. Cytometry 45: 206-213, 2001. (C) 2001 Wiley-Liss, Inc.