RAPID COMMUNICATION |
Correspondence to: Bernard Jacquet, Laboratoire TIMC, IMAG UMR CNRS 5525, Equipe RFMQ, Institut Albert Bonniot, Université Joseph Fourier, Domaine de la Merci, 38706 La Tronche Cedex, France. E-mail: bjacquet@imag.fr
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Summary |
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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 APaseFast 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. (J Histochem Cytochem 49:433437, 2001)
Key Words: AgNORs, flow cytometry, image cytometry, cell cycle time, growth fraction, cell proliferation, tumor prognosis
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Introduction |
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It has long been known that nucleolar size and shape vary widely among different cell types, and even for the same cell type, as a function of the proliferating status of the cells. Argyrophil nucleolar organizer regions (AgNORs) are nucleolar proteins that interact specifically with silver salts (
Two methods are now in use for AgNOR quantitation. The first consists of counting the number of silver-stained dots per nucleus. The second consists of automatic or semiautomatic measurement of the area occupied by the silver-stained structures per nucleus by computer-assisted image analysis (ICM) (
The aim of this study was to develop a new approach to AgNOR measurement based on flow cytometry (FCM). Silver-stained dots introduce into the organic environment of the cell a refractive index heterogeneity and are therefore expected to alter the scattered light pattern, such alteration being as pronounced as the volume of silver-stained dots is high. Combined with a marker of the cell cycle, the flow cytometry of AgNORs should then become a method of choice for the assessment of cell proliferation activity in advanced and routine pathology.
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Materials and Methods |
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To investigate the effect of silver precipitates on the scattered light, cells were treated by the usual procedure described by
For FCM analysis, well-dispersed cells were fixed in 1% paraformaldehyde in PBS for 2 min at room temperature (RT) and then diluted in PBS. Fixed cells were precipitated by centrifugation for 10 min at 1000 x g, then dispersed and fixed again in glacial methanol for 10 min at -20C. The fixation solution was progressively diluted in PBS and centrifuged for 10 min at 1000 x g. The pellet was washed two times in PBS and finally suspended in PBS 3%BSA 1%Tritton X-100 (Solution A) at a final concentration of 1 million cells per milliliter. After 10 min of blocking incubation, MIB1 antibody was added at a dilution of 1:50 and incubated for 1 hr at RT. After two washes in Solution A, the GAMalkaline phosphatase (APase) was added at a dilution of 1:50 in Solution A and incubated for 1 hr at RT. An aliquot of control cells was incubated only with the GAMAPase. All antibodies used were supplied by Immunotech (Marseille, France). The APase reaction was performed at RT for 8 min with the kit FR/Naphthol AS-MX (Sigma; Saint Quentin Fallavier, France). When cells were not stained for Ki-67/MIB1 detection, propidium iodide (PI) was incorporated by incubating fixed cells for 30 min in a solution of PBS, 10 µg/ml of PI, and 1 mg/ml of RNase A. For AgNOR staining, cells were washed two times in H2O UP and the pellet was suspended in 120 µl of a solution of 200% Ag(NO3) in H2O UP (Sigma) and transferred to 60 µl of a solution of 2% gelatin in 1% formic acid. After 12 min at 37C in the dark, 10 ml of H2O UP was added and slowly agitated for 10 min at RT in the dark. Cells were then precipitated, washed once in 5 ml of H2O UP for 10 min, precipitated, and suspended in 1 ml of PBS5% sodium thiosulfate. For controls, fixed cells were suspended directly in PBS5% sodium thiosulfate.
For ICM analysis, cell fixation and staining were carried out as described by
FCM was performed using a FACscan system (BectonDickinson; San Jose, CA) managed by CellQuest software (BectonDickinson). Acquisition was carried out on 5000 to 10,000 events and was restricted to a gap defined with the double parameter FL2-A and FL2-W density dot-blot to exclude impurities. Briefly, PI fluorescence was detected by the FL2 captor and signal was treated for its intensity value (FL2-A) and its duration (FL2-W). As a consequence, for a given intensity, a single cell had a lower FL2-W value than an aggregate. FL3 captor was used for MIB1-stained cells. For each cell line, one silver-stained and one unstained control population was measured. The forward (FSC) and side (SSC) scatter channels were calibrated with the control cells and the same settings were used during acquisition of the silver-stained cells. The difference between the FSC of the control cells and that of the silver-stained cells was selected as representative of the AgNOR quantity. The experiments, from staining to measurement, were performed three times for each culture bath to calculate the average effect of AgNORs on FSC and the standard deviation.
ICM was performed as described by Canet et al. (2001) using a SAMBA 2005 system (SAMBA Technologie; Meylan, France). Briefly, for each slide, 300 cells were measured with a x40 oil immersion objective for each culture bath. The AgNOR relative area (AgNOR area divided by nuclear area) was selected as representative of the AgNOR quantity.
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Results |
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Before FAC acquisition, the staining was verified by microscopy (data not shown). We could observe the expected AgNOR silver staining specifically localized in the nucleolus. Fig 1 shows the FCM results obtained with the T47-D cell line. The FSC plotted against the SSC showed that both the control and the stained cell populations were homogeneous (Fig 1B). The mean value of the intensity in the FSC, evaluated by statistical analysis, was 1445 for the control cells and 616 for the silver-stained population (Fig 1A). Therefore, the AgNORs could be detected by a significant decrease in the FSC. As shown in Fig 2, the effect of silver staining on the FSC was linearly correlated (r2=0.99) with the AgNOR relative area measured by ICM on the three different epithelial cell lines, MCF7, T47-D, and ZR-7530 (Fig 3). The coefficients of variation of the measurements were 51%, 65%, and 51%, respectively, by ICM compared to 13%, 15%, and 39%, respectively, by FCM. The FSC value obtained by FCM can therefore be considered as actually equivalent to the relative area of AgNOR, with a reproducibility better than that of ICM.
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Because AgNOR size has been shown by ICM to be negatively correlated with the cell doubling time, we verified whether similar results could be obtained by FCM using the FSC measurement. Fig 4 shows that the size of AgNORs measured by the FSC actually decreased as the cell population doubling time increased (r2=0.94).
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Unlike cells in culture, tumor cell populations are composed of cycling and non-cycling cells. Cycling cells are usually detected by Ki-67/MIB1 immunostaining (
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Discussion |
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The results obtained in the experiments reported here suggest, for the first time, that the AgNOR size, which is conventionally measured by ICM on cycling cells, can also be measured by FCM.
Effects of Silver Precipitates on Scattered Light
It might be questioned whether the decrease in FSC actually results from the silver precipitates in the nucleolus or from any other structural alterations caused by the treatment. The controls showed that cells treated as silver-stained cells but with replacement of silver salts by H2O UP did not significantly decrease the FSC (data not shown). Moreover, the AgNOR relative area measured by ICM was linearly correlated with the effect of silver salts on the FSC. Given these observations, it can be concluded that the decrease in the FSC is equivalent to the AgNOR relative area measured by ICM.
Relevance of FCM vs ICM Measurements
The advantage of measuring AgNORs by FCM instead of ICM is a better representation of the results. In our experiments, ICM analysis was conventionally performed on 300 cells while three separate specimens, from staining to measurement and involving 5000 to 10,000 cells each, were measured by FCM, and the analysis was less time-consuming. As reported, the CVs were on average three time better with FCM compared to ICM, and the representation was potentially improved because a much higher number of cells were analyzed. We noted that the CV between aliquots of one same experiment, from staining to measurement, was very similar to the CV between experiments (data not shown), thus indicating a satisfactory reproducibility of the AgNOR staining.
Potential of FCM for Cancer Prognosis
It has been repeatedly reported that the AgNOR size is significantly greater in groups of patients with a short survival and/or relapse-free time (
Received for publication November 9, 2000; accepted December 6, 2000.
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Literature Cited |
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