Quantitation of AgNORs by flow versus image cytometry.

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.

Correlation between silver-stained nucleolar organizer region area and cell cycle time.

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.

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytometry (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes fixed by the Boehm-Sprenger procedure optimal for preserving the chromatin pattern. The question was whether fluorochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2 c ratio was still equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the fixation conditions used. The intranuclear and the internuclear SD of the fluorescence intensities describing the fluorescence pattern of the 2c hepatocytes proved to vary according to both the basepair specificity and the binding mode of the fluorochromes. The results reported here argue in favor of an external binding of 7-AMD to DNA and an increased quantum yield of QM when bound to AT-rich DNA. For PI, EB, 7-AMD, and OM, the measured DNA content increased with the fluorescence distribution heterogeneity. This correlation was not observed with other fluorochromes and is suggested to result from decreased fluorochrome accessibility to DNA when the chromatin is condensed. This study demonstrates that under conditions that preserve chromatin organization, only HO for AT-rich DNA and MA for GC-rich DNA can be used, alone or in combination, to measure nuclear DNA content. With other fluorochromes, either the measured DNA content or the chromatin pattern is assessed in suboptimal conditions when fluorescent image cytometry is used.

Image analysis of lymphoid cell differentiation in rat thymus throughout development.

In order to identify subtle changes in cell morphology and nuclear pattern modification during thymus ontogenesis, cell image analysis using System for Analytical Microscopy in Biological Applications (SAMBA 200) was applied in 9 stages of rat thymus development. The morphometric and chromatin parameters made it possible not only to identify automatically between the two main cell populations in the thymus gland (lymphoid and epithelial cells), but also to classify automatically 5 lymphoid sub-populations (lymphoid stem cells, lymphoblasts, large lymphocytes, medium lymphocytes and small lymphocytes). The evaluation of the 18 parameters during the lymphoid cell differentiation was studied in detail. The nuclear texture parameters made it possible to discriminate, in each cell subpopulation, 4 phases of cell cycle (G0, G1, S-phase, and cells in G2). Evaluation of the nuclear parameters of the cell cycle in each lymphoid sub-population was studied in this investigation. The results illustrate the high majority of the lymphoid stem cells at the 14-day-old embryo stage while in the 20-day-old embryo the small lymphocytes become the main part of the whole lymphoid population. From the continuously renewed modification of lymphoid nuclear image analysis we discuss the origin of thymus lymphocytes. Lymphoid cells can be distinguished into different functional states and the striking morphological changes appearing during cell differentiation are related with drastic structural changes occurring in chromatin pattern from undifferentiated lymphoid stem cells to small lymphocytes. Terminal cell differentiation is associated with inhibited cell proliferation. The relative increase of chromatin condensation and nuclear pattern heterogeneity which reaches an extreme in small lymphocytes is accompanied by a progressive diminution of the nuclear area during the successive differentiation of the lymphoid population. Using one parameter of the nuclear texture features from the co-occurrence matrix (as LM or CON) and one parameter of the nuclear textures from the run-length section matrix (as GLD or RPC) the image analysis can discriminate between the different states of lymphoid cell differentiation.

Effects of gossypol on the cell cycle phases in T-47D human breast cancer cells.

Since gossypol, a naturally occurring component of cottonseed oil, exhibits a broad spectrum of activities, we have examined it as an antitumor agent on breast cancer. The effects of different concentrations of gossypol on the T-47D human breast cancer cell cycle phases were studied using cytometric image processing on Feulgen stained nuclei. The proportion of cells at different cell cycle phases was determined by discriminate analysis of the image parameters and gave good classification ranging from 86 to 100%. Gossypol was found to increase the G0/G1 fraction of the T-47D cells. This cell kinetic alteration by gossypol was shown to be dose dependent and reversible. Complete reversal of the effect of gossypol was observed after four days with a simple change to gossypol-free medium. The cells then progressed into S and G2/M phase, thus indicating that gossypol-treated cells remain viable. Gossypol was shown to have a strong inhibitory effect on cellular proliferation in T-47D cells. It was also found that this agent is only toxic to cells at the highest dose tested (10 microM). The results of this study may be of clinical significance in the treatment of breast cancer, since gossypol shows strong antiproliferative properties.

Reevaluation of optimal Feulgen reaction for automated cytology. Influence of fixatives.

The Feulgen reaction is used for cytophotometric quantification of nuclear DNA and texture studies of chromatin structure. It appears that fixative agents are responsible for the microscopic appearance of chromatin. In this investigation, different fixative agents mixed in various proportions were tested for their performance in automated quantitative cytology. It was determined that three factors have to be considered in the choice of a fixative: stain intensity, nuclear area and chromatin texture. In this respect, the Regaud fixative appears to be the best for automatic analysis of Feulgen-stained nuclei.